MEKK1 Activation of Human Estrogen Receptor {alpha} and Stimulation of the Agonistic Activity of 4-Hydroxytamoxifen in Endometrial and Ovarian Cancer Cells

Heehyoung Lee, Feng Jiang, Qiang Wang, Santo V. Nicosia, Jianhua Yang, Bing Su and Wenlong Bai

Department of Pathology (H.L., F.J., Q.W., S.V.N., W.B.) University of South Florida College of Medicine and H. Lee Moffitt Cancer Center Tampa, Florida 33612-4799
Department of Immunology (J.Y., B.S.) M. D. Anderson Cancer Center Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens are mitogens that stimulate the growth of both normal and transformed epithelial cells of the female reproductive system. The effect of estrogens is mediated through the estrogen receptors, which are ligand-regulated transcription factors. Tamoxifen, a selective estrogen receptor modulator, functions as an estrogen receptor antagonist in breast but an agonist in uterus. In the current study, we show that coexpression of a constitutively active MEKK1, but not RAF or MEKK2, significantly increases the transcriptional activity of the receptor in endometrial and ovarian cancer cells. The expression of wild-type MEKK1 and an active Rac1, which functions upstream of MEKK1, also increased the activity of the receptor while coexpression of dominant negative MEKK1 blocked the Rac1 induction, indicating that endogenous MEKK1 is capable of activating the receptor. Additional experiments demonstrated that the MEKK1-induced activation was mediated through both Jun N-terminal kinases and p38/Hog1 and was independent of the known phosphorylation sites on the receptor. p38, but not Jun N-terminal kinases, efficiently phosphorylated the receptor in immunocomplex kinase assays, suggesting a differential involvement of the two kinases in the receptor activation. More importantly, the expression of the constitutively active MEKK1 increased the agonistic activity of 4-hydroxytamoxifen to a level comparable to that of 17ß-estradiol and fully blocked its antagonistic activity. These findings suggest that the uterine-specific agonistic activity of the tamoxifen compound may be determined by the status of kinases acting downstream of MEKK1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogens are pleiotropic hormones that regulate the growth and differentiation of many diverse tissues. They are also mitogens that promote the formation and progression of cancers of breast, endometrium, and ovary, which account for 40% of cancer incidence among women. The action of estrogens is mediated through the estrogen receptor (ER), which belongs to the steroid/thyroid nuclear receptor superfamily, a group of ligand-regulated, zinc finger-containing transcription factors (1, 2). Two ER genes have been cloned thus far: the classical ER, which is now designated in the literature as ER{alpha}, and the recently cloned ERß (3, 4, 5). Both ERs contain activation domains located in the A/B region at the amino terminus and the E region at the carboxyl terminus, known as activation function 1 and 2 (AF-1 and AF-2), respectively. While AF-1 is active to a certain extent in the absence of ligands, the activity of AF-2 is highly hormone dependent. The AFs mediate the transcriptional activation or repression of target genes independently and/or synergistically in a cell- and promoter-specific manner (6, 7). So far, the most notable difference between ER{alpha} and ERß is that they are expressed in a tissue-specific manner. For example, in females, ER{alpha} is the predominant receptor expressed in uterus while ERß is the predominant type in ovary (8, 9).

In addition to steroids, molecules such as kinase activators, phosphatase inhibitors, neurotransmitters, cell cycle regulators, and growth factors also regulate the activity of steroid receptors including ERs (10, 11, 12). The non-ligand-induced ER activation has profound clinical implications. First, it may contribute to the hormone-independent growth of human cancers. Second, it may contribute to the tissue-specific agonistic activity of synthetic antiestrogens such as tamoxifen, the prototype of selective ER modulators (SERMs). Furthermore, the physiological relevance of the non-ligand-induced ER activation has been demonstrated in whole animal experiments. For example, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) increase progesterone receptor synthesis in fetal uterus and antiestrogens block this effect (13, 14, 15). In the mouse uterus, EGF promotes growth, increases lactoferrin production, and induces biochemical changes of ERs such as increased DNA binding and production of heterogenous forms of the receptor (11, 16). These effects of EGF, which mimic those of estrogens, do not occur in ER{alpha}-deficient mice and thus are ER{alpha} mediated (17). Other studies suggest that ER{alpha} activation by EGF in COS1 (18) and Hela (19) cells is mediated through the direct phosphorylation of Ser118 in the AF-1 region by the external signal-regulated kinases (ERKs), which are the prototype of mitogen-activated protein kinases (MAPKs).

The MAPK family includes not only the ERKs but also the more recently identified c-Jun N-terminal kinases (JNKs), also referred as stress-activated protein kinases (SAPKs) and p38/Hog-1. MAPKs are phosphorylated and activated by MAPK kinases (MAPKKs; e.g. MEKs and JNKKs), which in turn are phosphorylated and activated by MAPK kinase kinases (MAPKKKs; e.g. RAF, Mos, and MEKKs). Whereas RAF and Mos primarily activate the ERKs, MEKKs are known to preferentially regulate JNK pathways.

To assess the involvement of the different MAPK pathways in ER activation, we examined the transcriptional activity of ER{alpha} in endometrial and ovarian cancer cells transfected with constitutively active forms of three different MAPKKKs, MEKK1, MEKK2, and RAF. Our studies demonstrated that the transcriptional activity of the ER{alpha} was enhanced by the expression of full-length or constitutively active MEKK1 but not by its relatives, MEKK2 or RAF. We also found that MEKK1-induced ER{alpha} activation was mediated through both JNK and p38 and that MEKK1 activation fully blocked the antagonistic activity of 4-hydroxytamoxifen, a metabolite of tamoxifen that is a well established estrogenic mitogen for endometrial cancers.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MEKK1 Increased the Transcriptional Activity of Human ER{alpha} in Endometrial Cancer Cells
Ishikawa cells are derived from tissues naturally targeted by estrogens and, for this reason, were used in our studies examining the role of MAPKs in the transcriptional activation of ER{alpha}. Because Ishikawa cells are heterogeneous with respect to endogenous ER{alpha} expression (20), we first assessed the effect of endogenous as well as transfected ER{alpha} on the activity of an ER reporter gene, EREe1bCAT, in which synthetic estrogen response elements were placed in front of the simple promoter of adenovirus E1b gene and chloramphenicol acetyl transferase (CAT). As shown in Fig. 1AGo, 17ß-estradiol did not activate the ER reporter gene in the absence of the ER{alpha} expression vector. In contrast, when the cells were cotransfected with the ER{alpha} expression vector and treated with 17ß-estradiol, reporter activity increased about 5 fold. Ectopic ER{alpha} expression had little effect on reporter activity in the absence of 17ß-estradiol. These findings demonstrate that endogenous ER, if present, lacks detectable transcriptional activity. However, the transcriptional machinery required for estrogen-induced ER activation is intact in these cells.



