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Nucleic Acids Research, 2003, Vol. 31, No. 18 5266-5274
© 2003 Oxford University Press

Ligand-independent activation of estrogen receptor {alpha} by XBP-1

Lihua Ding1,2, Jinghua Yan1, Jianhua Zhu1, Hongjun Zhong1, Qiujun Lu3, Zonghua Wang2, Cuifen Huang1 and Qinong Ye*,1

1 Beijing Institute of Biotechnology, Beijing 100850, Peoples Republic of China, 2 Fujian Science and Technology University, Fuzhou 350002, Peoples Republic of China and 3 Beijing Institute of Radiation Medicine, Beijing 100850, Peoples Republic of China

*To whom correspondence should be addressed at Beijing Institute of Biotechnology, 27 Tai-Ping Lu Rd, Beijing 100850, Peoples Republic of China. Tel: +86 10 6818 0809; Fax: +86 10 6824 8045; Email: yeqn{at}nic.bmi.ac.cn

Received July 1, 2003; Revised and Accepted July 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen receptor (ER) is a member of a large superfamily of nuclear receptors that regulates the transcription of estrogen-responsive genes. Several recent studies have demonstrated that XBP-1 mRNA expression is associated with ER{alpha} status in breast tumors. However, the role of XBP-1 in ER{alpha} signaling remains to be elucidated. More recently, two forms of XBP-1 were identified due to its unconventional splicing. We refer to the spliced and unspliced forms of XBP-1 as XBP-1S and XBP-1U, respectively. Here, we report that XBP-1S and XBP-1U enhanced ER{alpha}-dependent transcriptional activity in a ligand-independent manner. XBP-1S had stronger activity than XBP-1U. The maximal effects of XBP-1S and XBP-1U on ER{alpha} transactivation were observed when they were co-expressed with full-length ER{alpha}. SRC-1, the p160 steroid receptor coactivator family member, synergized with XBP-1S or XBP-1U to potentiate ER{alpha} activity. XBP-1S and XBP-1U bound to the ER{alpha} both in vitro and in vivo in a ligand-independent fashion. XBP-1S and XBP-1U interacted with the ER{alpha} region containing the DNA-binding domain. The ER{alpha}-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, including the N-terminal basic region leucine zipper domain (bZIP) and the C-terminal activation domain. The bZIP-deleted mutants of XBP-1S and XBP-1U completely abolished ER{alpha} transactivation by XBP-1S and XBP-1U. These findings suggest that XBP-1S and XBP-1U may directly modulate ER{alpha} signaling in both the absence and presence of estrogen and, therefore, may play important roles in the proliferation of normal and malignant estrogen-regulated tissues.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen receptor (ER) belongs to a superfamily of nuclear receptors that act as ligand-activated transcription factors [for reviews see Klinge (1), Katzenellenbogen and Katzenellenbogen (2), and Aranda and Pascual (3)]. Based on structural and functional similarities (4,5), the nuclear receptors can be subdivided into six regions (A–F). Two domains are well conserved among nuclear receptors, the highly conserved C domain serving to direct DNA binding and the moderately conserved C-terminal E/F domain forming a pocket for ligand binding. The ligand-binding domain (LBD) contains a ligand-dependent transcriptional activation function (AF-2), whereas the quite divergent A/B domain contains another transactivation function (AF-1), which is constitutive in the absence of ligand. There are two isoforms of ERs, namely ER{alpha} and ERß [Kuiper et al. (6,7), and references therein]. Activation of ER{alpha} is responsible for many biological processes, including cell growth and differentiation, morphogenesis and programmed cell death (8,9). In addition, ER{alpha} plays an important role in the development and progression of breast cancer by regulating genes and signaling pathways involved in cellular proliferation. Regulation of gene expression by the ER{alpha} requires the coordinate activity of ligand binding, phosphorylation and cofactor interactions, with particular combinations probably resulting in the tissue-specific responses elicited by the receptor (10–13). However, the intracellular signaling pathways modulating these components and regulating ER{alpha} transcriptional activity are not fully understood.

