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Originally published In Press as doi:10.1074/jbc.M402148200 on April 12, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26184-26191, June 18, 2004
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The Relative Contribution Exerted by AF-1 and AF-2 Transactivation Functions in Estrogen Receptor {alpha} Transcriptional Activity Depends upon the Differentiation Stage of the Cell*

Yohann Mérot{ddagger}, Raphaël Métivier{ddagger}, Graziella Penot{ddagger}§, Dominique Manu§, Christian Saligaut{ddagger}, Frank Gannon§, Farzad Pakdel{ddagger}, Olivier Kah{ddagger}, and Gilles Flouriot{ddagger}

From the {ddagger}Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Université de Rennes I, 35042 Rennes cedex, France and the §EMBL, Meyerhofstraße 1, D-69117 Heidelberg, Germany

Received for publication, February 26, 2004 , and in revised form, April 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The activity of the transactivation functions (activation function (AF)-1 and AF-2) of the estrogen receptor {alpha} (ER{alpha}) is cell-specific. This study aimed to decipher the yet unclear mechanisms involved in this differential cell sensitivity, with particular attention to the specific influence that cell differentiation may have on these processes. Hence, we comparatively evaluated the permissiveness of cells to either ER{alpha} AFs in two different cases: (i) a series of cell lines originating from a common tissue, but with distinct differentiation phenotypes; and (ii) cell lines that undergo differentiation processes in culture. These experiments demonstrate that the respective contribution that AF-1 and AF-2 make toward ER{alpha} activity varies in a cell differentiation stage-dependent manner. Specifically, whereas AF-1 is the dominant AF involved in ER{alpha} transcriptional activity in differentiated cells, the more a cell is de-differentiated the more this cell mediates ER{alpha} signaling through AF-2. For instance, AF-2 is the only active AF in cells that have achieved their epithelial-mesenchymal transition. Moreover, the stable expression of a functional ER{alpha} in strictly AF-2 permissive cells restores an AF-1-sensitive cell context. These results, together with data obtained in different ER{alpha}-positive cell lines tested strongly suggest that the transcriptional activity of ER{alpha} relies on its AF-1 in most estrogen target cell types.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A wide range of physiological processes, including female reproductive function, are controlled by 17{beta}-estradiol (1). Most of the biological actions exerted by this steroid hormone are mediated by a specific receptor, the estrogen receptor {alpha} (ER{alpha})1 (2). ER{alpha} has a critical role in the control of the balance between cell proliferation and differentiation and is therefore intimately associated with the biology of endometrium and breast cancers (3). The biological effects of ER{alpha} often result from modifications in the pattern of expression of specific target genes. These transcriptional regulations are achieved through recruitment of ER{alpha} to the promoter region of the target gene, either directly through interaction with cognate DNA sequences (ERE or estrogen responsive elements), or through protein/protein interaction with other transcriptional factors (4-6).

ER{alpha} is a ligand-inducible transcription factor that belongs to the nuclear receptor (NR) subfamily whose members include steroid, thyroid hormone, retinoic acid, and orphan receptors. Based on structural and functional similarities, the sequences of NRs were divided into six functional domains designated A to F (4, 5). The central well conserved cysteine-rich C domain constitutes the signature of the NR superfamily, and mediates DNA binding. Hormone lodges into a hydrophobic pocket located within the C-terminal E/F domains that constitute the ligand binding domain. Ligand-induced transcription involves the action of distinct transactivation functions (AFs), located in the N-terminal A/B (AF-1) and the C-terminal E/F (AF-2) domains (7).

The respective contribution that these AFs make toward the activity of the full-length ER{alpha} is both promoter- and cell-specific (7-10). For instance, a maximal transcriptional activity of ER{alpha} can require both AFs in some cells, but only a specific one in others. This suggests that ER{alpha} does not interact with the transcriptional machinery in an identical manner in all cells. Functional and physical links between ER{alpha} and the transcriptional machinery involves the sequential recruitment by ER{alpha} of a group of proteins, called coactivators, on the target promoter, on which they build large protein complexes (11, 12). Structurally, these recruitments occur after ligand binding has induced specific conformational changes within the protein. So far, two classes of NR coactivator complexes, directly interacting with AF-2 in a ligand-dependent manner, have been identified. The first is formed by CBP/p300, the p160 nuclear receptor coactivator family (SRC-1/TIF2/AIB1), an RNA coactivator (SRA), and probably other unknown components (11, 12). This complex allows a strong decondensation of the chromatin, mainly because of the histone acetyltransferase activity of several of its components (13, 14). The second coactivator complex includes proteins of the SMCC/TRAP/DRIP/ARC/Mediator class, and allows the physical link between ER{alpha} and the general transcription apparatus, facilitating the activation of polymerase II (12).

A cell-type specific activity of both AFs was suggested to result from a specific expression of distinct coactivators. However, the majority of the coactivators are widely expressed in a similar amount in most cells (15, 16). Furthermore, several of the coactivators primarily identified as AF-2 specific have now been shown to also interact with the N-terminal region of ER{alpha} and to mediate AF-1 activity (17-20). Therefore, although considerable advances have been made in understanding the mechanisms allowing the receptor to modulate the transcription of a target gene, no clear scheme is emerging with regard to the differential sensitivity of cell types to AF-1 and AF-2.

