A Tumor-Specific Truncated Estrogen Receptor Splice Variant Enhances Estrogen-Stimulated Gene Expression

Sushela S. Chaidarun and Joseph M. Alexander

Neuroendocrine Unit Massachusetts General Hospital and Department of Medicine Harvard Medical School Boston, Massachusetts 02114


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study examines the cooperative effects of a human estrogen receptor-{alpha} (ER{alpha}) isoform on estrogen (E2)-mediated gene activation in U2-OS osteosarcoma cells. {Delta}5ER{alpha}, an alternatively spliced ER{alpha} variant lacking exon 5, is coexpressed with normal ER{alpha} in several E2-responsive neoplastic tissues. However, the potential interactions of {Delta}5ER{alpha} with normal ER{alpha} have not been functionally characterized. {Delta}5ER{alpha} encodes the hormone-independent trans-activating function (AF-1), as well as the constitutive receptor dimerization and DNA-binding domains. It is generated by an alternate splice event that omits exon 5 and alters the reading frame of the resulting mRNA. The {Delta}5ER{alpha} protein is prematurely truncated and lacks the majority of the hormone-binding and activating function-2 (AF-2) domains. When {Delta}5ER{alpha} mammalian expression vector was transfected alone in human ER{alpha}/ERß-negative osteosarcoma U2-OS cells, it had no effect on either basal or E2-mediated EREtk81Luc reporter transcriptional activity, while transfected cells expressing control normal ER{alpha} increased EREtk81Luc activity up to 20-fold in response to 10 nM E2. However, when {Delta}5ER{alpha} was cotransfected with normal ER{alpha}, both basal and E2-stimulated EREtk81Luc reporter activation were increased approximately 500% over levels observed when cells were transfected with ER{alpha} alone. Similar effects of {Delta}5ER{alpha} and normal ER{alpha} coexpression were observed using an E2-responsive human C3 promoter/luciferase reporter construct. The effects of {Delta}5ER{alpha} on normal ER{alpha} were further assessed in U2-OS cells stably transfected with normal ER{alpha}. Transfection of increasing amounts of {Delta}5ER{alpha} expression vector into [ER{alpha}+]OS cells resulted in potentiation of E2-stimulated ERELuc activity in a synergistic, dose-dependent manner. Moreover, coexpression of {Delta}5ER{alpha} in [ER{alpha}+]OS cells improved E2 sensitivity 100-fold over cells expressing ER{alpha} alone. Proliferation rates of stable U2-OS cell lines expressing {Delta}5ER{alpha} were significantly increased (P < 0.05), with cell doubling times reduced from 35 h in control parental U2-OS cells to 28 h in [{Delta}5ER{alpha}]OS cells. However, growth rates were not affected by either E2 or tamoxifen treatment. Electromobility shift/supershift assays using nuclear extracts of U2-OS cells stably transfected with ER{alpha} and {Delta}5ER{alpha} confirmed the constitutive binding of {Delta}5ER{alpha} and ER{alpha} protein to estrogen-response element (ERE) sequence independent of E2 and also showed an increase in {Delta}5ER{alpha}/ER{alpha}-ERE complexes with E2 treatment. These data are consistent with interactive effects of normal ER{alpha} and {Delta}5ER{alpha} on transcription from classic ERE gene promoters. {Delta}5ER{alpha} appears to therefore act as a dominant positive receptor that increases both basal and E2-stimulated gene transactivation of normal ER{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen (E2) has long been known to promote the growth of certain human neoplasms, notably tumors of the breast, endometrium, and pituitary. It also modulates the development and function of normal tissues, such as bone, as well as the cardiovascular and central nervous system (1, 2). The mitogenic and regulatory effects of E2 are mediated through its two nuclear receptors, estrogen receptor-{alpha} and -ß (ER{alpha} and ERß), ligand-activated transcription factors that are members of the steroid receptor superfamily (3, 4). The genomic structure of the ER{alpha} gene reveals strong homology to viral v-erbA, suggesting that ER{alpha} is a cellular homolog of this oncogene (3). ER{alpha} is encoded by eight exons within a genomic locus of greater than 140 kb (5). It has been well documented that ER{alpha} isoform variants generated by alternative splicing of ER{alpha} heteronuclear RNA are coexpressed in a number of human normal and neoplastic tissues (6, 7, 8, 9, 10, 11, 12, 13). Thus, ER{alpha} gene expression and alternative splicing have been postulated to create a heterogeneous population of ER{alpha} isoforms with differential transcriptional activity, which may help to potentiate the diverse action of E2 through a single gene.

The ER{alpha} protein is composed of several structural domains, each of which has a unique function in ligand binding, gene promoter activation, and association with other members of the general transcriptional apparatus (14). The A/B domain has a ligand-independent gene activation function (AF-1) and is encoded by exon 1. It has been shown to be important for stimulating transcription from certain E2-responsive genes such as pS2 (14), c-fos (15), and C3 (16). In addition, it is also critical for growth factor interactions with ER{alpha}-signaling pathways both in yeast and mammalian cells (17). However, because the A/B domain cannot bind DNA directly, it has been hypothesized to activate target genes by associating with components of the core transcriptional machinery, such as TFIID and other coactivators/repressors (18). The ER{alpha} DNA-binding domain (DBD) lies within the C region and is encoded by exons 2 and 3. The DBD is composed of two type II zinc (Zn) finger motifs that have been shown by structure/function studies to be directly responsible for DNA promoter sequence recognition (19). The D region (or variable hinge region) is thought to allow the ER{alpha} to alter conformation and is encoded by exon 4. This ER{alpha} domain may potentiate much of the allosteric regulation of the receptor after ligand binding (14). It also contains a constitutive nuclear localization signal as well as sequences required for dimerization of the ER{alpha}. Finally, the COOH-terminal E region is encoded by exons 5–8. It is functionally complex and is the most characterized domain in terms of structure and function (20, 21, 22). The E region contains protein sequences important for 1) heat-shock protein association in the cytoplasm, 2) nuclear localization, 3) ligand-dependent receptor dimerization and the AF-2 gene activation function, and 4) E2 and antiestrogen ligand binding.

This study focuses on the cooperative effects of human {Delta}5ER{alpha}. This variant encodes the A/B domain as well as the C and D domains critical for binding estrogen-response elements (EREs), receptor dimerization, and nuclear localization, but lacks the hormone-binding domain. It is generated by an exon 5 splice deletion, and the subsequent fusion of exons 4 and 6 results in an immediate frame shift, a short novel carboxyl terminus (GTRQNV), and termination codon. The exon-5 ER{alpha} spliced variant {Delta}5ER{alpha} has been shown by others to have approximately 10–15% constitutive transcriptional activity of normal ER{alpha} when expressed alone in yeast (23). However, little is known as to their potential cooperative effects when coexpressed with normal ER{alpha}. We and others have shown that in E2-sensitive human tissues, {Delta}5ER{alpha} variant isoform was typically found to be coexpressed with normal receptor. Both tamoxifen-resistant and primary breast tumors express {Delta}5ER{alpha} along with normal ER{alpha}. {Delta}5ER{alpha} expression was elevated in ER{alpha}+ tumors that were tamoxifen-resistant and in ER{alpha}- tumors that expressed the E2-responsive markers, PgR (progesterone receptor) and pS2 (24). In pituitary tumors, {Delta}5ER{alpha} was found to be tumor specific and coexpressed with normal ER{alpha} only in prolactinomas and gonadotroph tumors, but not in normal pituitary or other pituitary tumor phenotypes (13). Therefore, we hypothesized that {Delta}5ER{alpha} may interact with normal ER{alpha} and may play a role in tumor pathogenesis