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Figure 1. Enhancement of ER{alpha} activity by Constitutively Active MEKK1 in Endometrial Cancer Cells

A, Ishikawa cells were transfected with 0.5 µg EREe1bCAT and 0.5 µg pLENßgal with or without 0.1 µg pLEN-hER{alpha} and treated with or without 10-10 M 17ß-estradiol (E2) as described in Materials and Methods. CAT activity was determined and normalized to ß-gal activity. B, Cells were transfected with the same constructs as in panel A but together with 0.2 µg of SR{alpha}MEKK1(CT), SR{alpha}HAMEKK2(CT), SR{alpha}RAF(BxB) or parental vector SR{alpha}3. After treatment with indicated concentrations of 17ß-estradiol, CAT activity was determined, and normalized activity is expressed as fold activation relative to SR{alpha}3 activity in the presence of 10-10 M 17ß-estradiol. C, Cells were transfected with the same constructs as in panel A and 0.2 µg of SR{alpha}MEKK1(CT) or SR{alpha}3. Transfected cells were treated with or without 10-9 M 17ß-estradiol and 10-7 M ICI 182,780 (ICI) as indicated. CAT activity is expressed as in panel B. D, Cells were transfected with 0.5 µg EREe1bCAT or EREtkCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and 0.2 µg of SR{alpha}3 or SR{alpha}MEKK1(CT). Transfected cells were treated with 10-8 M 17ß-estradiol, and CAT activity is expressed as in panel B. E, Immunodetection of human ER{alpha} from lysates of MCF-7 (positive control) or Ishikawa cells transfected with SR{alpha}3, SR{alpha}MEKK1(CT), SR{alpha}HAMEKK2(CT), or SR{alpha}RAF(BxB) and with or without pLEN-hER{alpha}. Transfected cells were treated with (+E2) or without (-E2) 10-8 M 17ß-estradiol. Duplicated samples from the transfected cells were analyzed and shown.

 
The negligible transcriptional activity of endogenous ERs allowed us to examine the effect of different MAPKKKs on the activity of exogenously expressed ER{alpha} in Ishikawa cells. As determined by reporter gene assays, the ER{alpha} activity induced by 10-10 M 17ß-estradiol was further enhanced by coexpression of constitutively active MEKK1, but not by active MEKK2 or RAF (Fig. 1BGo). The extent of enhancement was comparable to that caused by a 100-fold increase in estrogen concentration, and thus apparently is significant. MEKK1 did not activate the reporter gene in the absence of exogenous ER{alpha} expression, confirming that activation of the reporter gene by MEKK1 is ER{alpha}-dependent. This conclusion is consistent with the capacity of ER antagonist ICI 182,780 to block the MEKK1 activation (Fig. 1CGo) and the promoter independency of the activation, which was similarly observed on both EREe1bCAT and EREtkCAT (Fig. 1DGo). EREtkCAT is a more complex ER reporter in which CAT expression is controlled by synthetic estrogen response elements linked to a portion of thymidine kinase (tk) promoter which, in contrast to E1b promoter, contains binding sites for multiple sequence-specific transcription factors.

As determined by Western blotting, expression of active MEKK2 or RAF increased ER{alpha} levels in the absence of estrogen, and estrogen treatment decreased the ER{alpha} level as expected (Fig. 1EGo). However, constitutive active MEKK1 did not increase ER{alpha} expression in either the absence or presence of estrogen, indicating that the increase in ER{alpha} activity induced by MEKK1 was not due to increased expression of the transfected ER{alpha}. In the presence of estrogen, the ER{alpha} level in MEKK1-transfected cells is much lower than the level in cells transfected with control vector. Since it is known that ER activation is associated with increased ER degradation (21), the data further indicate that MEKK1 activated ER{alpha}. Consistent with transcriptional data in Fig. 1AGo, little endogenous ER{alpha} protein was detected in cells transfected with SR{alpha}3 but without ER{alpha} expression vector and treated with or without estrogen (Fig. 1EGo). The ER{alpha} antibody detected ER{alpha} in MCF-7 cells but did not detect any signal at the predicted size in ER-negative COS cells (data not shown). These findings indicate that this antibody is ER{alpha}-specific.

To determine dosage effects of the different MAPKKKs on ER{alpha} activation, various amounts of active MEKK1, MEKK2, and RAF were cotransfected with the ER{alpha} expression plasmid and EREe1bCAT reporter into Ishikawa cells, and the cultures were treated with 10-8 M 17ß-estradiol. For comparative purposes, replicate cultures were transfected with similar amounts of the MAPKKKs and an AP1 reporter, AP-1-CAT, to determine the effect of the MAPKKKs on endogenous AP1. As shown in Fig. 2AGo, MEKK1 activated ER{alpha} at all three tested dosages, but the activation was highest at an intermediate dose of 0.2 µg DNA. A similar profile was observed for AP1 activity (Fig. 2BGo), suggesting that MEKK1 at 1 µg may result in a level of MEKK1 expression that is toxic to the cell. Although neither MEKK2 nor RAF activated ER{alpha} at any of the dosages examined, these kinases activated AP1 as effectively as did MEKK1. The finding indicates that the lack of ER{alpha} activation by MEKK2 or RAF was not due to lower kinase activity or level of protein expression in the transfected cells.



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Figure 2. Correlation of MEKK1-Induced ER{alpha} Activation with MEKK1-Induced Activation of Endogenous AP1

A, Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and the indicated amounts of SR{alpha}MEKK1(CT), SR{alpha}HAMEKK2(CT), SR{alpha}RAF(BxB), or SR{alpha}3. Transfected cells were treated with 10-8 M 17ß-estradiol. B, Ishikawa cells were transfected with 0.2 µg AP-1-CAT, 0.5 µg pLENßgal, and indicated amounts of SR{alpha}MEKK1(CT), SR{alpha}HAMEKK2(CT), SR{alpha}RAF(BxB), or SR{alpha}3; CAT activity was determined, and normalized activity is expressed as fold activation relative to SR{alpha}3 activity. The total amount of DNA used in transfections was equalized with SR{alpha}3.

 
The constitutively active MEKK1 is a truncated form of full-length MEKK1. To eliminate the possibility that the increased ER{alpha} activation by the active MEKK1 was due to artifacts resulting from truncation of the kinase, the effect of full-length MEKK1 on ER{alpha} activity was examined. As shown in Fig. 3AGo, the full-length MEKK1 activated the ER in a dose- dependent manner, albeit to a lesser extent than did active MEKK1 (compare with Figs. 1BGo and 2AGo). This is not surprising because full-length MEKK1 in its basal state is less active and depends on upstream activators such as Rac1 to induce its activity to a higher level.