In the present study, we demonstrate that the relative contribution exerted by AF-1 and AF-2 on the transcriptional activity of ER{alpha} varies in a cell differentiation stage-dependent manner. Precisely, we show that the more a cell is differentiated, the more this cell mediates ER{alpha} signaling through its AF-1. In contrast, AF-2 becomes the dominant AF involved in the transcriptional activity of ER{alpha} in undifferentiated or de-differentiated cells. These results, together with data obtained in different ER{alpha}-positive cell lines strongly suggest that the transcriptional activity of ER{alpha} relies on its AF-1 in most estrogen target cell types.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—The expression vectors used in this study were pSG5, pSG human (h)ER{alpha} (HEO), pSG hER{alpha} CF (21, 22), and pCMV-{beta}-galactosidase (Promega). Four luciferase reporter plasmids with different estrogen-sensitive promoters were employed in the transfection experiments: an artificial promoter containing one ERE upstream of the TK promoter (ERE-TK-LUC) (22), the promoter of the human complement C3 (C3-LUC) (23), the rainbow trout estrogen receptor promoter (rtER-LUC) (24), and the chicken vitellogenin promoter (cVg-LUC) (25).

Cell Culture—All cell lines (HeLa, HepG2, MCF7, T47D, BT20, MDA-MB 231, LNCaP, PC3, DU 145, TSU PR1, P19, Ishikawa, and {alpha}T3) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (Sigma), penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin (25 µg/ml) (Sigma) at 37 °C in a 5% CO2 incubator. The MDA-MB 231 cell line stably transfected with a hER{alpha} expression vector (26) were grown in Dulbecco's modified Eagle's medium containing 5% charcoal-stripped serum, antibiotics (see above), and hygromycin (0.4 mg/ml). The differentiation of the pluripotent stem cell line P19 into neurons and glial cells was achieved by treating P19 aggregates with retinoic acid (1 µM) during 48 h (27).

Transient Transfection Experiments—Cell lines were transfected with FuGENETM 6 as recommended by the manufacturer (Roche Diagnostics). One day before transfection, cells were plated in 24- or 6-well plates at a density previously determined as giving the best transfection efficiency. One hour prior to transfection, the medium was replaced with phenol red-free Dulbecco's modified Eagle's medium/F-12 containing 2.5% charcoal-stripped calf serum. Transfection was carried out with 250 ng of total DNA per well for the 24-well plates or with 1 µg of total DNA for the 6-well plates. Total DNA was composed by the expression vector (50 ng for the 24-wells plate or 200 ng for the 6-well plates), the reporter gene (100 or 400 ng, respectively), and the CMV-{beta}-galactosidase internal control (100 or 400 ng, respectively). Following an overnight incubation with the transfection mixture, the cells were treated with 10 nM estradiol (E2), 2 µM 4-hydroxytamoxifen (4-OHT), or ethanol (control). After 36 h of transient transfection, cells were harvested and luciferase and {beta}-galactosidase assays were performed as previously described (23). The reporter gene activity was obtained after normalization of the luciferase activity with the {beta}-galactosidase activity.

Western Blot Analysis—The expression of estrogen receptor, E-cadherin, vimentin, Tau, as well as actin, was examined by Western blot analysis. Subconfluent cells from 10-cm diameter dishes were washed with phosphate-buffered saline and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0,1% SDS) containing a mixture of protease inhibitors (Roche Diagnostics). Following sonication and quantification, 20 µg of these whole cell extracts were denatured in Laemmli buffer at 95 °C for 5 min, resolved on a 10% SDS-PAGE, and electrotransferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were blocked in phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat milk powder, during 1.5 h at room temperature. The blots were then incubated with either the polyclonal anti-hER{alpha} HC20 (1:1000, TEBU), the monoclonal anti-E-cadherin SHE78-7 (1:1000, Zymed Laboratories), the polyclonal anti-vimentin C-20 (1:500, TEBU), the polyclonal anti-Tau C-17 (1:500, TEBU), or the monoclonal anti-{beta}-actin AC-15 (1:5000, Sigma) in phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat milk powder for 1.5 h at room temperature. After three washings with phosphate-buffered saline, 0.1% Tween, the blots were incubated with peroxidase-conjugated goat anti-rabbit (1:5000, Pierce), peroxidase-conjugated horse anti-goat (1:5000, TEBU), or peroxidase-conjugated goat anti-mouse (1:5000, Pierce) for 1 h. Membrane-bound secondary antibodies were detected using the SuperSignal West Dura kit from Pierce according to the manufacturer's instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Distinct Sensitivities of HepG2 and HeLa Cell Lines to ER{alpha} AF-1 and AF-2: A Result from Their Divergent Phenotype?—Both ER{alpha} AF-1 and AF-2 have been shown to exert their transcriptional activity in a cell-specific manner. Accordingly, cell contexts can be defined as AF-1 or AF-2 permissive, depending upon which AF is principally involved in ER{alpha} activity: cell lines can be either equally or exclusively sensitive to either AFs (7-9, 22, 23, 28). Selection of an adequate cell line is therefore of primary importance when studying a specific AF of ERs. For instance, the hepatocarcinoma cell line HepG2 is frequently used to measure the AF-1 activity, whereas AF-2 function is mainly studied in HeLa cells that derive from a cervix carcinoma.

We first hypothesized that a distinct cell phenotype may explain the so far misunderstood cell preference for one given AF. Indeed, HeLa and HepG2 cells exhibit very distinct growth rates and phenotypes, despite their common epithelial origin. The cell proliferation rate of HeLa cells is in fact ~15-fold higher than for HepG2 cells, as demonstrated by a kinetic cell counting (Fig. 1A). Furthermore, contrasting to HeLa cells that exhibit a poorly differentiated phenotype associated with an elongated morphology and little cell-cell interactions, HepG2 cells appear more differentiated, establishing many cell contacts (data not shown). These observations are corroborated by the fact that HeLa cells do not express E-cadherin, a calcium-dependent cell-cell adhesion molecule considered as a marker for epithelial differentiation (29), as assessed by Western blot using HeLa whole cell protein extracts (Fig. 1B). Furthermore, HeLa cells express vimentin, an intermediate filament protein whose expression is associated with increased invasive and metastatic potency (30) (Fig. 1B). This is in sharp contrast to HepG2 cells that produce a high level of E-cadherin but no vimentin (Fig. 1B).



