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Endocrinology, doi:10.1210/en.2005-1314
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Endocrinology Vol. 147, No. 4 1986-1996
Copyright © 2006 by The Endocrine Society

Classical Genotropic Versus Kinase-Initiated Regulation of Gene Transcription by the Estrogen Receptor {alpha}

M. Almeida, L. Han, C. A. O’Brien, S. Kousteni and S. C. Manolagas

Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Stavros C. Manolagas, M.D., Ph.D., Division of Endocrinology and Metabolism, Slot 587, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, Arkansas 72205. E-mail: manolagasstavros{at}uams.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Elucidation of kinase-initiated routes by which the estrogen receptors {alpha} and ß (ER{alpha} and ERß) control gene transcription, along with evidence of distinct biologic outcomes in response to ligands that can selectively activate nongenotropic signaling of the ERs or the androgen receptor, suggest that the ERs control a range of genes wider than that regulated by their direct association with DNA. To ascertain the extent and significance of nongenotropic ER-mediated transcription, we employed transduced HeLa cells expressing wild-type ER{alpha} or the ligand binding domain of ER{alpha} localized to the cell membrane (E-Mem), the OB-6 osteoblastic cell line, MCF-7 breast carcinoma cells and uteri from mice treated with 17ß-estradiol (E2), or the nongenotropic signaling activator 4-estren-3{alpha},17ß-diol (estren). E2 and estren induced ERK1/2 and Akt phosphorylation in ER{alpha} or E-Mem stably transfected HeLa cells; however, the phosphorylation kinetics differed between the two cell lines. In all four models, nongenotropic ER actions regulated a population of genes distinct from those regulated by genotropic ER actions. Specifically, the expression of Wnt2, Frizzled10, Egr-1, and c-Fos was strongly up-regulated in E-Mem-containing HeLa cells treated with E2 or estren, or in ER{alpha}-containing HeLa cells treated with estren. Up-regulation of Frizzled10 by estren was reproduced in MCF-7 cells. Egr-1 was up-regulated by both estren and E2; but complement 3, only by E2 in the uteri. Estren had no effect on complement 3, cathepsin D, progesterone receptor, bcl-2, and cyclin D1 in MCF-7 cells, whereas E2 up-regulated all these estrogen response element or activating protein-1-containing genes. These results support an extensive divergence in gene expression depending on the mode of ER activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIKE OTHER STEROID hormones, estrogens and related compounds such as selective estrogen receptor (ER) modulators bind to and activate specific receptors, which then alter the transcription of a plethora of genes. Specifically, the binding of estrogen to its receptor protein causes dimerization and conformational changes that allow several coactivator proteins to interact with the receptor dimer. These complexes attach to specific DNA response elements, known as estrogen response elements (EREs), in target gene promoters (1). In addition, ER{alpha} and ERß can interact with other DNA-bound transcription factors, such as activating protein-1 or Sp1, to influence transcription activation (2, 3). Alternatively, the activated ERs may repress transcription by forming protein-protein complexes with transcription factors, such as nuclear factor-{kappa}B, thus preventing them from interacting with their target gene promoters (4).

In addition to these classical modes of action, several effects of estrogens have been attributed to the regulation of extranuclear signaling cascades. A truncated form of the ER{alpha} or a membrane receptor distinct from the ERs may be responsible for some of these effects (5, 6, 7). However, it is now well documented that a subpopulation of the classical ER is located outside the nucleus, in the cytoplasm or at caveoli of the cell membrane (8, 9, 10, 11), and in response to estrogens, rapidly activates or deactivates cytoplasmic kinases (12, 13, 14). The kinases, in turn, modulate the activity of transcription factors and thereby influence downstream gene transcription. Therefore, ligand-activated ERs can modulate the function of transcription factors both directly or indirectly: for simplicity purposes, the former has been described as genotropic and the latter as nongenotropic.

Kinase-initiated actions of estrogens seem to be particularly relevant to nonreproductive actions of estrogen. For example, in the central nervous system estrogens activate cytoplasmic kinases to influence electrical excitability, synaptic function, and morphological features. Estrogens also exert antiapoptotic effects on neuronal cells that are mediated by activation of MAPKs or protein kinase A and protein kinase C, down-regulation of the expression of neurotrophin receptors, or by altering free radical production or free radical action on cells. In cultured endothelial cells, 17ß-estradiol (E2) rapidly stimulates endothelial nitric oxide synthase via an ER{alpha}-dependent mechanism (15, 16), which leads to phosphorylation of the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways (12). Activation of endothelial nitric oxide synthase, in turn, increases the levels of nitric oxide and is a fundamental determinant of cardiovascular homeostasis (17).