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Figure 3. ER{alpha} Activation by Full-Length MEKK1 and Active Rac1

A, Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and indicated amounts of SR{alpha}3 or SR{alpha}51p1 which encodes full-length MEKK1. Transfected cells were treated with (+E2) or without (-E2) 10-8 M 17ß-estradiol. B, Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, 0.2 µg Rac1L61 or SR{alpha}3, and indicated amounts of dominant negative MEKK1 (DN-MEKK1) or dominant negative MEKK2 (DN-MEKK2). Transfected cells were treated with 10-8 M 17ß-estradiol; CAT activity was determined and normalized activity is shown.

 
To determine whether endogenous MEKK1 was capable of mediating ER activation in response to signals upstream of MEKK1, the effect of Rac1 on the ER activity was examined. As shown in Fig. 3BGo, expression of active Rac1 efficiently activated ER{alpha}. Cotransfection of a dominant negative MEKK1 inhibited the effect of Rac1 in a dose-dependent manner, thus indicating that Rac1 enhanced ER{alpha} activity via activation of endogenous MEKK1. As a negative control, we also show that a dominant negative MEKK2 did not affect the Rac1-mediated ER{alpha} activation.

Activation of ER{alpha} by MEKK1 Was Mediated through JNK and p38
To elucidate the mechanism of ER{alpha} activation by MEKK1, the involvement of MAPKKs downstream of MEKK1 in ER{alpha} activation was examined. Since some published studies (22) suggested that MEKK1 could also activate MEK in addition to JNKKs, the involvement of MEK and JNKK1 in MEKK1-induced ER{alpha} activation was investigated. As shown in Fig. 4AGo, coexpression of a dominant negative JNKK1 blocked ER{alpha} activation by MEKK1. On the other hand, the specific MEK inhibitor PD98059 did little to affect ER{alpha} activation by MEKK1 (Fig. 4BGo), although, as expected, it inhibited the ERK activation by RAF (Fig. 4CGo). These data suggest that MEKK1 activation of ER{alpha} is mediated through JNKK instead of MEK.



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Figure 4. Blockage of MEKK1-Mediated ER{alpha} Activation by Dominant Negative JNKK1 but Not by a MEK Inhibitor

A, Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and, as indicated, with or without 0.2 µg SR{alpha}MEKK1(CT) or SR{alpha}HAJNKK1(AL), which encodes dominant negative JNKK1 (DN-JNKK). The total amount of DNA used in transfections was equalized with SR{alpha}3. Transfected cells were treated with 10-8 M 17ß-estradiol and CAT activity was determined. B, Cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and either 0.2 µg SR{alpha}3 or 0.2 µg SR{alpha}MEKK1(CT). Transfected cells were treated with or without 10-8 M 17ß-estradiol (E2) and indicated concentrations of PD98059. PD98059 was dissolved in dimethylsulfoxide (DMSO), and the absolute amount of DMSO added to all samples was balanced. CAT activity was determined. C, Cells were transfected with 0.2 µg SR{alpha}3 or SR{alpha}RAF(BxB) and with or without 0.5 µg HA-ERK2. Transfected cells were treated with indicated concentrations of PD98059. HA-ERK2 was immunoprecipitated from cell lysates using anti-HA antibody, and in vitro immunocomplex kinase assays were performed using MBP (3 µg) as substrate (top panel). The level of HA-ERK2 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-MBP, Phosphorylated MBP. Duplicated samples were analyzed and shown for both assays.

 
To assess the involvement of different MAPKs in ER{alpha} activation by MEKK1, the effects of MEKK1 on different MAPKs, JNK1, ERK2, and p38 were first examined. These proteins were expressed in Ishikawa cells and JNK, ERK, and p38 activities were determined by in vitro kinase assay using appropriate substrates. In agreement with previous findings, MEKK1 activated JNK1 (Fig. 5AGo). In contrast to other studies which reported that MEKK1 activated ERK but not p38 (23), we found that MEKK1 did not activate ERK (Fig. 5BGo) but significantly increased the activity of p38 (Fig. 5CGo) in Ishikawa cells. As shown in Fig. 4CGo, ERK was, however, effectively activated by RAF, thus indicating that the lack of effect of MEKK1 on ERK activity was not artifactual. The capacity of MEKK1 to activate JNK1 and p38 suggests that both kinases may mediate the effect of MEKK1 on ER{alpha} activation.



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Figure 5. Activation of JNK and p38, but Not ERK, by MEKK1 in Ishikawa Cells

A, Ishikawa cells were transfected with 0.5 µg HA-JNK1 and 0.2 µg of either SR{alpha}MEKK1(CT) or SR{alpha}3. HA-JNK1 was immunoprecipitated from the cell lysates using anti-HA antibody, and in vitro kinase assays were performed using GST-c-Jun (3 µg) as substrate (top panel). The level of HA-JNK1 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-GST-c-Jun, Phosphorylated GST-c-Jun. B, Cells were transfected with 0.5 µg HA-ERK2 and 0.2 µg of either SR{alpha}MEKK1(CT) or SR{alpha}3. HA-ERK2 was immunoprecipitated from the cell lysates using anti-HA antibody, and in vitro immunocomplex kinase assays were performed using MBP (3 µg) as substrate (top panel). The level of HA-ERK2 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-MBP, Phosphorylated MBP. C, Cells were transfected with 0.2 µg of either SR{alpha}MEKK1(CT) or SR{alpha}3 and with or without 0.5 µg Flag-p38. Flag-p38 was immunoprecipitated from the cell lysates using anti-Flag antibody, and in vitro immunocomplex kinase assays were performed with GST-ATF2 (3 µg) as substrate (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). P-GST-ATF2, Phosphor-ylated GST-ATF2; duplicated samples were analyzed and shown for both Western blotting and kinase assays in all panels.

 
To determine whether JNK activity was required for ER{alpha} activation by MEKK1, we transfected cells with a dominant negative JNK1 together with active MEKK1, and the ER{alpha} activity was determined. As shown in Fig. 6AGo, expression of dominant negative JNK1 decreased the MEKK1-induced ER{alpha} activation in a dose-dependent manner. More interestingly, dominant negative JNK1 also prevented the estrogen-induced ER activation in the absence of ectopic MEKK1 expression (Fig. 6BGo). As expected, dominant negative JNK1 inhibited Jun activation by MEKK1 (Fig. 6CGo). In this study, Jun activation was determined by measuring the activity of transfected Gal-Jun fusion protein on a Gal4 reporter, 3x17 mer-Gal-CAT. In the Gal-Jun, Gal4 DNA binding domain was fused to c-Jun transcriptional activation domain of which the activity is known to depend on the phosphorylation of Ser63 and Ser73 by JNKs. The lack of MEKK1 effect on the Gal-Jun (Ala63/73) mutant confirmed that the MEKK1-induced increase in the Gal4 reporter activity accurately measured MEKK1 activation of JNK1. The dominant negative JNK1 did not affect the activation of endogenous AP1 by RAF (Fig. 6DGo), a process that is mediated through ERK pathway. Thus, dominant negative JNK1 specifically targeted the JNK pathway. These findings demonstrate that JNK1 activity is necessary for both MEKK1-induced and 17ß-estradiol-induced ER{alpha} activation.