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  		FIG. 1.
HepG2 and HeLa cells, commonly used as, respectively, AF-1 and AF-2 permissive contexts, present divergent phenotype. A, proliferation profile of HeLa and HepG2 cells. 24-Well plates were seeded with 5 x 104 cells per well in phenol red-free Dulbecco's modified Eagle's medium/F-12 containing 2.5% charcoal-stripped serum. Cell numeration was performed every 24 h during 3 days. Data represent the mean of three independent experiments performed in triplicate. B, Western blot analysis of E-cadherin and vimentin expression in HeLa and HepG2 whole cell extracts. Probing {beta}-actin expression controlled equal loading of the samples.

 
These results suggest a correlation between the distinct permissiveness of HepG2 and HeLa cells to either ER{alpha} AF-1 or AF-2 and the divergent phenotype of these cells. However, the different tissue origins of these cell lines may also account for the observed differences. We thus decided to verify whether the cell-dependent involvement of AF-1 or AF-2 in ER{alpha} activity is associated with the differentiation stage of cells from a similar tissue origin. However, this study was subordinated to the development of adequate test systems to define the relative cell sensitivity to either ER{alpha} transactivation functions.

Design of Accurate Test Systems to Probe Distinct Cell Sensitivity to ER{alpha} AF-1 and AF-2—Two different approaches were selected to define the relative sensitivity of a cell line to AF-1 and AF-2. In the first system, cell contexts were evaluated in transient transfection experiments by comparing the transcriptional activity of full-length ER{alpha} with that of an ER{alpha} deleted from the A/B domain (ER{alpha} CF) and thus devoid of AF-1 activity. A similar activity of both receptors would define a strict AF-2 permissive cell context, whereas an AF-1 permissive cell context would be inferred from the observation of a transcriptional activity of full-length ER{alpha} but not ER{alpha} CF. The respective involvement of the AFs of ER{alpha} is also promoter-specific (7-10). Therefore, the assessment of the permissiveness of different cell lines for AF-1 or AF-2 required the design of a reporter system having no intrinsic preference for a specific AF and whose response would therefore only reflect cellular variations in the relative contribution that both AF-1 and AF-2 make toward ER{alpha} transcriptional activity. From the different estrogen-sensitive promoters tested, a synthetic ERE-TK promoter and the promoters from the complement 3 (C3), rtER{alpha}, and cVg genes, the ERE-TK promoter only gave the expected responses (Fig. 2A). Indeed, on this promoter, whereas full-length and truncated ER{alpha} exhibit the same activity in the strictly AF-2 permissive HeLa cells, the truncated form is inactive in strictly AF-1 permissive HepG2 cells. Although to a lesser extent, the C3 promoter was also permissive to both AFs. Finally, as the ER{alpha} CF was transcriptionally inactive on the rtER and cVg promoters in both HeLa and HepG2 cell lines, these two promoters did not fulfill the criteria of selection for a promoter adequate to our aims. Western blots controlled similar expression of both full-length ER{alpha} and ER{alpha} CF proteins in the tested cells (Fig. 2B), confirming the relevance of the results. The ERE-TK promoter was therefore selected as a reporter system to probe the distinct cell sensitivity of the ER{alpha} AF-1 and AF-2.



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  		FIG. 2.
Design of accurate experimental approaches to define cell sensitivity to ER{alpha} AF-1 and AF-2. A, to select estrogen responsive systems assessing cell permissiveness to either ER{alpha} AF, we transiently transfected into AF-1 (HepG2) and AF-2 permissive (HeLa) cells several promoter constructs (100 ng of ERE-TK-LUC, 100 ng of C3-LUC, 400 ng of rtER-LUC, or 400 ng of cVg-LUC) together with 50 ng of the expression vectors pSG5, pSG hER{alpha}, or pSG hER{alpha} CF. 100 ng of CMV-{beta}-galactosidase was used as internal control. Cells were treated for 36 h with 10 nM E2 or with ethanol (EtOH) (control). Quantified luciferase and {beta}-galactosidase activities were normalized, and the values standardized to the reporter activity measured in the presence of pSG hER{alpha} without E2. B, Western blot analysis controlling the correct and similar expression of ER{alpha} and ER{alpha} CF in HeLa and HepG2 cells transiently transfected with the corresponding expression vectors. C, identification of cell sensitivity to either AFs through the ability of ER{alpha} CF to repress the AF-1 activity of the full-length ER{alpha}. HeLa and HepG2 cells were cotransfected with 100 ng of ERE-TK-LUC together with 50 ng (+) of pSG5, 200 ng (++) of pSG hER{alpha} CF, or 50 ng (+) of pSG hER{alpha} alone or with increasing amounts of pSG hER{alpha} CF (50 ng (+) and 200 ng (++)). 100 ng of CMV-{beta}-galactosidase was used as internal control. Results are expressed as the percentage of the reporter gene activity measured in the presence of pSG hER{alpha} and E2. D, use of the agonistic activity of the 4-OHT to characterize cell permissiveness to AF-1. HeLa and HepG2 cells were cotransfected with 200 ng of C3-LUC, 100 ng of CMV-{beta}-galactosidase, and 50 ng of pSG hER{alpha}. Cells were treated 36 h with EtOH (control), 10 nM E2, or 2 µM 4-OHT. Results are expressed as in panel A. All experiments were performed at least 3 times, and the values correspond to the average ± S.E. of the results.

 
Although this approach is particularly pertinent in defining the AF permissiveness of ER{alpha} negative cell lines, this test is not appropriate in cells endogenously expressing ER{alpha}, whose transcriptional activity is likely to mask that of the transfected receptor. Alternatively, the relative sensitivity of ER{alpha} positive cell lines to either AFs can be characterized through the ability of the ER{alpha} CF to repress the transcriptional activity of full-length ER{alpha} in cell contexts sensitive to AF-1 only (22). Indeed, as shown in Fig. 2C, whereas an increased expression of ER{alpha} CF does not impact ER{alpha} transcriptional activity in HeLa cells, ER{alpha} CF becomes a potent inhibitor of the full-length form in HepG2 cells. This variant of the first approach was therefore used to estimate the sensitivity of ER{alpha} positive cell lines for either AFs.