Human X box-binding protein 1 (XBP-1), originally identified as a protein binding to the cis-acting X box present in the promoter regions of target genes, is a basic region leucine zipper (bZIP) protein in the CREB/ATF (cAMP response element-binding protein/activating transcription factor) family of transcription factors (14). XBP-1 has been found to be ubiquitously expressed in adult tissues but preferentially expressed in fetal exocrine glands, osteoblasts, chondroblasts and liver (14,15). XBP-1 is essential for the growth of hepatocytes (16), as XBP-1-deficient embryos die in utero from severe liver hypoplasia and the resultant fatal anemia. In human multiple myeloma cells, XBP-1 has been shown to be implicated in the proliferation of malignant plasma cells (17). Since the cloning of XBP-1 over 10 years ago (14), there has been general acceptance that only one XBP-1 existed. More recently, however, XBP-1 mRNA has been shown to be unconventionally spliced by IRE1 in response to endoplasmic reticulum stress (18,19), resulting in production of a highly active transcription factor that can activate the mammalian unfolded protein response (UPR). The unconventional splicing of XBP-1 mRNA results in a frameshift at amino acid 165 of the unspliced XBP-1. Thus, unspliced and spliced XBP-1 mRNA encode proteins of 261 and 376 amino acids, respectively, with the identical N-terminal regions containing the bZIP domain. We refer to the spliced and unspliced forms of XBP-1 as XBP-1S and XBP-1U, respectively.

XBP-1 has been reported to be expressed at high levels in ER{alpha}-positive breast tumors (20–25), although the forms of XBP-1 are unknown. Using cDNA arrays, Perou et al. described that variation of the ER{alpha} gene in 65 surgical specimens of human breast tumors from 42 different individuals paralleled variation in the expression of the XBP-1 gene (20). Bertucci et al. compared the gene expression profiles in ER{alpha}-positive breast cancers (n = 23) versus ER{alpha}-negative breast cancers (n = 11) with cDNA array technology (22). The XBP-1 mRNA expression in ER{alpha}-positive breast cancers was 2.7-fold as much as that in ER{alpha}-negative breast cancers. SAGE (serial analysis of gene expression) showed that XBP-1 appeared to be highly expressed in cancerous mammary epithelial cells (26). Because of the importance of ER{alpha} signaling in the regulation of breast cancer development and progression, we investigated the potential role of XBP-1 in ER{alpha} transcriptional activity. Here, we show that both XBP-1S and XBP-1U enhance ER{alpha} transactivation in breast cancer and non-breast cells in a ligand-independent manner. We further present in vitro and in vivo evidence that XBP-1S and XBP-1U interact with ER{alpha}, and that deletion of the N-terminal portion of XBP-1S and XBP-1U fully abolishes the ER{alpha} transcriptional activity by XBP-1S and XBP-1U.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
The following plasmids have been described previously: pERE-LUC (estrogen-responsive element-containing luciferase reporter) (27), a kind gift from Dr Ming-jer Tsai (Baylor College of Medicine, Houston, TX); pcDNA3-ER{alpha} (human ER{alpha} expression vector) (27); pCMX-SRC-1 (28), a kind gift from Dr Rosalie M. Uht (University of Virginia, VA); pcDNA-hAR [human androgen receptor (AR) expression vector] (29); and PSA-LUC [androgen responsive element (ARE)-containing luciferase reporter] (29), a generous gift from Dr Chinghai Kao (University of Indiana, Indianapolis, IN). To construct pcDNA3-FLAG-XBP-1S, full-length human XBP-1S cDNA was obtained by standard PCR amplification from an ovary two-hybrid cDNA library (Clontech). Full-length human XBP-1U cDNA was obtained using recombinant PCR (27). The amplified XBP-1S and XBP-1U cDNAs were both cloned into pcDNA3 vector harboring FLAG epitope sequence (pcDNA3-FLAG), and the constructs were confirmed by sequencing. Deletion mutants of XBP-1S and XBP-1U were constructed by inserting PCR-generated fragments from the corresponding XBP-1 cDNAs into the pcDNA3-FLAG vector. The expression vectors for ER{alpha} AF-1 (amino acids 1–282) and ER{alpha} AF-2 (amino acids 178–595) were constructed by introducing the cDNAs into pcDNA3 (Invitrogen). Constructs encoding GST fusion proteins were prepared by amplification of each sequence by standard PCR methods, and the resulting fragments were cloned in-frame into pGEX-KG (Amersham Pharmacia Biotech), using appropriate restriction sites as described previously (30). The constructs were partially sequenced to confirm the correct orientation.