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Figure 6. Inhibition of MEKK1- and Estrogen-Induced ER{alpha} Activation by Dominant Negative JNK1

A, Inhibition of MEKK1-induced ER{alpha} activation by dominant negative JNK1. Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, 0.2 µg of either SR{alpha}3 or SR{alpha}3MEKK1(CT), and indicated amounts of SR{alpha}HAJNK1(APF), which encodes a dominant negative JNK1 (DN-JNK1). Transfected cells were treated with or without 10-8 M 17ß-estradiol (E2). B, Inhibition of estrogen-induced ER{alpha} activation by DN-JNK1 in the absence of ectopic MEKK1 expression. As in panel A except that cells were not transfected with SR{alpha}3MEKK1(CT) DNA. C, Inhibition of MEKK1-induced c-Jun activation by DN-JNK1. Cells were transfected with 0.2 µg 3x17 mer-Gal-CAT, 0.5 µg pLENßgal, 0 .1 µg of pGal-Jun(1–223) or pGal-Jun(1–223)(Ala63/73), 0.1 µg of SR{alpha}3 or SR{alpha}MEKK1(CT), and indicated amounts of SR{alpha}HAJNK1(APF). D, Lack of inhibition of RAF-induced activation of endogenous AP1 by DN-JNK1. The cells were transfected with 0.2 µg AP-1-CAT, 0.5 µg pLENßgal, 0.2 µg of SR{alpha}3 or SR{alpha}RAF(BxB), and indicated amounts of SR{alpha}HAJNK1(APF). CAT activity was assayed and normalized with ß-gal activity in all panels.

 
We next examined the role of p38 in ER{alpha} activation. In these experiments, Ishikawa cells transfected with active MEKK1 were treated with p38 inhibitor SB203580. As shown in Fig. 7Go, SB203580 inhibited MEKK1-induced ER{alpha} activation in a dose-dependent manner (Fig. 7AGo) and partially inhibited the estrogen-induced activation in the absence of ectopic MEKK1 expression (Fig. 7BGo), suggesting that p38 is also involved in the ER activation. As expected, SB203580 inhibited MEKK1-induced p38 activation in a dose-dependent manner (Fig. 7CGo) but had no effect on MEKK1-induced activation of endogenous AP1 (Fig. 7DGo), a process that is mainly mediated through JNK pathways. The data demonstrate that the effect of the inhibitor on MEKK1 activation of the ER was indeed due to the specific repression of p38 and that the inhibitor did not affect transcription in a nonspecific manner in the cells. These findings, coupled with those presented above, implicate both JNK1 and p38 in the activation of ER{alpha} by MEKK1 and estrogen.



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Figure 7. Attenuation of MEKK1- and Estrogen-Induced ER{alpha} Activation by a p38 Inhibitor, SB203580

A, Effect of SB203580 on MEKK1-induced ER{alpha} activation. Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and 0.2 µg of either SR{alpha}3 or SR{alpha}MEKK1(CT). Transfected cells were treated with or without 10-8 M 17ß-estradiol (E2) and indicated concentrations of SB203580. SB203580 was dissolved in DMSO, and the absolute amount of DMSO added to all samples was balanced. CAT activity was determined. B, Effect of SB203580 on estrogen-induced ER{alpha} activation in the absence of ectopic MEKK1 expression. As in panel A except that cells were not transfected with SR{alpha}MEKK1(CT). C, Inhibition of p38 activity by SB203580. Cells were transfected with 0.2 µg of either SR{alpha}3 or SR{alpha}MEKK1(CT) and with or without 0.5 µg Flag-p38. Transfected cells were treated with indicated concentrations of SB203580. Flag-p38 was immunoprecipitated with anti-Flag antibody, and in vitro immunocomplex kinase assays were performed using GST-ATF2 (3 µg) as substrates (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). Duplicated samples were analyzed and shown for both assays. P-GST-ATF2, Phosphorylated GST-ATF2. D, Effect of p38 inhibitor on MEKK1-induced activation of endogenous AP1. Cells were transfected with 0.2 µg AP-1-CAT, 0.5 µg pLENßgal, and 0.2 µg of SR{alpha}MEKK1(CT) or SR{alpha}3. Transfected cells were treated with indicated concentration of SB203580, harvested, and assayed for CAT activity.

 
p38, but Not JNK1, Phosphorylates ER{alpha}
ER{alpha} contains five known phosphorylation sites, some of which are Ser-Pro motifs that are part of the consensus sequence for phosphorylation by MAPKs. To test the requirement for each of these sites for MEKK1-induced ER{alpha} activation, we transfected into Ishikawa cells ER{alpha} mutants in which one or two of the known phosphorylation sites were changed to alanines together with active MEKK1, and reporter activity was determined. Although a difference in fold increase was observed, all of the ER{alpha} mutants were substantially activated by MEKK1 (Fig. 8Go). This finding indicates that MEKK1 activation either does not depend on phosphorylation of the receptor at any of the known phosphorylation sites or is mediated through redundant receptor phosphorylation of these sites.



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Figure 8. Effect of MEKK1 on the Activity of ER{alpha} Phosphorylation Site Mutants

Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.2 µg of SR{alpha}MEKK1(CT) or SR{alpha}3, and 0.1 µg pLEN vector encoding either wild-type or mutant ER{alpha}. Transfected cells were treated with or without 10-8 M 17ß-estradiol and CAT activity was determined. Solid bars, Cells were transfected with SR{alpha}3 and treated with 10-8 M 17ß-estradiol (SR{alpha}3 + E2); hatched bars, cells were transfected with SR{alpha}MEKK1(CT) and treated with 10-8 M 17ß-estradiol (MEKK1 + E2); double hatched bars, cells were transfected with SR{alpha}3 and treated with ethanol (SR{alpha}3 - E2).