The second system, designed to confirm the relative sensitivity of a cell line to ER{alpha} AFs, relies on the partial estrogen agonistic activity of 4-OHT. Actually, the estrogenic activity of 4-OHT exclusively depends upon the AF-1 of ER{alpha}, and is therefore observed only in cells sensitive to this AF (8). Transfection experiments carried out with the human complement C3 promoter, a well characterized 4-OHT-responsive promoter (31), confirmed that this molecule induces the reporter gene activity via the AF-1 of ER{alpha} only. Indeed, an OHT-induced response of the C3 promoter was observed in AF-1 permissive HepG2 cells but not in AF-2 permissive HeLa cells (Fig. 2D). We therefore used this test to further substantiate the AF-1 sensitivity of cells assayed by the first approach.

Correlation between the Relative Activity of ER{alpha} AFs and the Differentiation Status of Cell Lines Deriving from a Common Tissue—To verify whether the differentiation status of a given cell line influences the respective activity of both ER{alpha} transactivation functions, four breast cancer cell lines exhibiting distinct differentiation phenotypes were tested for their ability to mediate ER{alpha} transactivation through AF-1 or AF-2. According to the literature, the selected cell lines can be classified from a more to a less differentiated phenotype as follows: MCF7, T47D, BT20, and MDA-MB 231 (30, 32). We first wished to confirm these data by probing through Western blots the expression levels of two differentiation markers (ER{alpha} and E-cadherin), and one invasive and metastatic marker (vimentin) in these cells. Results of these experiments (Fig. 3A) confirm that MDA-MB 231 cells (ER{alpha} and E-cadherin negative; vimentin positive) and to a lesser extend BT20 cells (E-cadherin positive; ER{alpha} and vimentin negative) are less differentiated than MCF7 or T47D cells (ER{alpha} and E-cadherin positive; vimentin negative). Importantly, MCF7 and T47D cells are ER{alpha} positive, in contrast to BT20 and MDA-MB 231 cells.



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  		FIG. 3.
Correlation between the relative activity of ER{alpha} AFs and the differentiation status of MCF7, T47D, BT20, and MDA-MB 231 breast cancer cell lines. A, Western blot analysis probing the expression of endogenous ER{alpha}, E-cadherin, vimentin, and {beta}-actin in the four indicated cell lines. B, the permissiveness of MCF7 and T47D ER{alpha} positive cell lines to either ER{alpha} AFs was assessed using the dominant negative property of ER{alpha} CF in AF-1-sensitive cells. Cells were transfected with 100 ng of ERE-TK-LUC together with 50 ng of pSG5 or pSG hER{alpha} in the absence or presence of 200 ng of hER{alpha} CF. In the ER{alpha} negative BT20 and MDA-MB 231 cell lines, the direct comparison of ER{alpha} and ER{alpha} CF transcriptional activities evaluated the sensitivity of these cells to either AFs. Cells were transfected using 100 ng of ERE-TK-LUC with 50 ng of pSG5, pSG hER{alpha}, or pSG hER{alpha} CF. In both ER{alpha} positive and negative cell lines, 100 ng of CMV-{beta}-galactosidase was used as internal control. Cells were treated for 36 h with 10 nM E2 or with EtOH vehicle (control). Luciferase activities were normalized by {beta}-galactosidase values, and the results are figured as the percentage of the reporter gene activity measured in the presence of pSG hER{alpha} and E2. C, agonistic activity of 4-OHT in the four breast cancer cell lines. Cells were co-transfected with 200 ng of C3-LUC, 50 ng of pSG hER{alpha}, and 100 ng of CMV-{beta}-galactosidase. Cells were treated 36 h with EtOH or 2 µM 4-OHT. After normalization, the values were reported to the activity obtained in the absence of 4-OHT. Values are the average ± S.E. from at least three separate experiments.

 
To check for variations in the respective involvement of ER{alpha} AFs in the activity of the receptor in these four breast cancer cell lines, we used the first assay system, based on the ERE-TK reporter. As previously mentioned, in the specific case of the MCF7 and T47D cells that are ER{alpha}-positive, we used the variant of this system, which relies on ER{alpha} CF property to antagonize ER{alpha} activity in an AF-1 permissive cell context only. Results of these experiments clearly show in both MCF7 and T47D cells a repressive activity of the truncated ER{alpha} CF on the transcriptional activity of either endogenous or transfected full-length ER{alpha} (Fig. 3B). This indicates that AF-1 is the dominant transactivation function used by ER{alpha} in MCF-7 and T47D cell lines. To define the permissiveness of ER{alpha}-negative BT20 and MDA-MB 231 cell lines to either ER{alpha} AFs, we used the first test through comparing the transactivation activity of ER{alpha} and ER{alpha} CF proteins on the ERE-TK reporter. As shown in Fig. 3B, ER{alpha} CF transcriptional activity represented 50% of that of the full-length form in BT20 cells, defining a cell context sensitive to both AFs. In contrast, MDA-MB-231 cells allowed ER{alpha} CF to be as efficient as full-length ER{alpha} in activating the reporter gene, defining a strictly AF2 permissive cell context.

These results were next confirmed through the second approach. Accordingly, the agonistic activity of the 4-OHT was assessed by transfecting the four breast cancer cell lines with the ER{alpha} expression vector (pSG hER{alpha}) and the C3-LUC reporter gene. A 10-20-fold induction by 4-OHT-liganded ER{alpha} was observed in MCF7, T47D, and BT20 cells, indicating a cell context permissive to AF-1 (Fig. 2C). In contrast, no induction was detected in 4-OHT-treated MDA-MB 231 cells, confirming their strict AF-2 cell context. Altogether, these results, based on two complementary approaches, show a correlation between the differentiation status of the four breast cancer cell lines and the respective activities of AFs in these cells: the more a cell expresses markers for differentiated breast epithelium cells (ER{alpha} and E-cadherin positive; vimentin negative) the more it is sensitive to ER{alpha} AF-1.