Estrogens (and androgens) protect the adult skeleton against bone loss by suppressing the rate of bone turnover and maintaining a focal balance between bone formation and resorption (18, 19, 20). Suppression of bone turnover results from attenuating effects of sex steroids on the birth rate of osteoblast and osteoclast progenitors. Maintenance of a focal balance between formation and resorption results from opposite effects on the life span of osteoblasts/osteocytes and osteoclasts: an antiapoptotic effect on the former and a proapoptotic effect on the latter cell type. The antiapoptotic effect of estrogens or androgens on osteoblasts results from a mechanism that is distinct from that requiring direct interaction of their receptors with DNA (ERE or androgen response element), or protein/protein interaction between the receptor and other transcription factors. Instead, it results from an extranuclear action of the classical receptors that causes activation of the cytoplasmic kinases Src/Shc/ERKs and PI3K or repression of c-Jun N-terminal kinase signaling and kinase-dependent changes in the activity of transcription factors (21, 22). The proapoptotic effects of estrogens on osteoclasts also involve ERK activation. The mechanistic basis of the divergence of the biologic outcome on the survival of the two cell types downstream from ERKs is evidently dependent on the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus, perhaps by determining the activation of a distinct set of transcription factors (23).

Using 4-estren-3{alpha},17ß-diol (estren), a synthetic ligand of ER{alpha}, ERß, or the AR, which simulates the nongenotropic effects of estrogens on osteoblast/osteocyte apoptosis and has minimal effects on classical transcription, we demonstrated that the nongenotropic actions of sex steroids, specifically antiapoptosis, can be dissociated from the classical transcriptional activity of the receptor (21). Next, we demonstrated that estren reversed the loss of bone resulting from gonadectomy but had no effect on female or male reproductive organs, and it did not stimulate the proliferation of breast cancer cells (24). Nevertheless, the contribution of the nongenotropic (kinase initiated) effects on gene transcription to the overall effect of the sex steroids in bone vs. reproductive tissues is mostly unknown.

The studies described herein were designed to elucidate differential effects of the classical genotropic vs. the kinase-initiated mode of ER action on gene expression in several cell types. To this end, we transduced ER-negative HeLa cells with wild-type ER{alpha} or the ligand binding domain of ER{alpha} targeted to the membrane. Using the transduced HeLa cells, as well as an osteoblastic (OB-6) and a breast cancer cell line (MCF-7), and uteri of adult ovariectomized mice, we compared the effects of estren and estradiol on gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
E2, PD98059, and etoposide were purchased from Sigma-Aldrich (St. Louis, MO). Estren was purchased from Steraloids Inc. (Newport, RI). Slow-release pellets containing vehicle, E2, or estren were purchased from Innovative Research of America (Sarasota, FL).

Plasmids
The C3-Luc reporter plasmid consisted of the complement 3 promoter inserted upstream from the firefly luciferase gene. The generation of ER{alpha}-ECFP (wild-type ER{alpha} fused to an enhanced cyan fluorescent protein) and E-Mem-ECFP (ligand binding domain of ER{alpha} targeted to the membrane and fused to an enhanced cyan fluorescent protein) constructs in ECFP-containing vectors (CLONTECH, Palo Alto, CA) has been described previously (21). For the purposes of this study, the ER{alpha}-ECFP or E-Mem-ECFP DNA sequences were excised from their respective ECFP vectors and were subcloned in the BamHI restriction site of the pLEN retroviral vector (25).

Cell culture
MCF-7 cells were cultured in MEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 1% minimum essential amino acids, 1% each of penicillin, streptomycin, glutamine, and insulin. OB-6 cells were cultured in {alpha}-MEM as previously described (21). Before use in experiments, cells were grown in medium supplemented with 2% charcoal-dextran-treated fetal bovine serum for 24 h before start of E2 or estren treatment.

Retroviral infections
Plasmids harboring the above described retroviral constructs were transiently transfected into the Phoenix ampho packaging cell line (26) using LipofectAMINE (Invitrogen). Supernatants containing viral particles were collected between 48 and 72 h after transfection, filtered through a 45-µm filter and used immediately. Subconfluent HeLa cells were exposed to viral supernatants in the presence of polybrene (4 µg/ml) for 6–12 h and then incubated in fresh MEM culture medium containing 10% fetal bovine serum for 12–24 h. The cells were then exposed to aliquots of the same supernatant two additional times. Transduced HeLa cells were then selected in MEM supplemented with 800 µg/ml G418 (Invitrogen).

Transient transfections
The C3-Luc reporter plasmid was used to assay transcriptional effects of the steroids. Transient transfections of HeLa cells were performed in 6-cm culture plates using LipofectaminePlus reagent (Invitrogen), as described by the manufacturer. The day before transfection, cells were seeded in medium containing 10% fetal bovine serum. The next day, the cells were washed once with serum-free medium and each plate was incubated with serum-free medium containing the plasmids to be transfected and the LipofectaminePlus reagent for 3 h. The medium was replaced with serum-containing medium and the cells were allowed to recover for 24 h before treatment with the hormones. Lysate preparation and luciferase activity assays were performed using the Dual-Luciferase Reporter assay system (Promega, Madison, WI), according to the manufacturer’s instructions. Light intensity was measured with a luminometer and luciferase activity was divided by the Renilla activity (control reporter) to normalize for transfection efficiency.