 
Although mutation of the known ER{alpha} phosphorylation sites did not eliminate ER{alpha} activation by MEKK1, it is possible that p38 and/or JNK phosphorylates ER{alpha} at a yet unidentified site. To determine whether p38 phosphorylates ER{alpha}, we transfected Flag-tagged p38 into Ishikawa cells with or without active MEKK1. p38 was then immunoprecipitated from the cell extracts, and in vitro immunocomplex kinase assays were performed using full-length biologically active human ER protein as a substrate. As shown in Fig. 9AGo, ER{alpha} was phosphorylated by p38 immunocomplex prepared from cells both containing and lacking active MEKK1. Cotransfection of active MEKK1, however, increased ER{alpha} phosphorylation about 3-fold, an amount that correlates well with fold increase in ER{alpha} transcriptional activity that is typically induced by MEKK1. No phosphorylation of the ER{alpha} was detected in the immunoprecipitates prepared from the control vector-transfected cells, suggesting that the kinase that phosphorylated ER{alpha} in the immunoprecipitates is indeed Flag-p38. For comparative purposes, we also examined the phosphorylation of GST-ATF2 by p38 (Fig. 9BGo). Taking into consideration the fact that the mol wt of ER{alpha} is more than twice that of GST-ATF2 and that the amount of ER{alpha} used in the kinase assays was one-sixth that of GST-ATF2, p38 phosphorylated ER{alpha} at least 20-fold better than GST-ATF2 (Fig. 9BGo). Therefore, ER{alpha} is an excellent substrate for p38.



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Figure 9. Phosphorylation of Purified ER{alpha} Protein by p38

A, Ishikawa cells were transfected with 0.2 µg of either SR{alpha}MEKK1(CT) or SR{alpha}3 and where indicated, 0.5 µg Flag-p38. Flag-p38 was immunoprecipitated with anti-Flag antibody, and in vitro immunocomplex kinase assays were performed using recombinant human ER{alpha} protein (0.5 µg) as substrates (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). P-ER, Phosphorylated ER. B, Cells were transfected with 0.5 µg Flag-p38 and 0.2 µg of either SR{alpha}MEKK1(CT) or SR{alpha}3. Flag-p38 was immunoprecipitated, and in vitro immunocomplex kinase assays were performed using either recombinant ER{alpha} protein (0.5 µg) or GST-ATF2 (3 µg) as substrates (top panel). The level of Flag-p38 expression was determined by Western blotting using the same anti-Flag antibody (bottom panel). P-GST-ATF2, Phosphorylated GST-ATF2. C, Cells were transfected with or without 0.5 µg HA-JNK1 and 0.2 µg of either SR{alpha}MEKK1(CT) or SR{alpha}3. HA-JNK1 was immunoprecipitated with anti-HA antibody, and in vitro immunocomplex kinase assays were performed using either recombinant human ER{alpha} protein (0.5 µg), GST-c-Jun (3 µg), or both as substrates (top panel). The level of HA-JNK1 expression was determined by Western blotting using the same anti-HA antibody (bottom panel). P-GST-c-Jun, phosphorylated GST-c-Jun; duplicated samples were analyzed and shown for both assays in all panels.

 
The capacity of JNK1 to phosphorylate ER{alpha} was then determined by the same protocol used in the p38 studies described above. In contrast to p38, however, hemagglutinin (HA)-tagged JNK1 did not detectably phosphorylate ER{alpha} regardless of whether or not active MEKK1 was cotransfected (Fig. 9CGo). As shown in Fig. 9CGo, ER{alpha} was basally phosphorylated in immunoprecipitates lacking HA-JNK1, and the presence of MEKK1-activated HA-JNK1 in immunoprecipitates did not increase the ER{alpha} phosphorylation above this basal level. In contrast, MEKK1-activated HA-JNK1 efficiently phosphorylated GST-c-Jun. MEKK1-activated HA-JNK1 also phosphorylated GST-c-Jun in assays in which ER{alpha} protein was added to the reaction mixture, eliminating the possibility that the lack of ER{alpha} phosphorylation was due to any inhibition of activated JNK1 activity by buffer components used to dissolve ER{alpha}. The basal level of ER{alpha} phosphorylation detected in these kinase assays presumably reflects the presence of a contaminating kinase in the immunoprecipitates.

These studies suggest that JNK and p38 may have different roles in ER{alpha} activation.

MEKK1 Activates ER{alpha} in Endometrioid Ovarian Cancer Cells
To determine whether the ER{alpha} activation by MEKK1 is limited to endometrial cancer cells, ER{alpha} was transfected into epithelial ovarian cancer cells containing no endogenous ER{alpha}, and its activation by MEKK1 was examined. As determined by ER reporter assay, active MEKK1 increased ER{alpha} activity in the endometrioid ovarian cancer cell line, MDAH 2774 (Fig. 10AGo). The activation was inhibited by ICI 182,780, and neither MEKK2 nor RAF increased the ER{alpha} activity. On the other hand, none of these kinases, even at high doses, activated the ER{alpha} in the serous ovarian cancer cell, OV1063 (Fig. 10BGo). These data suggest that ER{alpha} activation by MEKK1 may be restricted to endometrial cancer and endometrioid ovarian cancer cells, which are morphologically and characteristically indistinguishable.



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Figure 10. Activation of ER{alpha} by MEKK1 in Endometrioid Ovarian Cancer Cells

A, MDAH-2774 endometrioid ovarian cancer cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and 0.15 µg of either SR{alpha}MEKK1(CT), SR{alpha}HAMEKK2(CT), SR{alpha}RAF(BxB), or SR{alpha}3. Transfected cells were treated with vesicle (-hormone), 10-9 M 17ß-estradiol (E2) or 10-9 M 17ß-estradiol plus 10-7 M ICI 182,780 (E2 + ICI) as indicated. B, OV1063 serous ovarian cancer cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and indicated amounts of SR{alpha}MEKK1(CT), SR{alpha}HAMEKK2(CT), SR{alpha}RAF(BxB), or SR{alpha}3. Transfected cells were treated with 10-9 M 17ß-estradiol; cells were harvested and assayed for CAT activity.

 
MEKK1 Affects the Agonistic as Well as Antagonistic Activity of 4-Hydroxytamoxifen
ER{alpha} is the major ER subtype in the uterus, and tamoxifen is known to act as an ER agonist in the development of endometrial tumors. Thus, the effect of MEKK1 on the agonistic activity of tamoxifen was examined in Ishikawa cells. We found that in the absence of active MEKK1, 4-hydroxytamoxifen only slightly activated ER{alpha} (Fig. 11Go) and thus is a poor agonist. Expression of active MEKK1 resulted in a significant increase in 4-hydroxytamofen-induced ER{alpha} activity, the level of which was comparable to that of cells stimulated with 10-8 M 17ß-estradiol. These findings show that MEKK1 activation allows 4-hydroxtamoxifen to function as a potent ER agonist.