Is the Correlation between the Relative Activity of Both AFs and the Differentiation Status of Cell Lines a General Rule?—To answer this question, we selected four prostatic carcinoma cell lines as another model: LNCaP, PC3, DU 145, and TSU PR1. We first classified these cells according to their differentiation status, as determined by probing cells for E-cadherin and vimentin expression (Fig. 4A). Within the four cell lines, TSU PR1 cells were determined as the most de-differentiated, with E-cadherin negative and vimentin positive phenotypes. In opposite, LNCaP appears as the most differentiated cell line, expressing high levels of E-cadherin (Fig. 4A). Corroborating these observations, within the prostatic cell lines tested, the LNCaP cells are the only cells known to express the androgen receptor, a prostatic differentiation marker (33, 34). The classification raised by the results of our assays is therefore in agreement with previous studies (33, 34).



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  		FIG. 4.
Correlation between the relative activities of ER{alpha} AFs and the differentiation status of LNCaP, PC3, DU 145, and TSU PR1 prostatic cancer cell lines. A, Western blot analysis probing the expression of endogenous E-cadherin, vimentin, and {beta}-actin in the four indicated cell lines. B and C, transient transfections were performed as described in the legend to Fig. 3. The asterisk indicates the absence of transcriptional activity of the reporter gene C3-LUC in LNCaP cells.

 
A comparison of ER{alpha} and ER{alpha} CF transcriptional activities in the different prostatic cell lines was next performed, using our first system test. These experiments showed that the most differentiated cell line, LNCaP, is strictly AF-1 permissive, as the ER{alpha} CF is transcriptionally inactive in these cells (Fig. 4B). The reduced activity of ER{alpha} CF observed in DU 145 indicates a mixed context permissive to both AF-1 and AF-2. Finally, TSU PR1 cells are defined as strictly permissive to AF-2, as ER{alpha} CF exhibits a similar activity to that of the full-length ER{alpha} (Fig. 4B). We next confirmed these data through the analysis of the 4-OHT agonistic activities on the C3-LUC reporter (Fig. 4C). No activity, reflecting an AF-1 sensitivity, was observed in TSU PR1, whereas a roughly 6-fold induction of the reporter gene was measured in 4-OHT-treated PC3 and DU 145 cells. Unfortunately, the C3-LUC reporter gene was not functional in LNCaP cells, preventing the determination of the agonistic property of 4-OHT in these cells.

Besides demonstrating a correlation between the differentiation status of prostatic cell lines and their relative sensitivity to ER{alpha} AFs, these results altogether also generalize our conclusions: the relative contributions that both AF-1 and AF-2 exert toward the activity of ER{alpha} are linked to the degree of differentiation of the cell considered. Precisely, ER{alpha} activity is mediated through AF-1 in differentiated cells and through AF-2 in well de-differentiated cells.

Correlation between the Relative Activity of Both ER{alpha} AFs during the Differentiation of a Given Cell Line—The above study relied on a comparative measurement of ER{alpha} AF activities in several cell lines with distinct differentiation phenotypes. To further confirm the correlation deduced from this study, the preferential use of either AFs in ER{alpha} transcriptional activity was probed during the differentiation process of a given cell line. We selected the mouse pluripotent P19 cell line for its ability to differentiate into neural cells when cell aggregates are treated with retinoic acid (1 µM) (27). Phenotypically, retinoic acid-treated P19 cells differentiate into neuroectoderm that yields cultures including neurons and glial cells, as shown in Fig. 5A. The differentiation process of these cells was confirmed by probing the expression of the microtubule-binding protein Tau, which stabilizes microtubule in growing axons (Fig. 5B).



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  		FIG. 5.
The relative activity of ER{alpha} AFs varies during the differentiation of the pluripotent P19 cells into neurons and glial cells. A, aggregated P19 cells were differentiated into a neural cell population through treatment with 1 µM retinoic acid during 48 h. B, Western blot checked Tau and {beta}-actin expression in undifferentiated and differentiated P19 cells. C, cell sensitivity to ER{alpha} AFs was evaluated through direct comparison of ER{alpha} and ER{alpha} CF transcriptional activities. Undifferentiated and differentiated P19 cells were transfected using 100 ng of ERE-TK-LUC, 100 ng of CMV-{beta}-galactosidase, and 50 ng of the expression vectors pSG5, pSG hER{alpha}, or pSG hER{alpha} CF. Cells were treated for 36 h with EtOH (control) or 10 nM E2. Normalized activities are shown as the percentage of the reporter gene activity measured in the presence of pSG hER{alpha} and E2. D, agonistic activity of 4-OHT in undifferentiated and differentiated P19 cells. Cells were transfected with 200 ng of C3-LUC, 100 ng of CMV-{beta}-galactosidase, and 50 ng of pSG hER{alpha} and then treated for 36 h with EtOH (control) or 2 µM 4-OHT. Histograms show the mean ± S.E. of values normalized to the reporter activity measured in the absence of 4-OHT, obtained in at least three separate experiments.

 
The sensitivity of undifferentiated P19 cells to ER{alpha} AFs was first determined by comparing ER{alpha} and ER{alpha} CF transcriptional activities. This first test depicts undifferentiated P19 cells to provide a mixed AF-1/AF-2 cell context. However, these cells are predominately AF-2 permissive, as ER{alpha} CF activity on the ERE-TK-LUC represented ~60% of that of the wild type receptor (Fig. 5C). The second approach confirmed the sensitivity of undifferentiated P19 cells to both ER{alpha} AFs, as revealed by the agonistic activity of the 4-OHT on the C3-LUC reporter (Fig. 5D).