Quantification of apoptotic cells
Apoptotic cells were quantified by trypan blue staining as described previously (21).

In vivo experiment
Six-month-old female Swiss Webster mice, were divided into four groups of 10 animals each. Mice were then sham-operated or ovariectomized (ovx). Ovx animals were left untreated or implanted sc with 60-d slow release pellets containing E2 (0.025 mg) or estren (7.6 mg). Six weeks later, total RNA from uteri (n = 6) was extracted using Ultraspec (Biotex Laboratories, Houston, TX). All animal procedures were approved by the Institutional Animal Care and Use Committee.

Real-time PCR
Total RNA was reverse-transcribed in 100 µl of a reaction mixture that contained reverse transcription buffer, deoxynucleotide triphosphate mixture, random primers and 5 U/µl MultiScribe RT, using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA), at 25 C for 10 min and 37 C for 2 h. The sequences of the PCR primers and probes are listed in Table 1Go. These primers and probes were designed using the Assay-by-Design service (Applied Biosystems). The TaqMan MGB probe was labeled at the 5' end with the reporter dye 6-carboxyfluorescein and a minor groove binder (MGB) and at the 3' end with a nonfluorescent quencher. The amplicon for each target gene spans an exon/exon boundary to minimize the signal generated by genomic DNA that may be contaminating the RNA sample.


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  		TABLE 1. Primers and probes used for real-time PCR

 
PCR was carried out in 20-µl reaction volumes of Gene Expression Assay Mix, TaqMan Universal Master Mix, and 60–80 ng of cDNA template. The PCR was performed in an ABI 7700 Prism (Applied Biosystems) using the following cycle parameters: 1 cycle of 50 C for 2 min, 1 cycle of 95 C for 10 min, and 40 cycles of 95 C for 15 sec and 60 C for 1 min. The fold change in expression was calculated using the {Delta}{Delta} Ct comparative threshold cycle method.

Western blot analysis
The phosphorylation status of ERK1/2 and Akt was analyzed by immunoblotting using a mouse monoclonal antibody recognizing tyrosine phosphorylated ERK1/2, or a rabbit polyclonal antibody recognizing total ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA); and, a rabbit monoclonal antibody recognizing serine 473 phosphorylated Akt, or a rabbit polyclonal antibody recognizing total Akt (Cell Signaling, Beverly, MA). Horseradish peroxidase-labeled secondary antibodies were added to the membranes and the blots were developed using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL).

Statistics
The data were analyzed by ANOVA, and the Student-Newman-Keuls method was used to estimate the level of significance of differences between means. All experiments shown were repeated at least three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation and functional characterization of HeLa cells stably expressing ER{alpha} or the E-Mem
To dissociate transcription resulting indirectly, i.e. via nongenotropic kinase-initiated signaling pathways, from classical transcription mediated by direct effects of the ER that require direct DNA binding, we generated HeLa cells (an ER-negative cell line) expressing either the wild-type ER{alpha} or a mutant comprising the ligand binding domain of ER{alpha} fused to a membrane localization sequence, designated E-Mem. We have previously shown that this mutant results in extranuclear localization of the receptor and thus eliminates all classical transcriptional events that require direct DNA binding. Yet, it is fully capable of mediating the nongenotropic actions of estrogen (21). The same approach has been successfully used by others (27). Constructs expressing ER{alpha} fused to a cyan fluorescent protein (ER{alpha}-ECFP) or the ligand binding domain of ER{alpha} (E) fused to ECFP and a membrane localization sequence (E-Mem-ECFP) were inserted into a retroviral vector and used to transduce HeLa cells. HeLa cells expressing ER{alpha}-ECFP or E-Mem-ECFP were obtained after approximately 3 wk of selection with G418. In these experiments, pools of cells stably transduced with ER{alpha} or E-Mem were used to avoid potential biases associated with the use of single clones such as different rates of cell proliferation or altered signaling pathways. The localization of the different fusion proteins to the appropriate cellular compartments was confirmed by visualization of the cells with a fluorescence microscope (data not shown).

Transduced HeLa cells were tested for the expression of ER{alpha} or the E domain mRNA by real-time PCR using primers designed to recognize the E domain of the receptor (Fig. 1AGo). Both the ER{alpha} and the E domain mRNA levels in the infected HeLa cells were similar to those of the MCF-7 human breast cancer cell line, which is known to contain elevated levels of endogenous ER{alpha}. As expected, ER{alpha} was not detected in control HeLa cells that had been transduced with the vector pLREN expressing enhanced green fluorescent protein.