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Figure 11. The Effect of Active MEKK1 on Both the Agonistic and Antagonistic Activity of 4-Hydroxytamoxifen

Ishikawa cells were transfected with 0.5 µg EREe1bCAT, 0.5 µg pLENßgal, 0.1 µg pLEN-hER{alpha}, and 0.2 µg of SR{alpha}MEKK1(CT) or SR{alpha}3. Transfected cells were treated with either vehicle (none), 10-8 M 17ß-estradiol (E2), 10-6 M 4-hydroxytamoxifen (4-HT), 10-8 M 17ß-estradiol plus 10-6 M 4-hydroxytamoxifen (E2 + 4-HT), or 10-8 M 17ß-estradiol plus 10-7 M ICI 182,780 (E2 + ICI) as indicated. CAT activity was determined.

 
To determine the effect of MEKK1 on the antagonistic activity of 4-hydroxytamoxifen, we also examined the effect of 4-hydroxytamoxifen on ER{alpha} activity when added to Ishikawa cells in combination with 17ß-estradiol (Fig. 11Go). In cells not expressing active MEKK1, 4-hydroxytamoxifen inhibited the estrogen-induced ER{alpha} activation as efficiently as did ICI 182,780. In contrast, in cells expressing active MEKK1, 4-hydroxytamoxifen did not inhibit the estrogen-induced ER{alpha} activation, and in fact, slightly enhanced the activation. These results demonstrate that 4-hydroxytamoxifen acts as an ER antagonist in the absence of MEKK1 and an agonist in the presence of MEKK1. ICI 182,780, on the other hand, ablated ER{alpha} activation by 17ß-estradiol and thus functions as an ER antagonist irrespective of whether or not active MEKK1 is expressed.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results of our study demonstrate that MEKK1, but not MEKK2 or RAF, activated ER genes in endometrial cancer cells. The activation was observed using both simple or complex promoter-based reporters. MEKK1- induced activation was inhibited by the estrogen antagonist ICI 182,780 and required cotransfection of the ER{alpha} expression plasmid, and thus is mediated through ER{alpha}. Since MEKK1 did not enhance the level of ER{alpha} expression, it must increase the reporter activity by enhancing the specific transcriptional activity of ER{alpha}. Although MEKK1 consistently enhanced ER{alpha} activity in cells treated with different concentrations of 17ß-estradiol, its effects on the ER{alpha} in the absence of ligands were variable. Thus, more studies are required to determine whether MEKK1 participates in the ligand-independent activation of ER{alpha}.

Our data in endometrial cancer cells show that MEKK1 activated both JNK1 and p38, and that ER{alpha} activation by MEKK1 was mediated through JNKK-JNK/p38 pathways. These results differ from those of previous studies which implicate the RAF-ERK pathway in ER{alpha} activation in Hela (19) and COS (18) cells. In these studies, the ER activation resulted from ERK-mediated phosphorylation of ER{alpha} at Ser118. In our studies, however, Ala118 ER{alpha} mutant was still activated by MEKK1. In addition, MEKK1 did not activate ERK in our system, and active RAF did not activate ER{alpha}. These results strongly argue against the involvement of the RAF-ERK pathway in MEKK1-induced ER{alpha} activation. It is important to note that ER{alpha} activation by various signals is both tissue- and promoter specific, and thus our results and others are not necessarily contradictory. In fact, they raise the interesting possibility that different cell types use different MAPK pathways to activate ER{alpha}.

Our studies suggest that none of the known phosphorylation sites on ER{alpha}, including Ser118, is absolutely required for ER{alpha} activation by MEKK1. This finding suggests that MEKK1-induced ER{alpha} activation may involve the phosphorylation of ER{alpha} at a novel site. p38 activity was required for ER{alpha} activation by MEKK1, and we found that p38 efficiently phosphorylated ER{alpha} in a MEKK1-regulated manner. p38 phosphorylated ER{alpha} to a greater extent than it phosphorylated GST-ATF2, a physiological substrate for p38; thus, ER{alpha} is an excellent substrate for p38. Although JNK activity was also needed for MEKK1-induced ER{alpha} activation, it did not detectably phosphorylate ER{alpha}. Overall, these data suggest that ER{alpha} activation by MEKK1 is mediated through two independently acting downstream kinases: p38, which directly phosphorylates ER{alpha}, and JNK1, which indirectly promotes ER{alpha} activation, perhaps by phosphorylating proteins interacting with ER{alpha}.

Our previous studies on the progesterone receptor (PR) showed that PR-A and PR-B are differentially activated by MEKK1 and MEKK2 in Hela cells (24). However, in contrast to the findings in the current study, PR activation by MEKKs was independent of JNK and p38. In the case of glucocorticoid receptor, JNK activation repressed receptor activity, and the repression was relieved by mutation of JNK sites (25). During the preparation of this manuscript, it was reported that reporter genes for androgen receptors were activated by MEKK1 in prostate and breast cancer cells (26). However, in this instance, it was not clear whether the observed activation resulted from increases in the expression level or the transcriptional capacity of the receptor. Moreover, the involvement of JNK and p38 in MEKK1-induced androgen receptor activation was not examined. We believe our data are the first to demonstrate that JNK and p38 signaling pathways activate steroid receptors.

As mentioned in the Introduction, the uterus exemplifies a system in which both steroid and nonsteroid signals impinge on the ER in a physiologically relevant manner and tamoxifen stimulates tumorigenesis. ER{alpha} activation by MEKK1 and the consequent increase in the agonistic activity of tamoxifen compounds suggest that MEKK1-JNK/p38 may be involved in mediating the stimulatory effect of estrogen and tamoxifen in tumorigenesis of endometrial epithelium. The MEKK1-induced activation of ER{alpha} transfected into MDAH 2774 cells suggests that MEKK1 may also play a similar positive role in the formation of endometrioid ovarian cancers, which are morphologically and characteristically indistinguishable from endometrial cancers and may have a similar sensitivity to estrogen and tamoxifen. Although some studies have implicated MEKK1 in apoptosis (27), a positive role for MEKK1-JNK pathways in tumorigenesis has been suggested by studies showing that cellular transformation by oncogenes such as Met (28), Abl (29) and certain mutant EGF receptors (30) depends on JNK activity and that the genetic deletion of MEKK1 from mice impaired the survival of murine embryonic fibroblasts to stress signals (23).

To our knowledge, our data showing that MEKK1 abrogates the antagonistic activity of 4-hydroxytamoxifen is the first demonstration of such an effect by an active kinase. Targeting the MEKK1-JNK/p38 pathway, therefore, represents a potential means of eliminating the undesired stimulatory effects of tamoxifen in endometrial tumorigenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Tissue culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). [3H]Chloramphenicol was from NEN Life Science Products (Boston, MA), and N-butyryl Coenzyme A from Pharmacia Biotech (Piscataway, NJ). 17ß-Estradiol, 4-hydroxytamoxifen, myelin basic protein (MBP), and M2 antibody were from Sigma (St. Louis, MO). PD98059 and SB203580 were from Calbiochem (La Jolla, CA). Purified hER{alpha} protein was from Affinity BioReagents, Inc. (Golden, CO). GST-ATF2 and GST-c-Jun proteins were from New England Biolabs, Inc. (Beverly, MA). The ECL reagents for immunoblotting were from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). Antibody for ER{alpha} (F-10) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 12CA5 and protease inhibitor cocktail tablets were from Roche Molecular Biochemicals (Indianapolis, IN). ICI 182,780 was from Tocris (Baldwin, MO). All other reagents were reagent grade.