In accordance with the correlation depicted above (AF-1/differentiated cells; AF-2/undifferentiated cells), our working hypothesis was that differentiated P19 cells should be more sensitive to ER{alpha} AF-1 than undifferentiated cells. Transient transfections of differentiated P19 cells were therefore performed to test this assumption. These experiments demonstrate that differentiated P19 cells are entirely sensitive to AF-1, as ER{alpha} CF is transcriptionally inactive in these conditions (Fig. 5C). Furthermore, and as expected from our hypothesis, the differentiation of P19 cells induced a 2-3-fold greater induction of the C3-LUC reporter gene by 4-OHT (Fig. 5D), reflecting an enhancement of AF-1 participation in ER{alpha} activity.

Interestingly, we obtained similar results in the PC12 pheochromocytoma cell line that differentiates into sympathetic neuron-like cells in the presence of nerve growth factor (data not shown). Altogether these data demonstrate that AF-1 becomes the predominant transactivation function used by ER{alpha} in differentiated cells.

Are Cells Expressing ER{alpha} Predominantly Permissive to AF-1?—In estrogen target tissues such as mammary gland or uterus, ER{alpha} expression is considered as a cell differentiation marker. We concluded from the experiments shown above that AF-1 is the main AF used by ER{alpha} in differentiated cells. We therefore speculated that an ER{alpha} positive cell type has to be mostly AF-1 permissive. Significantly, this hypothesis would be relevant to the previous observations that (i) both differentiated MCF7 and T47D ER{alpha} positive breast cancer are permissive to AF-1 (Fig. 3); and that (ii) differentiated LNCaP cells that are also strictly AF-1-sensitive (Fig. 4), express low levels of ER{alpha} (35). To substantiate these data, which are likely to indicate a general link between ER{alpha} expression and AF-1 permissiveness, we next analyzed two other ER{alpha} positive cell lines from distinct tissue origins: the endometrial adenocarcinoma Ishikawa and the pituitary gonadotrope {alpha}T3 cell lines. The sensitivity of these cells to AF-1 was initially assessed according to the variant of the first approach, aimed at measuring the dominant negative property of ER{alpha} CF. As expected, the truncated form ER{alpha} CF repressed the transcriptional activity of the endogenous or transfected full-length ER{alpha} in both cell lines (Fig. 6A). Corroborating the first test, an agonistic activity of the 4-OHT was also observed on the C3-LUC reporter (Fig. 6B). Therefore, both approaches demonstrate that two other ER{alpha} positive cell lines, Ishikawa and {alpha}T3, present cell contexts permissive to AF-1.



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  		FIG. 6.
ER{alpha} positive cells are predominantly permissive to AF-1. Two naturally ER{alpha} positive cell lines, Ishikawa and {alpha}-T3, as well as an MDA-MB 231 cell line stably expressing wild-type ER{alpha} (MDA-MB 231 hER{alpha}) were probed for their sensitivity to either ER{alpha} AFs by using in A and C, the dominant negative property of ER{alpha} CF or the AF-1 agonistic activity of 4-OHT in B and D.

 
Re-expression of ER{alpha} in AF-2-sensitive and De-differentiated Cells Induces AF-1 Permissiveness—Tumors expressing ER{alpha} are usually more differentiated and present lower aggressiveness and invasiveness than ER{alpha} negative tumors (36). Correspondingly, the stable re-expression of ER{alpha} into MDA-MB 231, an ER{alpha} negative breast cancer cell line, induces a reversion of the invasive property of these cells in conjunction with a more differentiated phenotype (37). We therefore anticipated that the stable expression of a functional ER{alpha} in the MDA-MB 231 cells that are strictly AF-2 permissive (Fig. 3) might restore an AF-1-sensitive cell context. A subline of the MDA-MB 231, stably transfected with an expression vector encoding wild-type ER{alpha}, was then analyzed through our tests. Results illustrated in Fig. 6, C and D, clearly show that these cells are AF-1 permissive. Indeed, the overexpression of ER{alpha} CF inhibited the activity of endogenous or transfected full-length ER{alpha} on the ERE-TK-LUC reporter (Fig. 6C). Moreover, an agonistic activity of the 4-OHT was also obtained on the C3-LUC reporter (Fig. 6D). In conclusion, these data indicate that AF-1 is the main transactivation function used by the ER{alpha} in ER{alpha} positive cells, and that AF-1 is linked to cell differentiation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although essential in generating the pleiotropic effects of estradiol, the mechanisms underlying the cell-specific activities of ER{alpha} AFs are yet poorly understood. To address this problem, several cell lines mediating ER{alpha} activity through either AF are commonly used to study individual AF activity. For instance, we and others frequently use HeLa and HepG2 cell lines, which represent strict AF-2 and AF-1 permissive contexts, respectively. We initially observed that AF-2-sensitive HeLa cells exhibit a poorly differentiated phenotype and a change from epithelial to fibroblastic characteristics combined with the acquisition of a motile behavior. In contrast, AF-1-sensitive HepG2 cells have preserved a more differentiated phenotype, close to the epithelial cell morphology exhibiting intercellular junctions. Our working hypothesis was therefore that cell preference for a specific AF is correlated with the differentiation status of the cells.