Figure 1
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  		FIG. 1. Characterization of HeLa cells stably expressing ER{alpha} or E-Mem. A, mRNA levels of ER{alpha} and E-Mem were evaluated in HeLa stable transfectants by real-time PCR as described in Materials and Methods. Bars indicate mean ± SD of triplicate determinations. *, P < 0.05 vs. HeLa vector by one-way ANOVA. B, HeLa-ER{alpha} or HeLa-E-Mem cells were treated for 1 h with 10–8 M E2 or vehicle followed by 6 h treatment with etoposide. The percentage of apoptotic cells was quantified by trypan blue staining. Bars indicate mean ± SD of triplicate determinations. *, P < 0.05 vs. vehicle by one-way ANOVA. C, HeLa-ER{alpha} or HeLa-E-Mem cells were transfected with C3-Luc and treated with 10–8 M E2 for 24 h. Bars represent the mean ± SD of the relative luciferase units (RLU) normalized to the Renilla luciferase activity. *, P < 0.05 vs. vehicle by ANOVA.

 
To confirm that the transduced cells were faithful models of full-length ER{alpha} or E-Mem action, we compared, in the two models, the effects of E2 on a kinase-mediated effect and on ERE-dependent transcription. Specifically, we assayed the ability of E2 to attenuate the proapoptotic effect of etoposide or to activate the transcription of a reporter construct carrying luciferase under the control of the promoter of the C3 gene (C3-Luc). This experimental design was based on earlier findings that estrogens protect ER{alpha}- or E-Mem-transiently transfected HeLa cells as well as osteoblasts and osteocytes from apoptosis induced by the DNA topoisomerase inhibitor etoposide by rapidly activating the Src/Shc/ERK signaling cascade (21); and that C3 transcription is stimulated by estrogens as a result of direct interaction of the ER with the three ERE sites contained within the C3 promoter. We found that both ER{alpha}-ECFP and E-Mem transfected HeLa cells were protected from etoposide-induced apoptosis after treatment with E2 (Fig. 1BGo). On the other hand, only the full-length ER{alpha} was able to stimulate transcription of the C3-Luc reporter within 24 h of treatment of the cells with E2 (Fig. 1CGo), confirming the expectation that extranuclear localization of the ER{alpha} cannot accommodate the classical action of estrogens.

Regulation of endogenous gene expression by E2 and estren in HeLa-ER{alpha} and HeLa-E-Mem: demonstration of selective activation of kinase-mediated actions of ER{alpha} by estren
Whereas E2 stimulated transcription of the C3 gene results from direct receptor-DNA interaction on ERE sites, E2 suppression of IL-6 gene expression results from protein/protein interaction of the ER with nuclear factor-{kappa}B (4). Based on this, we searched for the effects of E2 or estren on the expression of endogenous C3 and IL-6 in ER{alpha}- or E-Mem stably transfected HeLa cells using real-time PCR. As shown in Fig. 2Go, A and B, the expression of C3 and IL-6 were up- and down-regulated, respectively, by E2 in ER{alpha}-HeLa but not E-Mem-HeLa cells after 24 h treatment (Fig. 2AGo). Estren had no effect on C3 expression in either cell type.


Figure 2
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  		FIG. 2. Differential effects of E2 and estren on gene expression in HeLa-ER{alpha} and HeLa-E-Mem cells. A, The HeLa-ER{alpha} and HeL-E-Mem stable cell lines were treated with vehicle, 10–8 M E2 or estren for 1 and 24 h. Subsequently, cells were harvested, total RNA was extracted and then reverse transcribed to cDNA. The expression of C3 (A), IL-6 (B), Egr-1 (C), and c-Fos (D) was analyzed by real-time PCR. E, HeLa-ER{alpha} cells were treated with the ERK inhibitor PD98059 for 1 h before the addition of 10–8 M E2 for an additional 24 h. Cells were harvested, total RNA was extracted and the expression of Egr-1 was measured with real time PCR. Bars indicate means ± SD of triplicate determinations. *, P < 0.05 vs. vehicle by ANOVA.