Plasmids
The pLEN-hER{alpha}, EREe1bCAT, and EREtkCAT were kindly provided by Dr. Carolyn L. Smith (31). pLENßgal (24), pGal-ATF2 (32), HA-JNK1 (32), HA-ERK2 (32), Flag-p38 (32), AP-1-CAT (33), pGal-Jun(1–223) (32), pGal-Jun(1–223)(Ala63/73) (32), and 3x17 mer-Gal-CAT (34) have been described in previous studies. Dominant negative MEKK2 is an unpublished construct (B. Su, unpublished). Constitutively active MEKK1 [SR{alpha}MEKK1(CT)] (35), MEKK2 [SR{alpha}HAMEKK2(CT)] (36), RAF1 [SR{alpha}RAF(BxB)] (37), Rac1 (37) as well as dominant negative JNK1 [SR{alpha}HAJNK1(APF)] (38), MEKK1 (35), JNKK1 [SR{alpha}HAJNKK1(AL)] (36), and full-length MEKK1 (SR{alpha}51p1) (39) are all in the SR{alpha}3 vector and have also been described previously.

The receptor phosphorylation site mutants in pCMV5 were kindly provided by Dr. Benita Katzenellenbogan (40). Due to our unpublished observation that the cytomegalovirus (CMV) promoter was strongly activated by constitutively active MEKK1, which interferes with our transcriptional assays, all mutants were subcloned into the same pLEN vector used to express the wild-type receptor as well as ß-galactosidase, the internal standard for normalization of the reporter activity in all of our transcription assays. The Ala537 mutant was removed from pCMV5 by BamHI and ligated into the BamHI site in pLEN. pCMV5 expressing the remainder of the mutants was first digested with EcoRI at the 5'-end of the mutant ER cDNA and a BamHI-EcoRI linker was then ligated to it. The plasmids were further cut by BamHI at the 3'-end, and the mutant cDNAs were ligated into the BamHI site of the pLEN vector.

Transfections
Ishikawa cells were grown in DMEM containing 10% FBS. One day after plating, cells were transfected by a lipofectamine-mediated transfection procedure following the protocol from Life Technologies, Inc. In brief, the medium was removed, and phenol red-free DMEM was added to the cells. The DNA-lipofectamine complexes were prepared and added dropwise to the cells. After 5 h incubation, cells were washed with HBSS, and phenol red-free DMEM containing 1% charcoal-stripped FBS was added to the cells. On the following day, hormones and inhibitors were added to the cells. After 24 h, cells were harvested and assayed for CAT and ß-galactosidase (ß-gal) activity as indicated below. For in vitro kinase assays and Western blots, cells were treated on the third day after transfection with hormone or kinase inhibitors for 30 min and lysed.

Transcriptional Assays
The transfected cells were harvested by scraping into TEN buffer (40 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8.0), collected by centrifugation, resuspended in 0.25 M Tris, pH 7.5, and lysed by freeze-thawing. CAT activity (41, 42) and ß-gal activity (41) were assayed as previously described. CAT activity was normalized to ß-gal activity. Duplicate samples were analyzed for each data point.

In Vitro Immunocomplex Kinase Assays
Transfected cells were washed with ice-cold PBS and lysed in buffer containing 20 mM Tris (pH. 7.6), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 5 mM ß-glycerophosphate, 100 µM Na3VO4, 0.5% NP-40, and protease inhibitor cocktail (1 tablet/10 ml) for JNK1 or ERK2 kinase assays. For p38 kinase assays, the transfected cells were lysed in buffer containing 10 mM HEPES (pH 7.6), 250 mM NaCl, 3 mM EDTA, 100 mM Na3VO4, 1% Triton X-100, and protease cocktail (1 tablet/10 ml). After centrifugation, half of the supernatant was immunoprecipitated with 12CA5 antibody for HA-ERK2 and HA-JNK1 or M2 antibody for Flag-p38. Kinase reactions were performed at 30 C in 20 µl buffer containing 20 mM HEPES (pH 7.6), 10 mM PNPP, 20 mM MgCl2, 2 mM EDTA, 2 mM EGTA, 100 µM Na3VO4, 10 µM ATP, 10 µCi {gamma}-32P-ATP with the appropriate substrate. For MBP, GST-c-Jun, and GST-ATF2, 3 µg were used and, for the purified ER{alpha} protein, 0.5 µg was used. Reactions were terminated by adding 2x SDS-PAGE sample buffer, analyzed by SDS-PAGE, and visualized by autoradiography. For each kinase assay, duplicate samples were analyzed.

Immunoblotting Analysis
To determine ER{alpha} expression, transfected cells were washed with PBS and lysed by adding 0.5 ml hot 2x SDS-PAGE sample buffer directly to the plate. The cells were scraped into 1.5-ml microcentrifuge tubes, heated at 100 C for 10 min, and spun for 10 min at room temperature in a microcentrifuge set at maximum speed. Supernatants (50 µl) was separated on a 7.5% SDS gel.

To determine the expression of HA-ERK2, HA-JNK1, and Flag-p38, half of the cell lysate prepared for kinase assays was mixed with one sixth of 6x SDS-PAGE sample buffer, heated for 10 min at 100 C, and separated on a 10% SDS gel.

After separation on SDS-PAGE, all samples were transferred to nitrocellulose membrane, probed with the corresponding antibodies, and visualized using an ECL kit following the instructions of the manufacture. For each Western blot, duplicate samples were analyzed.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Carolyn L. Smith for the ER expression and reporter vectors, Dr. Benita S. Katzenellenbogen for phosphorylation site mutants of the ER{alpha} and Drs. Doug Cress, Nancy Olashaw, and Warren J. Pledger for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Wenlong Bai, Department of Pathology, University of South Florida College of Medicine and H. Lee Moffitt Cancer Center, 12901 Bruce B. Downs Boulevard, MDC 11, Tampa, Florida 33612-4799. E-mail: wbai{at}com1.med.usf.edu

This work was supported by NIH Grant R29 CA-79530 to W.B.