The Relative Importance of AF-1 and AF-2 in ER{alpha} Activity Varies in a Cell Differentiation Stage-dependent Manner—To substantiate our hypothesis, we probed the relative importance of each AFs in ER{alpha} signaling in (i) a series of cells originating from a common tissue (mammary and prostatic adenocarcinomas) and covering different stages of tumor development or epithelial-mesenchymal transition; and (ii) in cells undergoing differentiation processes following specific treatments (P19 or PC12 cells). This choice was reasoned by the fact that these approaches would help circumvent the problems emerging from the different tissue origins of cells generally used to analyze AF-1 and AF-2 activities. To define the relative sensitivity of the different cell lines to AF-1 and AF-2, we arranged two different tests. The first one uses a comparison of the transcriptional activity of transfected wild type ER{alpha} protein with that of an AF-1 deleted form (lacking the 173 first amino acids; ER{alpha} CF). As the ER{alpha} CF also behaves as an inhibitor of the AF-1 of the full-length ER{alpha} (22), we used this variant technique to define the AF sensitivity of cell lines naturally expressing ER{alpha}. To confirm the corresponding contribution of AF-1 in different cells, a good optional test would have been to use deletions or impeding mutations of AF-2. Unfortunately, these mutant proteins exhibited a dramatically reduced transcriptional activity in HepG2 or other cell lines strictly sensitive to AF-1 (data not shown). These data are consistent with reports showing the existence of functional links and/or direct physical interactions between N- and C-terminal domains of ER{alpha} and other NRs (23, 38, 39). To design the second test, we therefore tried to preserve as much as possible the estrogen receptor structure. Approaches potentially leading to less accurate results, such as fusions of isolated ER{alpha} AF domains to the DNA binding domain of the yeast activator GAL4 were therefore not considered. In contrast, we used the agonistic activity of the 4-OHT on the C3 promoter to reveal an AF-1-sensitive context. This second approach was used to confirm the results obtained through comparing ER{alpha} and ER{alpha} CF activities, a test which, alone, might be open to criticism because of the use of a truncated ER{alpha}. Our analysis shows that the more the cell is differentiated, the more it mediates ER{alpha} transactivation through AF-1. In contrast, AF-2 becomes the only active AF in cells that have achieved their epithelial-mesenchymal transition, such as MDA-MB 231 and TSU PR1 cells. This observation was confirmed by the switch from a predominant AF-2 to a major AF-1 activity occurring after differentiation of P19 or PC12 cells into neuronal-like cells. Altogether, our results demonstrate that the relative importance of AF-1 and AF-2 in ER{alpha} activity varies in a cell differentiation stage dependent manner.

AF-1 Versus AF-2 Preference: A Role for Cofactor Expression and Post-translational Modifications?—Variations in the relative contributions that AF-1 and AF-2 make toward ER{alpha} activity point to modifications of the interactions that ER{alpha} make with the transcriptional apparatus and coactivators. Several mechanisms may explain such changes. First, the expression of coactivators recruited to promoters by ER{alpha} may vary, as exemplified by the change from DRIP/mediator to SRC/p160 family coactivators as the major vitamin D receptor binding complex during keratinocyte differentiation (40). This switch is correlated with a decrease in DRIP205 expression and an increase in SRC-3/AIB1/RAC3 intracellular amounts during the differentiation process. Alterations in the expression pattern of coactivators such as SRC-3 are therefore mechanisms likely relevant to an up-regulation of AF-1 and/or a down-regulation of AF-2 during cell differentiation. For instance, SRC-3 expression in epithelial cells of breast and prostate tissues is correlated with the tumorization of these tissues. Correspondingly, SRC-3/AIB1/RAC3 is overexpressed in ER-positive breast tumors, in AR-positive prostate tumors, in most of the breast ER-positive cell lines (MCF7, ZR-75-1, BG-1, and BT-474), and AR-positive LNCaP cells, whereas SRC3/AIB1/RAC3 is weakly expressed in different ER-negative MDA-MB cell lines or AR-negative PC3 and DU 145 cells (41, 42). Further studies are now obviously required to examine the apparent correlation between the expression levels of SRC-3 and the varying permissiveness to AF-1 and AF-2 of cells at differentiation stages. Notably, SRC-3 mediates both the AF-1 and AF-2 of ER{alpha}, leading to a synergism between both AFs. Therefore, if the cell specific activities of AF-1 and AF-2 are controlled by the relative cellular level of SRC-3, the underlying mechanism remains to be defined.

In contrast to SRC-3, a majority of ER{alpha} coactivators are ubiquitously expressed. Consequently, cell-dependent modifications of ER{alpha} activity may also result from a differential coactivator activity and/or recruitment, as exemplified by the p68/p72 RNA helicases (43, 44). Indeed, conflicting with its ubiquitous expression, p68 enhanced ER{alpha} transcriptional activity in AF-1-sensitive HepG2 cells but not in strict AF-2 permissive HeLa cells (Ref. 22, data not shown). Physical interaction between ER{alpha} and p68 is tightly controlled through the phosphorylation of the Ser118 of ER{alpha} (43). Cell-type variations in the phosphorylation of this residue may therefore be a determinant in the cell differentiation stage dependent activity of ER{alpha} AFs. Unfortunately, no strict correlation has yet been made between the status of Ser118 phosphorylation and the cell permissiveness to either ER{alpha} AF. However, we recently evidenced that the formation of a tight complex between ER{alpha} and the orphan receptor chicken ovalbumin upstream promoter-transcription factor 1 allows an increased recruitment of ERK2/p42 MAPK. The subsequent phosphorylation of ER{alpha} Ser118 residue results in an enhanced ER{alpha} AF-1 activity (45). Restriction of this process to cells sensitive to AF-1 suggests that AF-2-sensitive cells do not allow ER{alpha} Ser118 phosphorylation. This might explain the absence of functional interaction between AF-1 and p68/p72 in AF-2-sensitive cells (23). The tissue-specific pattern of post-translational modification of ER{alpha} and coactivators, such as phosphorylation, is likely to determine selective receptor/coactivators interactions. In addition to Ser118, the ER{alpha} AF-1 domain contains other multiple phosphorylation sites susceptible to influence AF-1 activity (46-48). Furthermore, the enzymatic activity and/or recruitment of several coregulators are mediated through post-translational modifications. For instance, ER{alpha} transcriptional activity is potentiated by the targeting of TIF2/GRIP1, a member of the p160 family, through the MAPK signaling pathway (49). Phosphorylation of SRC-3/AIB1 by MAPK also increases the half-life of its association with p300 (50). Last, phosphorylation events enhance the acetyltransferase activity of various coactivators and determine the subcellular localization of some of these proteins (11, 12). Altogether, these data lead us to postulate that the cell differentiation state dependent activity of ER{alpha} AFs is likely a combination of several mechanisms involving specific cofactors expression and post-translational modifications.