The present study examined the hypothesis that the {Delta}5ER{alpha} tumor-specific splice variant is capable of regulating E2-responsive genes, which, in turn, control cellular phenotype and growth. These experiments investigated 1) the transcriptional effects of coexpression of {Delta}5ER{alpha} and normal ER{alpha} on E2-responsive gene promoter/reporter constructs, 2) the ability of {Delta}5ER{alpha} and normal ER{alpha} to stably bind ERE complexes in electrophoretic mobility shift/supershift assays (EMSAs) using nuclear extracts from stable cell lines harboring either normal ER{alpha} or {Delta}5ER{alpha}, and 3) the effects of {Delta}5ER{alpha} on cellular proliferation rates in stably transfected human U2-OS osteosarcoma cell lines.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Characterization of Human ER{alpha} and {Delta}5ER{alpha}
Normal ER{alpha} and {Delta}5ER{alpha} full-length cDNA were cloned by RT-PCR from pooled human gonadotroph and lactotroph tumor mRNA. These pituitary tumor subtypes have been previously shown to express the {Delta}5ER{alpha} variant in a tumor-specific manner along with full-length ER{alpha} (13). Figure 1AGo shows the exon/intron structure and functional domains of normal ER{alpha} as well as {Delta}5ER{alpha}. {Delta}5ER{alpha} is created by an RNA splice omission of exon 5, causing a fusion of exons 4 and 6, which acts to shift the open-reading frame and creates a truncated carboxy terminus. As a result of this altered splice event, {Delta}5ER{alpha} lacks almost the entire ligand-binding and AF-2 region and cannot bind E2. Figure 1BGo shows the expected in vitro translation protein products from normal human ER{alpha}- and {Delta}5ER{alpha}-cloned cDNAs. [35S]methionine-labeled protein products from ER{alpha} and {Delta}5ER{alpha} cDNAs migrated at the expected sizes of 67 and 41 kDa, respectively.



View larger version (37K):
[in this window]
[in a new window]
 
Figure 1. In Vitro Translation of cDNAs Encoding Human ER{alpha} and Splice Variant {Delta}5ER{alpha}

A, Schematic structure of normal ER{alpha} and the {Delta}5ER{alpha} alternative splice variant. {Delta}5ER{alpha} is generated by alternative splicing of exon 5, joining exons 4 and 6. This exon 5 skip alters the open-reading frame and results in a novel seven-residue COOH terminus followed by a termination codon encoded by the 5'-end of exon 6. {Delta}5ER{alpha} encodes the AF-1 gene activation, DNA-binding, and hinge region domains of the ER, but lacks the AF-2 ligand-binding domain. B, In vitro translated [35S]methionine-labeled ER protein from pBKCMV expression plasmids. Kilodalton markers are shown to the right of the gel. Lower molecular weight translation products in the CMV/ER{alpha} lane correspond to internal methionine residues in the full-length ER cDNA.

 
Cloned normal human ER{alpha} and {Delta}5ER{alpha} cDNAs were ligated into the pBKCMV expression plasmids to study their action in mammalian cells. U2-OS cells were stably transfected with either normal ER{alpha} or {Delta}5ER{alpha} to generate cell lines which harbor and constitutively express each ER{alpha} isoform. Geneticin (G418)-resistant clonal U2-OS cell lines were isolated and human ER{alpha} isoform expression was examined by immunocytochemistry using a human ER{alpha}-specific antibody which recognized an epitope within the A/B domain (residues 22–43) of both {Delta}5ER{alpha} and normal ER{alpha}. Immunocytochemical data for these stable cell lines are shown in Fig. 2Go. Figure 2AGo shows negative control parental U2-OS cells, which, based on RT-PCR analyses, do not express any form of ER{alpha} or ERß. No staining could be seen in these cells with a specific human ER{alpha}-1 antibody directed against the amino terminus of ER{alpha}, while control in ER{alpha}-positive MCF-7 breast cancer cells exhibited demarcated nuclear staining (data not shown). Figure 2Go, B and C, shows immunocytochemical localization of {Delta}5ER{alpha} and ER{alpha}, respectively, within the nuclear/perinuclear compartment. The pBKCMV-driven expression of each ER{alpha} isoform appeared to be at equivalent levels in the [ER{alpha}]OS and [{Delta}5ER{alpha}]OS stable cell lines used in this analysis. No staining was seen in any stable cell line when primary antibody was omitted or preabsorbed to in vitro translated ER{alpha} protein (data not shown). Together, the in vitro transcription/translation and immunocytochemical data demonstrate the predicted protein size and cellular protein expression of both ER{alpha} and its {Delta}5ER{alpha} splice variant in stably transfected U2-OS cell lines.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 2. Intracellular Localization of ER{alpha} and {Delta}5ER{alpha} in Stably Transfected U2-OS Cells

Immunocytochemical localization of ER{alpha} and {Delta}5ER{alpha} in stably transfected U2-OS cells. Mouse monoclonal antibody ER{alpha}1 against the A/B domain was detected using a secondary biotinylated goat antimouse IgG and tertiary avidin-biotinylated horseradish peroxidase conjugate. Control immunostaining in which primary antibody was preabsorbed to in vitro translated normal ER{alpha} protein was negative (data not shown). A, parental U2-OS cells; B, pBKCMV/{Delta}5ER{alpha} U2-OS cells; C, pBKCMV/ER{alpha} U2-OS cells.

 
Coexpression of {Delta}5ER{alpha} Enhances Both Basal and E2-Stimulated Gene Expression by Normal ER{alpha}
U2-OS cells have been reported to be unresponsive to E2 and fail to express normal ER{alpha} or ERß as measured by RT-PCR. We confirmed the reported U2-OS cell estrogen-resistant phenotype by transiently transfecting the E2-responsive human EREtk81Luc reporter construct with or without pBKCMV/ER{alpha} mammalian cell expression vector. Figure 3Go demonstrates that U2-OS cells are unresponsive to E2 levels as high as 0.1 µM, with no significant increases in ERE-driven luciferase activity above control wells. However, when transiently transfected with pBKCMV/ER{alpha}, U2-OS cells exhibited significant (P < 0.05) up-regulation of EREtk81Luc activity at E2 doses as low as 1 nM. Normal ER{alpha} activation of the reporter construct was found to be E2 dose-dependent, ranging from 9- to 15-fold activation with administration of 10-9 to 10-7 M E2, respectively. No activation was seen at any E2 dose in cells that were transiently transfected with empty pBKCMV vector. These data confirm that U2-OS cells lack endogenous ER{alpha} or ERß with a concomitant inability to activate ERE transcription. Moreover, the nonresponsive phenotype can be rescued by transfection of pBKCMV/ER{alpha}, which enables U2-OS cells to up-regulate E2-dependent gene transcription in response to physiological levels of exogenous E2.