 
In distinction to C3 and IL-6, Egr-1 and c-Fos represent genes that can be induced by activating ERK-dependent phosphorylation of the transcription factor Elk-1 and binding of Elk-1 to serum response elements (SREs) in their promoters; and both genes are rapidly up-regulated by E2 as a result of ERK activation (22, 28, 29, 30). Based on this evidence and the fact that both E2 and estren activate ERKs (see Refs.22 and 23 and Fig. 3AGo), we examined the effect of E2 or estren on the expression of these genes. Both c-Fos and Egr-1 expression was up-regulated in ER{alpha}-transduced HeLa cells at 1 h after exposure of the cells to 10–8 M E2 or estren (Fig. 2Go, C and D). However, after 24 h of treatment, only E2 stimulated c-Fos or Egr-1 expression. Strikingly, the effects of E2 or estren on c-Fos and Egr-1, after 1 h of treatment, were reproduced in the HeLa-E-Mem cells, demonstrating that the rapid effect of either ligand on the transcription of these two genes is indeed the result of a an extranuclear, i.e. nongenotropic, function of the receptor. In full support of this conclusion, the E2-induced increase in Egr-1 expression in E-Mem transduced cells was abrogated by pretreatment with the MEK1 inhibitor PD98059, demonstrating that the E2-induced up-regulation of Egr-1 expression was indeed the result of an ERK-mediated activation of the SRE site on the promoter of this gene (Fig. 2EGo).


Figure 3
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  		FIG. 3. Stimulation of ERK and Akt activity by E2 and estren in HeLa-ER{alpha} and HeLa-E-Mem cells. HeLa-ER{alpha} or HeLa-E-Mem cells were cultured in the absence of serum for 4 h before the addition of 10–8 M E2 or estren for the indicated periods of time. ERK1/2 (A) or (B) Akt (B) phosphorylation (p) was assessed by Western blot analysis. All the results shown here were reproduced in at least one additional experiment.

 
Activation of ERKs and Akt by E2 or Estren in HeLa-ER{alpha} and HeLa-E-Mem cells
We have previously shown that activation of kinases like Src/ERK and PI3K, are required for the prosurvival effect of sex steroids and estren on osteoblasts in vitro and in vivo (21, 22, 23). In line with these observations, as well as their ability to activate the expression of ERK-dependent gene targets (Fig. 2Go), we found that both E2 and estren induced within 5 min ERK phosphorylation in HeLa cells stably expressing ER{alpha} or the E-Mem (Fig. 3AGo). Subsequently, in either type of cells treated with E2 or in HeLa-E-Mem cells treated with estren, ERK phosphorylation reached a zenith at 15–30 min and was decreased to basal levels at 4 h. In contrast to this transient mode of ERK phosphorylation, in HeLa-ER{alpha} cells estren-induced ERK phosphorylation was prolonged for at least 4 h after treatment. Similar to the results obtained for ERK phosphorylation, E2 induced rapid, within 5 min, phosphorylation of Akt in ER{alpha} or E-Mem transfectants and estren in E-Mem transfectants (Fig. 3BGo). However, in HeLa-ER{alpha} transfectants estren stimulated a progressive increase in Akt phosphorylation until at least 4 h after treatment.

Up-regulation of Wnt2 and FZD10 expression by extranuclear actions of ER{alpha}
Estrogens can up-regulate the expression of several Wnt signaling family members and induce activation of canonical Wnt signaling in breast cancer cells (31, 32, 33, 34, 35). In fact, the ability of E2 to induce breast cancer cell proliferation may be linked to its Wnt-activating properties (36, 37, 38). We took advantage of our HeLa cell models to investigate whether extranuclear actions of the receptor are sufficient for the effects of E2 on a representative Wnt or Wnt receptor (Frizzled) protein. Neither E2 nor estren had an effect on Wnt-2 expression in HeLa-ER{alpha} cells after 1 h of treatment (Fig. 4AGo). However, in HeLa-E-Mem cells, estren, but not E2, up-regulated Wnt2 expression both at 1 and 24 h. Additionally, neither E2 nor estren had any effect on the expression of the Wnt receptor Frizzled10 (Fzd10) in HeLa-ER{alpha} cells. Nonetheless, both compounds up-regulated mRNA levels of Fzd10 in HeLa-E-Mem cells after 1 h of treatment (Fig. 4BGo). Furthermore, Fzd10 expression was elevated in HeLa-E-Mem cells treated with estren, but not E2, for 24 h.


Figure 4
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  		FIG. 4. Up-regulation of Wnt2 and Fzd10 expression by extranuclear actions of ER{alpha}. HeLa-ER{alpha} and HeLa-E-Mem cells were treated with vehicle, 10–8 M E2 or estren for 1 and 24 h. The expression of (A) Wnt2 and (B) Fzd10 was analyzed using real-time PCR. Bars indicate means ± SD of triplicate determinations. *, P < 0.05 vs. vehicle by ANOVA.

 
Demonstration of kinase-mediated control of gene transcription by the ERs in cells from bone and reproductive tissues
To establish the relevance of kinase-initiated vs. direct transcriptional actions of the ERs beyond the HeLa cell models, used thus far, we next compared the ability of E2 and estren to regulate gene expression in the OB-6 osteoblastic cell line, derived from murine mesenchymal bone marrow cells, and the MCF-7 breast cancer cell line in vitro, as well as in the murine uterus in vivo.