Role of AF-1 in ER{alpha} Activity in Vivo—Most of the cells present in adult mammalian organisms are differentiated. Consequently, the data obtained in this study strongly suggest that ER{alpha} transcriptional activity essentially relies on AF-1 in estrogen target cells. This is consistent with previous reports proposing AF-1 as the major transactivation function of ER{alpha}, with AF-2 primarily acting as a structural switch that senses ligand (9, 51). Correspondingly, a full AF-1 activity is required for the estradiol-dependent proliferation of MCF-7 cells (52). The importance of AF-1 in ER{alpha} activity is further emphasized by the critical deficiencies observed on the reproductive functions of ER{alpha}-/- mice generated through an insertional disruption of the ER{alpha} gene in the first coding exon (53). Indeed, although totally abolishing the production of the full-length ER{alpha}, this disruption does not suppress the expression of a naturally occurring N-terminal truncated isoform that originates from the deletion of the exon targeted by the insertional disruption (54). Because this isoform, referred to ER{alpha} 46, is structurally identical to the truncated mutant ER{alpha} CF used in this study, the severe phenotypes observed in the ER{alpha}-/- mice have to be considered as reflecting an ER{alpha} AF-1 knock-out. Such a physiological importance of AF-1 also questions the relevance of using strictly AF-2 permissive cell lines such as HeLa cells as models to study ER{alpha} transcriptional activity.

In conclusion, we provide in this study evidence for a correlation between the relative activity of ER{alpha} AFs and the differentiation state of the cell considered. Our hypothesis suggests that ER{alpha} transcriptional activity is influenced, for instance during the epithelial-mesenchymal transition, by (i) modifications in the pattern of cofactor expression and/or (ii) by post-translational modifications through precise signaling pathways. Such a model also implies that ER{alpha} target genes may, at least partially, differ according to the cell context defined as AF-1 or AF-2 permissive. Our data therefore join the comprehension of the molecular mechanisms underlying the cell specific transcriptional activity of the estrogen receptor, contributing to the diversity of E2 actions in vivo.


   FOOTNOTES
 
* This work was supported by the Association pour la Recherche contre le Cancer, the Ligue contre le cancer, CNRS, and the University of Rennes 1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Université de Rennes I, 35042 Rennes cedex, France. Tel.: 33-0-2-23-23-68-04; Fax: 33-0-2-23-23-67-94; E-mail: gilles.flouriot{at}univ-rennes1.fr.

1 The abbreviations used are: ER{alpha}, estrogen receptor {alpha}; ERE, estrogen responsive element; TK, thymidine kinase; NR, nuclear receptor; AF, activation function; rtER, rainbow trout estrogen receptor; cVg, chicken vitellogenin; CMV, cytomegalovirus; E2, estradiol; 4-OHT, 4-hydroxytamoxifen; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase. Back


   ACKNOWLEDGMENTS
 
We thank P. Chambon for the gift of expression vectors pSG5 and HEO, P. Webb for the reporter gene ERE-TK-LUC, and G. Lazennec for the cell line BT20.



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G. Penot, C. Le Peron, Y. Merot, E. Grimaud-Fanouillere, F. Ferriere, N. Boujrad, O. Kah, C. Saligaut, B. Ducouret, R. Metivier, et al.
The Human Estrogen Receptor-{alpha} Isoform hER{alpha}46 Antagonizes the Proliferative Influence of hER{alpha}66 in MCF7 Breast Cancer Cells
Endocrinology, December 1, 2005; 146(12): 5474 - 5484.
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 J Mol EndocrinolHome page
Y. Merot, F. Ferriere, E. Debroas, G. Flouriot, D. Duval, and C. Saligaut
Estrogen receptor alpha mediates neuronal differentiation and neuroprotection in PC12 cells: critical role of the A/B domain of the receptor
J. Mol. Endocrinol., October 1, 2005; 35(2): 257 - 267.
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 Mol. Cell. Biol.Home page
J. Matthews, B. Wihlen, J. Thomsen, and J.-A. Gustafsson
Aryl Hydrocarbon Receptor-Mediated Transcription: Ligand-Dependent Recruitment of Estrogen Receptor {alpha} to 2,3,7,8-Tetrachlorodibenzo- p-Dioxin-Responsive Promoters
Mol. Cell. Biol., July 1, 2005; 25(13): 5317 - 5328.
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 Mol. Endocrinol.Home page
D. G. Monroe, F. J. Secreto, M. Subramaniam, B. J. Getz, S. Khosla, and T. C. Spelsberg
Estrogen Receptor {alpha} and {beta} Heterodimers Exert Unique Effects on Estrogen- and Tamoxifen-Dependent Gene Expression in Human U2OS Osteosarcoma Cells
Mol. Endocrinol., June 1, 2005; 19(6): 1555 - 1568.
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V. Gburcik, N. Bot, M. Maggiolini, and D. Picard
SPBP Is a Phosphoserine-Specific Repressor of Estrogen Receptor {alpha}
Mol. Cell. Biol., May 1, 2005; 25(9): 3421 - 3430.
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 Mol. Cell. Biol.Home page
A. Fritah, C. Saucier, J. Mester, G. Redeuilh, and M. Sabbah
p21WAF1/CIP1 Selectively Controls the Transcriptional Activity of Estrogen Receptor {alpha}
Mol. Cell. Biol., March 15, 2005; 25(6): 2419 - 2430.
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