View larger version (24K):
[in this window]
[in a new window]
 
Figure 3. Dose-Dependent Responses to E2 in U2-OS Cells Require Transfected Normal ER{alpha}

One microgram of pBKCMV/ER expression vector was transiently transfected into U2-OS cells, followed by E2 adminstration at the indicated doses for 24 h. Control nonresponsive U2-OS cells were transfected with empty pBKCMV vector. one microgram of EREtk81Luc was cotransfected into all wells. *, P < 0.001 vs. control wells transfected with empty pBKCMV vector.

 
To examine the effects of normal ER{alpha} and {Delta}5ER{alpha} on E2-responsive gene transcription, U2-OS cells were transiently transfected with ER{alpha} and/or {Delta}5ER{alpha} along with EREtk81Luc, followed by treatment with E2. The results of these cotransfection studies are shown in Fig. 4AGo. {Delta}5ER{alpha} had no constitutive or E2-stimulated effects on EREtk81Luc transcription when expressed alone in U2-OS cells. However, coexpression of {Delta}5ER{alpha} with normal ER{alpha} resulted in significant up-regulation of both basal (P < 0.01) and E2-stimulated (P < 0.001) EREtk81Luc activity 4- and 33-fold, respectively, over basal levels in the control osteosarcoma (OS) cells, or approximately 5-fold compared with cells transfected with ER{alpha} alone. Transfection of 2 µg of ER{alpha} increased basal and E2-stimulated luciferase reporter activity approximately 2-fold over levels seen with transfections using 1 µg of ER{alpha}. The dose-dependent effect of {Delta}5ER{alpha} on the regulation of EREtk81luc gene transcription was also examined in stably transfected [ER{alpha}+]OS cells (Fig. 4BGo). Coexpression of {Delta}5ER{alpha} with normal ER{alpha} significantly (P < 0.001) increased E2-mediated trans-activation of the classic ERE promoter in a dose-dependent manner, up to 90-fold with 3 µg {Delta}5ER{alpha} compared with [ER{alpha}+]OS cells transfected with empty pBKCMV vector. Basal levels of EREtk81luc activity also significantly increased (P < 0.001) in a dose-dependent manner up to 314% of baseline.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 4. Effect of {Delta}5ER{alpha} on Transactivation of ERE Promoters in [ER{alpha}-] and [ER{alpha}+] Cells

A, [ER{alpha}-]OS cells were transiently cotransfected with 1 µg of EREtk81Luc reporter vector, with or without the indicated amount of ER{alpha} and/or {Delta}5ER{alpha} expression vectors, followed by 10 nM E2 in treatment wells for 24 h. Luciferase activity was determined, and representative data shown are the mean ± SEM, n = 3. ***, P < 0.001 vs. control wells; *, P < 0.05 vs. basal levels with 2 µg ER{alpha}. B, {Delta}5ER{alpha} dose response: Modulation of normal ER{alpha} transactivation on ERE promoter. [ER{alpha}+]OS cells were transiently transfected with 1 µg EREtk81Luc and varying amounts of {Delta}5ER{alpha}, followed by 10 nM E2 in treatment wells for 24 h. pBKCMV vector without insert was used to keep constant stoichiometric levels of CMV promoter in each transfected well (total CMV vector DNA = 3 µg/well). Data are the mean ± SEM, n = 3. All basal and E2-stimulated levels were significantly elevated (P < 0.001) over {Delta}5ER{alpha}-negative basal values as measured by Student’s t-test.

 
To determine whether coexpression of {Delta}5ER{alpha} with normal ER{alpha} enhances OS cells sensitivity to lower E2 dose administration, E2 dose-response curves were conducted in [ER{alpha}+]OS cells that were cotransfected with either empty pBKCMV or cytomegalovirus (CMV)/{Delta}5ER{alpha} along with the EREtk81Luc reporter. Cells were then treated with increasing [10-13 M to 10-7 M] E2 doses for 24 h, and EREtk81Luc activity was subsequently assayed. Figure 5Go shows an E2 dose-response curve in [ER{alpha}+]OS cells. Cells that coexpressed {Delta}5ER{alpha} were 100-fold more sensitive to E2 administration than OS cells expressing normal ER{alpha} alone. Moreover, the magnitude of the transcriptional response was on average 550% at higher E2 doses [10-10 M to 10-7 M].



View larger version (22K):
[in this window]
[in a new window]
 
Figure 5. U2-OS Cells That Coexpress {Delta}5ER{alpha} and Normal ER{alpha} Are More Sensitive to E2 Administration Than Cells That Express ER{alpha} Alone

Cells stably expressing CMV/ER{alpha} were transiently transfected with 1 µg/well CMV/{Delta}5ER{alpha} and EREtk81Luc. Wells were then treated with 10 nM E2 for 24 h and assayed for luciferase activity. Values are the mean ± SEM, n = 3. *, P < 0.001 compared with minus {Delta}5ER{alpha} control by Student’s t-test.

 
We repeated and confirmed our cotransfection studies using an E2-responsive human complement 3 promoter/luciferase reporter construct, C3T1Luc. The C3 promoter contains three E2-responsive regions, one of which resembles the consensus ERE sequence while the other two do not show significant homology to known EREs and act as weak EREs on heterologous promoters. However, in the context of the C3 promoter, these three EREs act synergistically to create a strong ERE enhancer (16). The results of this experiment are shown in Fig. 6AGo. The C3T1luc reporter exhibited an identical pattern of activation as the heterologous EREtk81luc reporter in U2-OS cells. Basal levels of C3T1luc activity in wells cotransfected with both ER{alpha} and {Delta}5ER{alpha} were enhanced by 5.5-fold over those seen in wells transfected with either ER{alpha} or {Delta}5ER{alpha} alone. E2-stimulated C3T1luc activity was increased 33-fold over the basal levels and 8.7-fold over E2-treated cells transfected with ER{alpha} alone. In contrast, when a c-fosLuc reporter containing imperfect ERE sequences was used, both ER{alpha} and {Delta}5ER{alpha} showed very little effect on c-fos transcription activity (Fig. 6BGo), suggesting that the classic palindromic ERE sequences are necessary for the cooperative transcriptional effect of ER{alpha} and {Delta}5ER{alpha}.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 6. Coexpression of {Delta}5ER{alpha} and ER{alpha} Confers Constitutive Activation and Enhances E2 Sensitivity to the C3T1luc Promoter in U2-OS Cells

Cells were transiently transfected with 1 µg of CMV/ER{alpha} and/or CMV/{Delta}5ER{alpha} along with 1 µg of C3T1luc (A) or -2000 c-fosLuc (B) reporter vectors, followed by 24-h 10 nM E2 treatments. Values are the mean ± SEM, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t-test.

 
Electrophoretic Mobility Shift/Supershift Assay (EMSA) Studies with Nuclear Extracts from Normal ER{alpha} and {Delta}5ER{alpha} U2-OS Stable Cell Lines
To further characterize the DNA-protein interaction between classic ERE sequences and normal ER{alpha} and/or its {Delta}5ER{alpha} protein variant, gel-shift assays were performed on nuclear extracts from normal ER{alpha} and {Delta}5ER{alpha} stable cell lines, and results from these EMSAs are shown in Fig. 7Go. There was a faint, but detectable, band shift of labeled ERE oligonucleotide in nuclear extracts prepared from parental ER{alpha}-negative U2-OS cells (lanes 1 and 2). As expected, nuclear extracts derived from ER{alpha}-stably transfected cells showed a marked increase in protein-ERE complexes with E2 treatment (lanes 3 and 4). Conversely, {Delta}5ER{alpha} nuclear extracts were capable of binding ERE oligonucleotide, independent of E2 (lanes 5 and 6). In addition, in ER{alpha}/{Delta}5ER{alpha} mixed extracts, this shifting pattern was increased in intensity in the presence of E2 (lanes 7 and 8). The specificity of the gel-shift was demonstrated by competition assays with excess, unlabeled ERE DNA (Fig. 7Go, lanes 9–14).