Neither estradiol nor estren affected c-Fos expression at 1 h in OB-6 cells. However, as in the HeLa cells, E2 stimulated the expression of c-Fos after 24 h of treatment in the OB-6 cells. Interestingly, estren had a similar effect at 24 h (Fig. 5Go). On the other hand, estradiol, but not estren, stimulated C3 and IGF-I expression after 24 h treatment in OB-6 cells. The effects of the two compounds on the IGF-I mRNA are consistent with evidence that IGF-I is regulated by estrogens through the protein/protein interaction mechanism (39), and the contention that estren could not mimic the effects of E2 on classical transcription, at least at the concentrations used in these in vitro experiments.


Figure 5
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  		FIG. 5. c-Fos expression is up-regulated by kinase-mediated actions of the ERs in OB-6 osteoblastic cells. OB-6 osteoblastic cells were treated with vehicle, or 10–8 M E2 or estren for 1 or 24 h. Expression of c-Fos, C3 and IGF-I was determined by real-time PCR. Bars indicate means ± SD of triplicate determinations. *, P < 0.05 vs. vehicle by ANOVA.

 
Also in agreement with the results from the HeLa transfectants, in MCF-7 breast cancer cells, which express high levels of ER{alpha}, both E2 and estren up-regulated the expression of Fzd10 and c-Fos within 1 h (Fig. 6Go). At 24 h, E2 had a considerably smaller effect on c-Fos compared with its effect at 1 h; and estren was completely ineffective. On the other hand, treatment of MCF-7 cells for 24 h with E2, but not estren, resulted in up-regulation of cathepsin D, cyclin D1, bcl-2, and progesterone receptor mRNA levels. The inability of estren to stimulate cyclin D1 expression may provide a mechanistic explanation for our previous observation that estren, as opposed to E2, fails to induce proliferation of MCF-7 cells (24).


Figure 6
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  		FIG. 6. Differential regulation of gene expression by E2 and estren in MCF-7 Cells. MCF-7 breast cancer cells were treated with vehicle, or 10–8 M E2 or estren. The mRNA levels of c-Fos were determined 1 or 24 h after treatment whereas the expression of progesterone receptor (PR), cathepsin D cyclin D1, Bcl-2 and Fzd10 was measured after 24 h of treatment with either compound. Gene expression was determined by real-time PCR. Bars indicate means ± SD of triplicate determinations. *, P < 0.05 vs. vehicle by ANOVA.

 
We had previously reported that estren reversed the ovariectomy-induced loss of bone in 6-month-old Swiss Webster mice without affecting the weight of their uteri (24). For the purpose of the present studies, we used stored mRNA from these older experiments, and compared the effects of the in vivo treatment with either E2 or estren for 6 wk on the expression of C3, IGF-I, creatine kinase B, and Egr-1 (Fig. 7Go). Consistent with the in vitro findings, E2 but not estren administration to ovariectomized mice increased the expression of IGF-I and the ERE-containing C3 and creatine kinase B genes. In contrast, treatment with both E2 and estren caused an increase in the abundance of the Egr-1 mRNA. These data support the notion that it is the classical transcriptional actions of ER{alpha}, rather than its kinase-mediated signaling, that are mainly responsible for the uterotrophic effects of estrogens.


Figure 7
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  		FIG. 7. Up-regulation of kinase-regulated gene expression but not classical transcription by estren in the murine uterus. Six-month-old Swiss Webster mice were sham-operated or ovx (OVX). The ovx animals were then left untreated or treated immediately with E2 or estren. Six weeks later, total RNA from uteri (n = 6) was isolated. C3, IGF-I, creatine kinase B (CKB) and Egr-1 expression was quantified by real-time PCR. Bars indicate means ± SD of the six determinations. *, P < 0.05 vs. ovx by ANOVA

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A large body of rapidly growing evidence has established that estrogens modify cellular responses in their numerous reproductive and nonreproductive target tissues, in part, by receptor-mediated activation of kinase-initiated signaling cascades (40, 41, 42, 43). Although these effects have been referred to in the literature at large as nongenotropic—as opposed to classical genotropic actions mediated via direct interactions of the ER/coactivator complex with DNA or via protein/protein interactions of the ERs with other transcription factors—in several well-documented instances altered kinase activity resulting from an extranuclear function of the ER{alpha} or ERß ultimately leads to downstream changes in transcription (22, 28, 31).

In the studies reported herein, we have tested directly the hypothesis that kinase-mediated actions of ER{alpha} on transcription control a different set of genes than those regulated by protein/DNA or protein/protein interactions of this receptor. To do this, we used stable transfectants of HeLa cells harboring either the wild-type ER{alpha} or only the ligand binding domain of the ER{alpha} localized to the cell membrane. In addition, we used a synthetic ligand of the ER{alpha} or ERß that stimulates kinase-mediated functions of either receptor at concentrations that are at least 4 orders of magnitude lower than those at which it can stimulate classical transcription, and compared the effects of this compound to those of E2 on the transcription of selected genes in bone and breast cancer cells in vitro and in the uterus of mice in vivo. We have obtained compelling evidence that, depending on the mode of ER activation, ER{alpha}, and most likely ERß, regulates the transcription of a divergent set of genes in cells of reproductive (uterus and breast) as well as nonreproductive (bone) target tissues. In other words, direct DNA interaction of the ER{alpha} with promoters of various genes or with other transcription factors influences the expression of a set of genes distinct from those activated by the kinase-mediated actions of the receptor.