Received for publication April 12, 2000. Revision received August 1, 2000. Accepted for publication August 7, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  2. Tsai M-J, O’Malley BW 1995 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  3. Kuiper GGJM, Enmark E, Peito-Huikko M, Nilson S, Gutafsson J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  4. Mosselman S, Polman J, Dijkema R 1996 ER ß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  5. Tremblay GB, Tremblay A, Copeland NC, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353–363[Abstract/Free Full Text]
  6. Tora L, Whote J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation domains. Cell 59:477–487[Medline]
  7. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike WJ, McDonnell DP 1994 Human estrogen receptor transactivation capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  8. Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gutafsson J 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor {alpha} and ß. Mol Endocrinol 138:863–870
  9. Saunders PTK, Maguire SM, Gaughan J, Miller MR 1997 Expression of estrogen receptor beta (ER ß) in multiple rat tissues visualized by immunohistochemistry. J Endocrinol 154:R13–R16
  10. Bai W, Weigel NL 1995 Phosphorylation and steroid hormone action. Vitam Horm 51:289–313[Medline]
  11. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, Machlachlan JA, Korach KS 1992 Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci USA 89:4658–4662[Abstract]
  12. Kazenellenbogen BS, Norman MJ 1990 Multihormonal regulation of the progesterone receptor in MCF-7 human breast cancer cells: interrelationships among insulin/ insulin-like growth factor-I, serum, and estrogen. Endocrinology 126:891–898[Abstract]
  13. Aronica SM, Kazenellenbogen BS 1993 Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-1. Mol Endocrinol 7:743–752[Abstract]
  14. Sumida C, Pasqualini JR 1990 Stimulation of progesterone receptors by phorbol ester and cyclic AMP in fetal uterine cells in culture. Mol Cell Endocrinol 69:207–15[CrossRef][Medline]
  15. Sumida C, Pasqualini JR 1989 Antiestrogens antagonize the stimulatory effect of epidermal growth factor on the induction of progesterone receptor in fetal uterine cells in culture. Endocrinology 124:591–7[Abstract]
  16. Nelson KG, Takahashi T, Bossert NL, Walmer DK McLachlan JA 1991 Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation. Proc Natl Acad Sci USA 88:21–5[Abstract]
  17. Curtis SW, Washburn T, Sewall C, DiAugustine R, Lindzey J, Couse JF Korach KS 1996 Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor. Proc Natl Acad Sci USA 93:12626–30[Abstract/Free Full Text]
  18. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige H, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract]
  19. Bunone G, Briand P, Miksicek RJ, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphor-ylation. EMBO J 15:2174–2183[Abstract]
  20. Nishida M, Kasahara K, Oki A, Satoh T, Arai Y, Kubo T 1996 Establishment of eighteen clones of Ishikawa cells. Hum Cell 9:109–116[Medline]
  21. Nawaz Z, Lonard DM, Dennis AP, Smith CL, O’Malley BW 1999 Proteasome-dependent degradation of the human estrogen receptor. Proc Natl Acad Sci USA 96:1858–1862[Abstract/Free Full Text]
  22. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL 1993 A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260:315–319[Medline]
  23. Yujiri T, Sather S, Fanger GR, Johnson GL 1998 Role of MEKK1 in cell survival and activation of JNK1 and ERK pathways by targeted gene disruption. Science 282:1911–1914[Abstract/Free Full Text]
  24. Bai W, Bingman III WE, Yang J, Edwards DP, Su B, Weigel NL, Differential activation of PR-A and PR-B by MEKKs. Program of the 80th Annual Meeting of The Endocrine Society. New Orleans, LA, 1998 (Abstract OR14–5)
  25. Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ 1997 Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol 17:3947–3954[Abstract]
  26. Abreu-Martin MT, Chari TA, Palladino A, Craft NA, Sawyers CL 1999 Mitogen-activated protein kinase kinase kinase 1 activates androgen receptor-dependent transcription and apoptosis in prostate cancer. Mol Cell Biol 19:5143–5154[Abstract/Free Full Text]
  27. Widmann C, Johnson NL, Gardner AM, Smith RJ, Johnson GL 1997 Potentiation of apoptosis by low dose stress stimuli in cells expressing activated MEK kinase 1. Oncogene15:2439–2447
  28. Rodrigues GA, Park M, Schlessinger J 1997 Activation of the JNK pathways is essential for transformation by the Met oncogene. EMBO J 16:2634–2645[Abstract/Free Full Text]
  29. Raitano AB, Halpern JR, Hambuch TM, Sawyers CL 1995 The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation. Proc Natl Acad Sci USA 92:11746–50[Abstract]
  30. Antonyak MA, Moscatello DK, Wong AJ 1998 Constitutive activation of c-Jun N-terminal kinase by a mutant epidermal growth factor receptor. J Biol Chem 273:2817–2822[Abstract/Free Full Text]
  31. Smith CL, Conneely OM, O’Malley BM 1993 Modulation of the ligand-independent activation of the human estrogen receptor by hormone and antihormone. Proc Natl Acad Sci USA 90:6120–6124[Abstract]
  32. Yang J, New L, Jiang Y, Han J, Su B 1998 Molecular cloning and characterization of a human protein kinase that specifically activates c-Jun N-terminal kinase. Gene 212:95–102[CrossRef][Medline]
  33. Aronheim A, Engelberg D, Li N, al-Alawi N, Schlessinger J, Karin M 1994 Membrane target of nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell 78:949–961[Medline]
  34. Onate SA, Tsai SY, Tsai M-J, O’Malley BW 1995 Sequence and Characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  35. Minden A, Lin A, Mcmahon M, Lange-Carter C, Derjard B, Davis RJ, Johnson GL, Karin M 1994 Differential activation of ERK and JNK mitogen activated protein kinases by Ras-1 and MEKK. Science 266:1719–1723[Medline]
  36. Cheng J, Yang J, Xia Y, Karin M, Su B 2000 Synergistic interaction of MEKK2, JNKK2, JNK1 results in efficient, specific JNK1 activation. Mol Cell Biol 20:2334–2342[Abstract/Free Full Text]
  37. Minden A, Lin A, Claret F-X, Abo A, Karin M 1995 Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPase Rac and Cdc42Hs. Cell 81:1147–1157[Medline]
  38. Kallunki T, Su B, Tsigelny I, Sluss HK, Derijard B, Moore G, Davis R, Karin M 1994 JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation. Genes Dev 8:2996–3007[Abstract]
  39. Xia Y, Wu Z, Su B, Murray B, Karin M 1998 JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension. Genes Dev 12:3369–3381[Abstract/Free Full Text]
  40. Lee Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of human estrogen receptor. J Biol Chem 269:4458–4466[Abstract/Free Full Text]
  41. Bai W, Weigel NL 1996 Phosphorylation of Ser 211 in the chicken progesterone receptor modulates its transcriptional activity J Biol Chem 271:12801–12806[Abstract/Free Full Text]
  42. Brian S, Sheen J 1988 A simple phase-extraction assay for chloramphenicol acetyltransferase activity. Gene 67:271–277[CrossRef][Medline]