View larger version (54K):
[in this window]
[in a new window]
 
Figure 7. EMSAs of ERE Promoter Sequence by {Delta}5ER{alpha} and Normal ER{alpha} OS Cell Nuclear Extracts

Lanes 1–8, Nuclear extracts (2 µg/lane) from each stable OS cells grown in the presence or absence of 10 nM E2, or mixed {Delta}5ER{alpha}/ER{alpha} extracts were coincubated with labeled ERE and fractionated by 5% PAGE. Lanes 9–14, Binding specificity of OS[ER{alpha}+] nuclear extract was tested by increasing the relative concentrations of cold competitor ERE in the presence of 1.0 pM of 32P-labeled ERE per lane. Bound ERE DNA is demarcated by the arrow (sDNA). Unbound ERE was electrophoresed off the gel and is not shown.

 
Supershift experiments with antibodies specific to the amino and carboxy termini of normal ER{alpha} and {Delta}5ER{alpha} were carried out to determine whether the observed EMSA patterns were due to direct interactions of normal and {Delta}5ER{alpha} variant with ERE complexes. Figure 8Go shows the results of these experiments with nuclear extract from each stable U2-OS cell line. There was no detectable supershift with either antibody in parental OS extracts, while both antibodies supershifted ERE DNA in the normal ER{alpha} extracts. As expected, the amino terminus antibody supershifted ERE in the {Delta}5ER{alpha} extracts. However, the carboxy-terminus antibody, which recognizes an epitope in the hormone-binding domain distal to the {Delta}5ER{alpha} premature termination codon, failed to supershift {Delta}5ER{alpha}/ERE complexes. Finally, normal ER{alpha}/{Delta}5ER{alpha} mixed nuclear extracts, as expected, displayed supershifted patterns of ER{alpha}/ERE complexes with both antibodies.



View larger version (78K):
[in this window]
[in a new window]
 
Figure 8. EMSAs of ERE Promoter Element DNA by ER{alpha} and {Delta}5ER{alpha} Variant

EMSAs were performed with 2 µg of nuclear extracts from the appropriate U2-OS stable cell line grown in the presence of 10 nM E2, or mixed {Delta}5ER{alpha}/ER{alpha} extracts. The position of protein-ERE (sDNA) and Ab-ER{alpha}-ERE (ssDNA) complexes are indicated. N, ER{alpha} antibody specific for the amino- terminal AF-1 domain; C, ER{alpha} antibody specific for the carboxyl terminal AF-2 domain absent in the {Delta}5ER{alpha} variant. 32P-labeled free DNA probes were electrophoresed off the gel and are not shown.

 
Proliferation Studies Utilizing Stable U-2 OS Cells Expressing ER{alpha} and {Delta}5ER{alpha} Variant
To assess the potential growth effects of {Delta}5ER{alpha} variant, we conducted growth studies of each stable cell line. Stable transfectant U2-OS cell clones were screened for the expression of ER{alpha} and {Delta}5ER{alpha} mRNA by Northern blot analysis (data not shown) and immunocytochemistry (ICC) using a monoclonal antibody against the A/B domain common to each ER{alpha} receptor studied (as shown in Fig. 2Go). Figure 9Go shows representative growth curves of each OS cell line. Cell-doubling times derived from the log phase of the growth curve are the average of three proliferation studies. Cell doubling time for parental OS cells was 35 ± 3.5 h (mean ± SD). OS cells stably transfected with normal ER{alpha} exhibited slightly slower cell-doubling rates of 39 ± 4.0 h, but the difference was not statistically significant by two-way ANOVA analysis. However, stable [{Delta}5ER{alpha}]OS cells exhibited a decrease in cell doubling time of 28 ± 1.2 h, a change that was also statistically significant (P < 0.05) over both parental and [ER{alpha}+]OS cell lines. Addition of either 10 nM E2 or 1 µM tamoxifen to culture medium had no significant effect on the growth rate of OS, [ER{alpha}+]OS, and [{Delta}5ER{alpha}]OS cell lines (data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
Figure 9. Growth of ER{alpha}-Negative and Stably Transfected ER{alpha}-Positive Cell Lines: Effects of Variant ER{alpha} Expression on U2-OS Cell Proliferation

Cells were plated at a density of 1 x 104 cells per well (day 0) and were grown in phenol red-free DMEM containing 5% charcoal-stripped FCS. Cell number was determined by Coulter cell counting, and representative data shown are the mean ± SEM of triplicate wells. *, Statistically significant differences in the growth curve by two-way ANOVA in cell growth as compared with parental U2-OS cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study examines the cooperative effects of the human {Delta}5ER{alpha} isoform with normal ER{alpha} on estrogen (E2)-mediated gene activation in U2-OS osteosarcoma cells. It demonstrates that the human {Delta}5ER{alpha} isoform can markedly enhance the transcriptional effects of normal ER{alpha} on E2-responsive genes in this human cell line. {Delta}5ER{alpha}, an alternatively spliced ER{alpha} variant lacking exon 5, is coexpressed with normal ER{alpha} in several E2-responsive neoplastic tissues, including primary pituitary (13), endometrial (25), and breast tumors (26, 27). These studies provide direct, functional evidence demonstrating that {Delta}5ER{alpha} can markedly up-regulate both basal and E2-stimulated classical ERE promoter gene activation when coexpressed with normal ER{alpha} in vitro. It also details experiments describing the subcellular distribution, DNA-binding ability, and proliferative potential of {Delta}5ER{alpha} variant in the human U2-OS cell line.

As predicted by its structure, the {Delta}5ER{alpha} variant has similarities to normal ER{alpha} as well as important differences. It encodes the A/B domains as well as the C and D domains critical for DNA binding and constitutive nuclear localization, respectively. EMSA experiments demonstrate that nuclear extracts from cells stably expressing {Delta}5ER{alpha} can bind ERE sequences in the presence and absence of E2. Immunocytochemistry utilizing stable cell lines harboring the {Delta}5ER{alpha} expression vector confirm that the truncated protein is expressed and at least partially compartmentalized in the cell nucleus. However, due to an alternate splice event that omits exon 5 and alters the reading frame of the resulting mRNA, the {Delta}5ER{alpha} protein is prematurely truncated and lacks the majority of the hormone-binding and activating function-2 (AF-2) domains. Coupled in vitro transcription/translation of {Delta}5ER{alpha} cDNA demonstrates that the cloned isoform encodes the expected 41-kDa protein product lacking the hormone binding and AF-2 domains. Transient transfection of {Delta}5ER{alpha} demonstrates that it is unable to trans-activate E2-responsive reporter vectors in the presence of E2.