In full agreement with our observations, Rai et al. (28) have reported distinct actions of membrane-localized, as opposed to wild-type ER{alpha}, on gene expression and the proliferation of the ER-negative MDA-MB-231 breast cancer cell line; and Pedram et al. (44) have shown that E2 rapidly (40 min) stimulates over 200 genes, including c-Fos and Egr-1, in a PI3K-dependent manner in human endothelial cells. Similarly, Beyer et al. (45) showed that E2 conjugated with BSA—a membrane impermeable estrogen analog—regulates a different set of genes compared with unconjugated E2 in neuronal and astroglial cell cultures .

Safe and colleagues (46) have suggested that regulation of the immediate early response gene c-Fos by E2 in MCF-7 cells is mediated by the raf-MAPK-dependent activation of SREs in its promoter. In agreement with these earlier findings, we show herein that c-Fos expression was up-regulated by both E2 and estren in MCF-7 cells. c-Fos as well as Egr-1 expression was also up-regulated in ER{alpha}-transduced HeLa cells at 1 h after the addition of E2. However, 24 h later this induction was lost in cells treated with estren but not in cells treated with E2. We suspect that, in the case of Egr-1, this difference reflects the fact that the Egr-1 promoter in addition to SRE elements also contains a half ERE site (32). Be that as it may, although rapid (within 1 h) and transient induction of Egr-1 expression with estren resulted from rapid and transient activation of ERK phosphorylation, long-term effects (24 h) may result from classical genotropic actions of E2 on the ERE site.

In our studies with HeLa-ER{alpha} and HeLa-E-Mem cells, the expression of Wnt2 and Fzd10 was differentially regulated by E2 and estren in the two cell models. Furthermore, whereas neither E2 nor estren were able to regulate Fz10 expression in HeLa-ER{alpha} cells, both steroids up-regulated Fzd10 mRNA levels at 1 or 24 h of treatment in HeLa-E-Mem cells. Additionally, the duration of ERK and Akt phosphorylation by estren in HeLa-E-ER{alpha} transfectants was different compared with HeLa-E-Mem cells or compared with HeLa-ER{alpha} and HeLa-E-Mem cells treated with E2. These results suggest that there is more that one signaling pathway that can be activated by the extranuclear actions of the ER{alpha}. Indeed, it is likely that different ligands may induce the recruitment of a distinct set of kinases or other signaling molecules such as G proteins or G protein-coupled receptors to the ligand-receptor complex, perhaps as a result of their ability to induce distinct conformations of the bound receptor. The formation of such extranuclear complexes will in turn dictate the specificity in the regulation of different transcriptional targets and eventually the biological outcome of distinct extranuclear ER{alpha}, and perhaps ERß, actions.

The divergence in gene expression resulting from extranuclear compared with classical genotropic function of the ER{alpha} may explain the distinct biological outcomes of ER{alpha} or ERß activation by natural ligands that activate kinase-mediated and genotropic signals alike compared with synthetic ligands that preferentially activate the former. In studies not shown here, we have determined that in pluripotent mesenchymal progenitors, derived from the bone marrow, estren decreases the Wnt antagonist Dickkopf-1, increases Wnt-1 expression, potentiates Wnt/ß-catenin-mediated transcription, and also increases bone morphogenetic protein-2 expression and Smad phosphorylation (47, 48). Consistent with these effects, estren induces commitment of these pluripotent mesenchymal progenitors to the osteoblastic lineage and also promotes the differentiation of committed osteoblastic cells, in a Src-, PI3K-, and of c-Jun N-terminal kinase-dependent manner. Importantly, in these in vitro systems, E2, or classical androgens including dihydrotestosterone and 19-nortestosterone, at concentrations as much as 5 orders of magnitude higher than estren, do not exhibit these properties, perhaps because of counter-regulatory genotropic actions.

The distinct biologic profile of estren’s action on osteoblast progenitors is entirely consistent with the demonstration herein of divergent target gene populations in response to estren compared with E2, in osteoblastic, breast, and uterine cell lines as well as in uteri in vivo. Importantly however, as we showed here, estren regulated the expression of the same set of genes, i.e. Egr-1, c-Fos, and Fzd10, in an identical manner in all the different cell types and tissues tested. Therefore, it is reasonable to expect that ligands which, similar to estren, can preferentially activate kinase-mediated signaling of the ERs may have a function-specific action, as opposed to the tissue-specific action of selective ER modulators, which is also distinct from that of classical sex steroids.