Previous studies have conclusively demonstrated that {Delta}5ER{alpha} has minimal constitutive activity when expressed either in yeast or mammalian cells (6, 7, 23, 28). Experiments in which {Delta}5ER{alpha} was transfected with either the EREtk81Luc or C3Luc reporter constructs confirmed these results. When {Delta}5ER{alpha} cDNA was transfected alone in human ER{alpha}/ERß-negative osteosarcoma U2-OS cells, it had no effect on either basal or E2-mediated EREtk81Luc or C3T1luc reporter transcriptional activity. However, this cell line clearly contains the basic transcriptional apparatus to up-regulate E2-stimulated gene activity, because transfected cells expressing control normal ER{alpha} increased EREtk81Luc activity up to 20-fold in response to 10 nM E2. These findings with normal ER{alpha} are in agreement with other studies that examine normal ER{alpha} activation of E2-responsive gene transcription in various OS cell lines (29, 30, 31, 32, 33). Our data also confirm previous studies that show that the {Delta}5ER{alpha} isoform has minimal activity when expressed alone. However, when {Delta}5ER{alpha} was cotransfected with normal ER{alpha}, both basal and E2-stimulated EREtk81Luc reporter activation were increased approximately 500% over levels observed when cells were transfected with ER{alpha} alone. Similar effects of {Delta}5ER{alpha} and normal ER{alpha} coexpression were observed using an E2-responsive human C3 promoter/luciferase reporter construct.

Because these data were obtained using transient transfection of U2-OS cells, we chose to extend and confirm these observations in stable cell lines expressing normal ER{alpha}. This approach allowed us to quantitatively assess the impact of {Delta}5ER{alpha} coexpression on normal ER{alpha} transactivation. Transfection of increasing amounts of {Delta}5ER{alpha} expression vector into [ER{alpha}+]OS cells resulted in potentiation of both basal and E2-stimulated ERELuc activity in a synergistic, dose-dependent manner. Collectively, these transient and stable transfection studies confirm what the structure of {Delta}5ER{alpha} predicts: When expressed alone in an ER{alpha}-negative cell, {Delta}5ER{alpha} is unable to bind E2 and fails to up-regulate either EREtk81luc or C3T1luc gene activity either in the presence or absence of E2. However, the unexpected result in this study is that {Delta}5ER{alpha} can act as a dominant positive receptor isoform and facilitate both basal and E2-stimulated ERE-mediated transcription of normal ER{alpha} when coexpressed in U2-OS cells.

The mechanism underlying the observed constitutive and E2-stimulated transcriptional activation of the classic ERE promoter by {Delta}5ER{alpha} and normal ER{alpha} may be due to formation a {Delta}5ER{alpha}/ER{alpha} heterodimer capable of binding ERE promoter sequences and activating transcription independent of E2. Several studies utilizing site-specific mutagenesis of ER{alpha} in both yeast expression systems and MCF-7 cells have led to the general hypothesis that ER{alpha} activation of gene transcription is facilitated by an interaction between AF-1 in the A/B domain and AF-2 in E domain (34, 35, 36). However, both AF-1 and AF-2 can also activate transcription independently, and each can bind the basal transcription components of the preinitiation complex directly, notably TFIIB and TFIID, in a ligand-independent manner in vitro (37, 38). In addition, {Delta}5ER{alpha} encodes a recently described third activation domain, AF2a, located between residues 282 and 351 of the D region (39). Data from electromobility shift/supershift assays using nuclear extracts of U2-OS cells stably transfected with ER{alpha} and {Delta}5ER{alpha} confirmed the constitutive binding of {Delta}5ER{alpha} protein to ERE-containing complexes in the presence and absence of E2. Parallel with transcriptional studies, EMSA also showed the expected increase in ER{alpha}-or ER{alpha}/{Delta}5ER{alpha}-ERE complexes with E2 treatment. The observed increase in the receptor-ERE bound complex with E2 treatment has been described as a result of the formation of ER{alpha} DBD-ERE complexes with greater stability (19, 40). These findings suggest the possibility that altered structure of {Delta}5ER{alpha} truncated receptor may promote enhanced binding to ERE promoters even in the absence of E2. Taken together, the results from functional transfection studies on the classic ERE transcription and gel shift assays of direct DNA-protein interactions in vitro are consistent with both constitutive and cooperative transcriptional activation of the classic ERE promoter by the coexpressed {Delta}5ER{alpha} and normal ER{alpha}.

In this study we found that human osteosarcoma cell lines stably transfected with a {Delta}5ER{alpha} expression vector exhibit E2-independent increases in cellular growth rates. Proliferation rates of stable U2-OS cell lines expressing {Delta}5ER{alpha} were significantly increased (P < 0.05), with cell-doubling times reduced from 35 h in control parental U2-OS cells to 28 h in [{Delta}5ER{alpha}]OS cells. Tamoxifen or E2 treatment had no significant effect on cellular proliferation rates in any of the U2-OS stable cell lines studied. This is not unexpected in {Delta}5ER{alpha} stable lines because of a lack of ligand-binding domain in the truncated receptor. In contrast to our proliferation data in U2-OS cells, {Delta}5ER{alpha} has been shown to have little effect on MCF-7 breast cancer cell growth in response to E2 or tamoxifen (41). These differences in {Delta}5ER{alpha} growth effects in MCF-7 cells may be confounded by endogenous production of {Delta}5ER{alpha} as well as a number of other ER variants in that cell line (8). Alternatively, these data may represent cell-specific mechanisms of {Delta}5ER{alpha} action on cellular proliferation in human osteosarcoma cells.

In contrast to growth effects seen with {Delta}5ER{alpha}, U2-OS cells stably transfected with normal ER{alpha} showed insignificant alterations in proliferative potential when compared with parental U2-OS cells and were not affected by treatment with either E2 or tamoxifen. These findings are consistent with several characterized human osteosarcoma cell lines which either fail to respond or inhibit growth in the presence of E2 in vitro. These proliferative responses may depend, in part, on exogenous ER{alpha} expression levels. For example, in the HTB 96 human OS cell line, E2 caused a growth-inhibitory response in lines that were stably transfected with ER{alpha}, when compared with a parental HTB 96 OS cell line (32). Human SaOS-2 cell lines stably transfected with ER{alpha} also exhibit growth inhibition when treated with E2 (29). However, similar growth studies with HOS TE85 human osteoblastic cells exhibited no proliferative response to exogenous E2 (30). Thus, although the proliferative effects of E2 in other human cell lines derived from E2-sensitive tissue (i.e. uterus and breast) are well-characterized, the data in osteoblast-like osteosarcoma cell lines suggest E2 effects on proliferative potential are limited in this in vitro cellular system.

It is unclear how the truncated {Delta}5ER{alpha} lacking the ligand-binding domain can enhance cellular proliferation in stable U2-OS cells. Several studies have demonstrated that the A/B domain containing AF-1 of the ER{alpha} is important for peptide growth factor interaction, independent of E2 (18, 37). Moreover, a mutagenesis-generated ER{alpha} isoform lacking much of the hormone- binding domain E has also been shown to activate c-fos promoter independent of E2 administration in HeLa cells (42). Therefore, the naturally occurring {Delta}5ER{alpha}, which encodes only the A/B (AF-1) and DBD domains, might function to up-regulate growth factor-induced proliferative responses. One potential hypothesis is that this isoform is a potent target for peptide growth factor kinase-signaling pathways that influence its interactions with other coregulatory proteins. This may result in enhanced cellular growth in the absence of stimulation of ERE-dependent gene activation, and an E2-independent growth phenotype.

Studies examining potential interactions of structurally altered ER{alpha} isoforms may have important implications for the well described ER{alpha} isoform coexpression observed in human E2-sensitive neoplastic tissues. If ER{alpha} isoform expression plays a role in growth regulation of E2-sensitive tumor cells, dysregulation of mRNA-splicing mechanisms that give rise to these variants in neoplastic cells may be selective during tumor progression to a more aggressive phenotype. These data are consistent with interactive effects of normal ER{alpha} and {Delta}5ER{alpha} on transcription from classic ERE gene promoters and suggest that coexpression of these ER{alpha} isoforms may alter cellular phenotype, growth, and E2-mediated gene activation. Given the observed effects of {Delta}5ER{alpha} on normal ER{alpha} function, we hypothesize that this {Delta}5ER{alpha} variant may have pathophysiological consequences in these tissues. Based on these data, we hypothesize that {Delta}5ER{alpha} potentiates E2 and normal ER{alpha} actions on cellular growth, differentiation, and/or neoplastic progression in human E2-responsive tissues expressing both {Delta}5ER{alpha} and normal ER{alpha}.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
Full-length normal human ER{alpha} and its variant {Delta}5ER{alpha} were cloned from human pituitary tumor cDNA into the eukaryotic pBK-CMV expression vector (Stratagene, La Jolla, CA). Briefly, pooled pituitary first-strand cDNA obtained from human lactotroph and gonadotroph tumors by reverse transcription served as template for PCR amplification using a high-fidelity Pfu DNA polymerase (Stratagene) as previously described (13). Primer pairs SpeI-ER{alpha} (5'-gga cta gtc cat gac cat gac cct CCA-3') and ER{alpha}4L (5'-ttc gcc cag ttg atc atg tg-3') were used to amplify the N-terminal portion of ER{alpha} (nucleotides 231-1298, GenBank Accession no. X03635) while ER{alpha}4U (5'-gcc ccc cat act cta ttc-3') and ER{alpha}-ClaI (5'-gga tcg atg cag cag gga tta tct ga-3') were used to amplify the C-terminal part of ER{alpha} (nucleotides 1201–2063). PCR products of the appropriate sizes for normal and variant ER{alpha} fragments were purified from 1% agarose gel using GlasPac/GS purification kit (National Scientific, San Rafael, CA). The N-terminal portion of ER{alpha} fragment was digested with SpeI and HindIII while the C-terminal portion of ER{alpha} and {Delta}5ER{alpha} fragments were digested with HindIII and ClaI before cloning into pBKCMV plasmid vector. Expression plasmids containing the complete protein-coding region of ER{alpha} or {Delta}5ER{alpha} were constructed by ligating the common N-terminal portion of ER{alpha} to the isoform-specific C-terminal portion via an overlapping unique HindIII site. Positive clones were identified and confirmed by dideoxy sequencing using primers covering the entire translated region of ER{alpha}. Reporter plasmid EREtk81Luc contains two copies of consensus ERE palindromic sequence (aggtcacagtgacct) upstream of the minimal thymidine kinase (tk) promoter in pA3Luc (courtesy of R. Pestell, Albert Einstein college of Medicine, Bronx, NY). C3T1Luc of the human C3 promoter (-1030 to +58) contains one copy of consensus ERE palindromic sequences and two non-ERE E2 response regions (courtesy of D. P. McDonnell, Duke University Medical School, Durham, NC). The human c-fosLuc reporter contains -2000 bp upstream of the transcriptional start site to 42 bp of human c-fos promoter in pGLBasic (courtesy of C. Chen, University of Queensland, Queensland, Australia).

In Vitro Translation of ER{alpha} and {Delta}5ER{alpha} Variant
pBKCMV-ER{alpha} or -{Delta}5ER{alpha} and the control pBKCMV (1 µg) were used as DNA templates in coupled reticulocyte lysate-transcription/translation reactions in the presence of T3 RNA polymerase, amino acid mixture minus methionine, and [35S]methionine in a final volume of 25 µl according to the manufacturer protocols (Promega, Madison, WI). The synthesized protein products were analyzed by fractionation on a 8.5% SDS-PAGE, and the gels were dried down and autoradiographed for 24 h.

Stable Transfection and Screening
The expression vectors pBKCMV-ER{alpha} and its {Delta}5ER{alpha} variant were stably transfected in ER{alpha}/ERß-negative human osteosarcoma cell line U2-OS (ATCC, Rockville, MD) to generate [ER{alpha}]OS and [{Delta}5ER{alpha}]OS cell lines. One x 106 OS cells were washed with phenol red-free reduced serum OptiMEM and incubated with the mixture of 30 µl lipofectamine (GIBCO BRL, Grand Island, NY) and 10 µg of pBKCMV-ER{alpha} or its {Delta}5ER{alpha} variant expression plasmid at 37 C for 24 h. Medium was replaced with serum-free phenol red-free DMEM and incubated for an additional 48 h. Cells were subcultured in a series of cell-plating density ranging from 5,000–50,000 cells per 10-cm culture dish, in phenol red-free DMEM containing 5% charcoal-treated FCS and geneticin G418 (GIBCO BRL) at 500 µg/ml for selection. Media were then changed every 4–5 days. In 3 weeks, visible colony foci were isolated and propagated in medium containing G418.

Immunocytochemistry
Parental U-2 OS cells and stably transfected [ER{alpha}]OS and [{Delta}5ER{alpha}] OS cells were grown on four-chamber cell culture slides (Nunc Inc., Naperville, IL) and fixed with 2% paraformaldehyde in PBS pH 7.2, for 24–48 h at 4 C. Before immunostaining, cells were washed with PBS, pH 7.2, containing 10 mM glycine for 15 min to quench unreacted aldehyde groups and rinsed twice with PBS. Cell membranes were permeabilized by 5-min incubation at room temperature with 0.2% Triton X-100 in PBS, and cells were washed twice with PBS without detergent. Nonspecific binding sites for IgG were blocked by incubating cells for 20 min with nonimmune serum (5%). Immunostaining by avidin-biotin-peroxidase complex technique was performed according to manufacturer’s instructions using Vectastatin Elite ABC kit (Vector Laboratories, Burlingame. CA). The ER{alpha}-monoclonal mouse antibody, ER{alpha}-1 (Babco, Richmond, CA) against the common N terminus (amino acids 22–43 of the A/B domain)) of ER{alpha} and {Delta}5ER{alpha}, was diluted to the optimal concentration in PBS. Cells were incubated with the primary antibody for 1 h at 37 C and were washed twice with PBS for 15 min each. The diluted biotinylated secondary antibody was applied for 30 min at room temperature, and cells were washed twice with PBS before a 30-min incubation with Vectastatin Elite ABC reagent. Slides then were incubated in 1 µg/ml diaminobenzidine-0.3% H2O2 for 5 min, rinsed, and counterstained with hematoxylin. Coverslips were mounted to the chamber slides with immumount (Shandon, Pittsburgh, PA). Immunoabsoption controls were performed by preabsorption of the ER{alpha} antibody ER1 overnight at 4 C with the excess in vitro translated normal ER{alpha} protein. Method controls included substitution of nonimmune serum for primary antibody, elimination of secondary antibody, or streptavidin-biotin-peroxidase complexes, and dilution of primary antibodies. All controls verified the specificity of the ER1 antibody against the N terminus of ER{alpha} and {Delta}5ER{alpha}.