In full agreement with a greater than 4 orders of magnitude difference in the potency of E2 vs. estren on MCF-7 cell proliferation we had reported earlier (24), in the present studies, we found that estren did not affect the expression of cyclin D1, a major regulator of entry into the proliferative stage of the cell cycle (49, 50, 51, 52, 53). It is reasonably well documented that E2 activates MCF-7 cell proliferation through ER-dependent up-regulation of cyclin D1; and that the Sp1, activating protein-1 and cAMP response element sites in the cyclin D1 promoter contribute to the estrogenic response (54, 55, 56). More recently, it was shown that both the PI3K/Akt and the ERK signaling cascades are required for the ability of E2 to up-regulate cyclin D1 expression and proliferation in MCF-7 cells (57, 58). Whether the lack of an effect of estren on cyclin D1 expression results from its greatly reduced classical transcriptional actions will, of course, require additional work.

In the studies reported here, estradiol, but not estren, stimulated the expression of the bcl-2 gene. An increase in bcl-2 mRNA levels has been correlated with the ability of E2 to inhibit apoptosis of MCF-7 cells (59, 60). Moreover, bcl-2 gene or protein expression in breast cancer patients correlated positively with ER{alpha}-positive tumors and seemed to play a role in drug resistance due, in part, to inhibition of drug-induced apoptosis (60, 61, 62, 63). Two EREs located in the coding region of bcl-2 (64) and Sp1 and cAMP response element motives (65) have been implicated in E2-mediated transactivation of bcl-2 in MCF-7 cells. Based on this evidence, it is possible that the lack of an effect of estren on bcl-2 expression may reflect its weak classical transcriptional activity.

Studies with mice carrying disrupted ERs indicate that ER{alpha} mediates the major uterotrophic effects of estrogen (66). Thus, female mice in which ER{alpha} has been deleted ({alpha}ERKO) have atrophic uterus, cervix, and vagina (66, 67, 68). We and others have shown that mice (ER{alpha}NERKI/–) carrying an ER{alpha} mutant that lacks ERE activity (69) while preserving kinase-mediated signaling have an atrophic uterus (70, 71). These observations are in agreement with our findings that estren, under conditions that do not affect the weight of the uterus, has also no effect on the expression of IGF-I and the ERE-containing genes C3 and creatine kinase B. On the other hand, estren stimulates the expression of the SRE-regulated gene c-Fos. Therefore, whereas in breast cancer cells the proliferative actions of E2 require both extranuclear (kinase-initiated) and classical nuclear genotropic actions of the ER{alpha}, its uterotrophic effects result mainly from the latter.

In conclusion, the results reported in this manuscript suggest that at least some of the kinase-initiated actions of the ER{alpha}, and perhaps ERß, result in the transcription of a different set of genes than those affected by direct DNA or protein/protein interactions of the ER with other transcription factors. Nonetheless, it has been recently shown that a protein, designated modulator of nongenomic activity of the ER{alpha} (MNAR), couples ER{alpha} and Src to the MAPK signaling pathway and elicits downstream ERE transcriptional activity (72). In addition, various kinases, including MAPK and inhibitor {kappa}B kinase, phosphorylate coactivators like the steroid receptor coactivator 3 (SRC-3) and this event leads to, or at least potentiates, E2-induced nuclear translocation of the ER{alpha}-SRC-3 complex and activation of classical transcription (73, 74, 75). The extent and biological significance of such interactions remains to be explored.


   Acknowledgments
 
The authors thank Robert L. Jilka and Teresita Bellido for helpful discussions; Verenda G. Lowe and Aaron D. Warren for their technical assistance; and Robyn I. Dewall for assistance with the preparation of this manuscript.


   Footnotes
 
This work was supported by the National Institutes of Health (P01 AG013918 to S.C.M. and R01 AR049794 to C.A.O.) and the Department of Veterans Affairs (Merit Review and REAP to S.C.M.).

Disclosure statement: M.A. and L.H. have nothing to declare. C.A.O., S.K., and S.C.M. have equity interests in Nuvios, Inc. S.C.M. is member of the Scientific Advisory Board for Nuvios, Inc.

First Published Online December 29, 2005

Abbreviations: C3, Complement 3; E2, 17ß-estradiol; ECFP, enhanced cyan fluorescent protein; E-Mem, ligand binding domain of ER{alpha} localized to the cell membrane; ER, estrogen receptor; estren, 4-estren-3{alpha},17ß-diol; ERE, estrogen response element; ovx, ovariectomized; PI3K, phosphatidylinositol 3-kinase; Sp1, specificity protein-1; SRE, serum response element.

Received October 17, 2005.

Accepted for publication December 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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