Transient Transfection and Luciferase Assay
[ER{alpha}-]OS and [ER{alpha}+]OS cells were plated in six-well plates at a density of 2 x 105 cells per well and allowed to adhere overnight. One hour before transfection, cells were washed and incubated in phenol red-free reduced serum OptiMEM. A lipid transfection mixture was prepared using a 1:400 mixture of Dioleoyl-a-phosphatidylethanolamine to demethyldioctadecylammonium bromide dissolved in 100% ethanol (both lipids were purchased from Sigma Chemical Co., St. Louis, MO). The DNA-lipid mix (1:5 ratio) containing the appropriate pBKCMV/ER{alpha} expression plasmid along with Luciferase reporter plasmid was prepared in phenol red-free reduced serum OptiMEM (1 ml/well) for 30 min at room temperature before addition to triplicate wells containing U2-OS cells. Whenever applicable, empty pBKCMV plasmid was used for experiments using pBKCMV-ER{alpha} or its variant to ensure that stiochiometrically equal amounts of CMV promoter, plasmid DNA, and lipid mix were applied to each well. Rous sarcoma virus/ß-galactosidase was used to monitor the variability of transfection efficiency. After 5 h incubation at 37 C, the transfection medium was replaced with serum-free phenol red-free DMEM and incubated for an additional 24 h, in the presence or absence of the indicated amount of E2. Transfected cells were lysed with 300 µl lysis buffer containing 1% Triton X-100, 10% glycerol, 2 mM EDTA, 2 mM dithiothreitol (DTT), and 25 mM Tris-phosphate (pH 7.8), and the cellular debris was removed by centrifugation. One hundred microliters of cell lysate were assayed for luciferase activity by measuring light emission with Luminometer (EG&G Berthold, Gaithersburg, MD) in the presence of luciferin and ATP.

EMSAs
Cell nuclear extracts were prepared from the parental U-2 OS, stable [ER{alpha}+]OS, and [{Delta}5ER{alpha}]OS cells grown in the absence or presence of 10 nM E2 using the miniextraction method with salt concentration modified for ER{alpha} extracts (43). Briefly, 5 x 106 to 1 x 107 cells were collected, washed with ice-cold Tris-borate-saline, and pelleted by centrifugation at 1,500 x g for 5 min. The pellet was resuspended in 400 µl cold buffer A containing 10 mM HEPES, pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 50 µl protease inhibitor cocktail (Sigma). Cells were placed on ice for 15 min, followed by addition of 25 µl 10% Nonidet NP-40, and were vortexed for 10 sec. After centrifugation for 30 sec in a microfuge, the nuclear pellet was resuspended in 50 µl ice-cold buffer C (20 mM HEPES, pH 7.9, 0.1 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride) and lysed for 15 min at 4 C on a shaking platform. The nuclear extract was centrifuged for 5 min at 4 C, and the supernatant was stored at -80 C. Protein concentrations of the nuclear extracts were determined by Bradford microassay (Sigma).

Consensus and mutant ERE oligonucleotides were end-labeled with [{gamma}32P]ATP and polynucleotide kinase (50,000 cpm/ng). Mutant ERE was identical to consensus ERE oligonucleotide with the exception of an AG-to-CC substitution in the ERE consensus sequence. Labeled probe (10,000 cpm) was added to 20 µl reaction mixture containing 1–2 µg nuclear extract in EMSA binding buffer (20 mM HEPES, pH 7.6, 400 mM KCL, 1 mM DTT, and 20% glycerol) and 1 µg poly(deoxyinosinic-deoxycytidylic)acid (Pharmacia Biotech, Piscataway, NJ). Binding reactions were allowed to proceed at 25 C for 20 min. For competition studies, cold ERE was incubated with the appropriate nuclear extract before addition of labeled ERE probe. DNA-protein complexes were resolved by electrophoresis (150 V at 25 C) through a nondenaturing polyacrylamide gel containing 5% glycerol in 0.5x Tris-borate-EDTA and were visualized by autoradiography.

For gel supershift analysis, 1–2 µl of appropriate gel supershift antibodies were added per 20 µl reaction volume subsequent to addition of 32P-labeled probe and incubated for 30 min at 25 C. Gel supershift antibodies included two monoclonal ER{alpha} supershift antibodies (Babco) against the N terminus (ER{alpha}-2, directed to amino acids 29–43 of the A/B domain) and the C terminus (ER{alpha}-6, directed against amino acids 575–595 of the EF region).

Cell Growth and Doubling Time Determination
Stably transfected [ER{alpha}+]OS cells, [{Delta}5ER{alpha}+[{rho}{varsigma}{theta}ß]OS cells, and parental [ER{alpha}-]Os cells were seeded in 12-well plates at a density of 10,000 cells per well in phenol red-free DMEM containing 5% charcoal-treated FCS and antibiotics. Cells in triplicate wells were harvested every 2 days, and nuclei were released with two drops of Zapoglobin per well (Coulter Co., Miami, FL) and Coulter counted (Coulter Electronics, Hialeah, FL), as previously described (44). The cell-doubling time (D), obtained during log phase of the growth curve, was calculated using a formula (45): D = 0.693/y (in hours), where y = 2.3 x (log ct1 - log ct0)/t1 - t0; ct1 = cell number at time 1; ct0 = cell number at time 0; t1 = time (h) at measurement of cells at time 1; t0 = time (h) at measurement of cells at time 0.


    FOOTNOTES
 
Address requests for reprints to: J. M. Alexander, Ph.D., Division of Bone and Mineral Metabolism, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Room 944, 330 Brookline Avenue, Boston, Massachusetts 02215.

This work was supported in part by an American Cancer Society, Massachusetts Division, Research Grant Award (to J.M.A.), The Jarislowsky Foundation, and a Massachusetts General Hospital Medical Discovery Award (to S.S.C.).

Portions of this work were presented at the 80th Annual Meeting of The Endocrine Society, Minneapolis MN, June 1997.

Received for publication March 6, 1998. Revision received May 7, 1998. Accepted for publication May 26, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Horowitz MC 1993 Cytokines and estrogen in bone: anti-osteoporotic effects. Science 260:626–627[Medline]
  2. Nabulsi AA, Folsom AR, White A, Patsch W, Heiss G, Wu KK, Szklo M 1993 Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. The Atherosclerosis Risk in Communities Study Investigators [see comments]. N Engl J Med 328:1069–1075[Abstract/Free Full Text]
  3. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139[Medline]
  4. Katzenellenbogen BS, Korach KS 1997 Editorial: a new actor in the estrogen receptor drama–enter ER-beta. Endocrinology 138:861–862[Free Full Text]
  5. Ponglikitmongkol M, Green S, Chambon P 1988 Genomic organization of the human oestrogen receptor gene. EMBO J 7:3385–3388[Abstract]
  6. Fuqua SA, Wolf DM 1995 Molecular aspects of estrogen receptor variants in breast cancer. Breast Cancer Res Treat 35:233–241[Medline]
  7. Fuqua SA, Allred DC, Auchus RJ 1993 Expression of estrogen receptor variants. J Cell Biochem Suppl 17G:194–197
  8. Pfeffer U, Fecarotta E, Vidali G 1995 Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells. Cancer Res 55:2158–2165[Abstract]
  9. Gotteland M, Desauty G, Delarue JC, Liu L, May E 1995 Human estrogen receptor messenger RNA variants in both normal and tumor breast tissues. Mol Cell Endocrinol 112:1–13[CrossRef][Medline]
  10. Rice LW, Jazaeri AA, Shupnik MA 1997 Estrogen receptor mRNA splice variants in pre- and postmenopausal human endometrium and endometrial carcinoma. Gynecol Oncol 65:149–157[CrossRef][Medline]
  11. Skipper JK, Young LJ, Bergeron JM, Tetzlaff MT, Osborn CT, Crews D 1993 Identification of an isoform of the estrogen receptor messenger RNA lacking exon four and present in the brain. Proc Natl Acad Sci USA 90:7172–7175[Abstract]
  12. Koehorst SG, Jacobs HM, Thijssen JH, Blankenstein MA 1993 Wild type and alternatively spliced estrogen receptor messenger RNA in human meningioma tissue and MCF7 breast cancer cells. J Steroid Biochem Mol Biol 45:227–233[CrossRef][Medline]
  13. Chaidarun SS, Klibanski A, Alexander JM 1997 Tumor-specific expression of alternatively spliced estrogen receptor messenger ribonucleic acid variants in human pituitary adenomas. J Clin Endocrinol Metab 82:1058–1065[Abstract/Free Full Text]
  14. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[Medline]
  15. Ambrosino C, Cicatiello L, Cobellis G, Addeo R, Sica V, Bresciani F, Weisz A, Tora L 1993 Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription. Mol Endocrinol 7:1472–1483[Abstract]
  16. Fan JD, Wagner BL, McDonnell DP 1996 Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity. Mol Endocrinol 10:1605–1616[Abstract]
  17. Metzger D, Ali S, Bornert JM, Chambon P 1995 Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells. J Biol Chem 270:9535–9542[Abstract/Free Full Text]
  18. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  19. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156[Medline]
  20. Pierrat B, Heery DM, Chambon P, Losson R 1994 A highly conserved region in the hormone-binding domain of the human estrogen receptor functions as an efficient transactivation domain in yeast. Gene 143:193–200[CrossRef][Medline]
  21. Tasset D, Tora L, Fromental C, Scheer E, Chambon P 1990 Distinct classes of transcriptional activating domains function by different mechanisms. Cell 62:1177–1187[Medline]
  22. Webster NJ, Green S, Jin JR, Chambon P 1988 The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell 54:199–207[Medline]
  23. Fuqua SA, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O’Malley BW, McGuire WL 1991 Variant human breast tumor estrogen receptor with constitutive transcriptional activity. Cancer Res 51:105–109[Abstract]
  24. Daffada AA, Johnston SR, Smith IE, Detre S, King N, Dowsett M 1995 Exon 5 deletion variant estrogen receptor messenger RNA expression in relation to tamoxifen resistance and progesterone receptor/pS2 status in human breast cancer. Cancer Res 55:288–293[Abstract]
  25. Fujimoto J, Ichigo S, Hirose R, Sakaguchi H, Tamaya T 1997 Expression of estrogen receptor wild type and exon 5 splicing variant mRNAs in normal and endometriotic endometria during the menstrual cycle. Gynecol Endocrinol 11:11–16[Medline]
  26. Leygue E, Huang A, Murphy LC, Watson PH 1996 Prevalence of estrogen receptor variant messenger RNAs in human breast cancer. Cancer Res 56:4324–4327[Abstract]
  27. Zhang QX, Borg A, Fuqua SA 1993 An exon 5 deletion variant of the estrogen receptor frequently coexpressed with wild-type estrogen receptor in human breast cancer. Cancer Res 53:5882–5884[Abstract]
  28. Castles CG, Fuqua SA, Klotz DM, Hill SM 1993 Expression of a constitutively active estrogen receptor variant in the estrogen receptor-negative BT-20 human breast cancer cell line. Cancer Res 53:5934–5939[Abstract]
  29. Huo B, Dossing DA, Dimuzio MT 1995 Generation and characterization of a human osteosarcoma cell line stably transfected with the human estrogen receptor gene. J Bone Miner Res 10:769–781[Medline]
  30. Ikegami A, Inoue S, Hosoi T, Kaneki M, Mizuno Y, Akedo Y, Ouchi Y, Orimo H 1994 Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells. Endocrinology 135:782–789[Abstract]
  31. Migliaccio S, Davis VL, Gibson MK, Gray TK, Korach KS 1992 Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor. Endocrinology 130:2617–2624[Abstract]
  32. Watts CK, Parker MG, King RJ 1989 Stable transfection of the oestrogen receptor gene into a human osteosarcoma cell line. J Steroid Biochem 34:483–490[CrossRef][Medline]
  33. Watts CK, King RJ 1994 Overexpression of estrogen receptor in HTB 96 human osteosarcoma cells results in estrogen-induced growth inhibition and receptor cross talk. J Bone Miner Res 9:1251–1258[Medline]
  34. Pakdel F, Reese JC, Katzenellenbogen BS 1993 Identification of charged residues in an N-terminal portion of the hormone-binding domain of the human estrogen receptor important in transcriptional activity of the receptor. Mol Endocrinol 7:1408–1417[Abstract]
  35. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  36. Wooge CH, Nilsson GM, Heierson A, McDonnell DP, Katzenellenbogen BS 1992 Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells. Mol Endocrinol 6:861–869[Abstract]
  37. Beato M, Candau R, Chavez S, Mows C, Truss M 1996 Interaction of steroid hormone receptors with transcription factors involves chromatin remodelling. J Steroid Biochem Mol Biol 56:47–59[CrossRef][Medline]
  38. Tsai MJ, BWO M 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  39. Norris JD, Fan D, Kerner SA, McDonnell DP 1997 Identification of a third autonomous activation domain within the human estrogen receptor. Mol Endocrinol 11:747–754[Abstract/Free Full Text]
  40. Christman JK, Nehls S, Polin L, Brooks SC 1995 Relationship between estrogen structure and conformational changes in estrogen receptor/DNA complexes. J Steroid Biochem Mol Biol 54:201–210[CrossRef][Medline]
  41. Rea D, Parker MG 1996 Effects of an exon 5 variant of the estrogen receptor in MCF-7 breast cancer cells. Cancer Res 56:1556–1563[Abstract]
  42. Weisz A, Rosales R 1990 Identification of an estrogen response element upstream of the human c-fos gene that binds the estrogen receptor and the AP-1 transcription factor. Nucleic Acids Res 18:5097–5106[Abstract]
  43. Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17:1[Abstract]
  44. Chaidarun SS, Eggo MC, Stewart PM, Barber PC, Sheppard MC 1994 Role of growth factors and estrogen as modulators of growth, differentiation, and expression of gonadotropin subunit genes in primary cultured sheep pituitary cells. Endocrinology 134:935–944[Abstract]
  45. Stanier RV, Adekberg EA, Ingraham JE 1976 The Microbial World. Prentice Hall, Upper Saddle River, NJ, pp 276