Mammalian cell transfection and luciferase assay
MDA-MB-435, MCF-7 and 293T cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) containing 10% newborn calf serum at 37°C in a humidified atmosphere of 5% CO2 in air. For transfection, MDA-MB-435, MCF-7 and 293T cells were seeded in 12-well plates containing phenol red-free RPMI 1640 medium (Invitrogen) supplemented with 10% charcoal dextran-treated fetal bovine serum (Hyclone). The cells were transfected using Lipofectamine 2000 (Invitrogen) with 0.2 µg of ERE-LUC or PSA-LUC reporter plasmid, 50 ng of ER{alpha} or AR expression vector, 0.1 µg of ß-galactosidase reporter, and 20 ng–0.5 µg of the expression vectors for XBP-1S or XBP-1U, and the respective empty vector was used to adjust the total amount of DNA. After treatment with 10 nM 17ß-estradiol (E2; for ER{alpha}) or 2 nM R1881 (for AR) for 24 h, the transfected cells were harvested, and luciferase and ß-galactosidase activities were determined as described previously (27). ß-Galactosidase activity was used as an internal control for transfection efficiency. All experiments were repeated at least three times with similar results.

GST pull-down assay
GST and GST fusion proteins were expressed and purified according to the manufacturer’s instructions (Pharmacia), with the induction of protein expression performed at 20°C overnight (30). The expression vector for ER{alpha}, XBP-1S or XBP-1U was used for in vitro transcription and translation in the TNT Reticulocyte Lysate System (Promega). The 35S-labeled ER{alpha}, XBP-1S or XBP-1U was mixed with 10 µg of GST derivatives bound to glutathione–Sepharose beads in 0.5 ml of binding buffer [50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3 mM dithiothreitol (DTT), 0.1% NP-40 and protease inhibitor tablets from Roche]. The binding reaction was performed at 4°C overnight and the beads were subsequently washed four times with the washing buffer (the same as the binding buffer), 30 min each time. The beads were eluted in 10 µl of 2x SDS–PAGE sample buffer and the proteins were resolved on a 10% denaturing gel. The gel was then dried and exposed to X-ray films overnight.

Co-immunoprecipitation
293T cells were transfected using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cells were washed twice with phosphate-buffered saline (PBS) and lysed in 0.5 ml of lysis buffer (50 mM Tris at pH 8.0, 500 mM NaCl, 0.5% NP-40, 1 mM DTT and protease inhibitor tablets from Roche). After brief sonication, the lysate was centrifuged at 14 000 r.p.m. for 15 min at 4°C. The supernatant was used for subsequent co-immunoprecipitation (30). A 15 µl aliquot of 50% slurry of the anti-FLAG agarose beads (Sigma-Aldrich) was used in each immunoprecipitation. Immunoprecipitation was performed overnight at 4°C. The beads were centrifuged at 3000 r.p.m. for 2 min, and washed four times with the washing buffer (the same as the lysis buffer), with each wash lasting at least 30 min. The precipitates were then eluted in 30 µl of 2x SDS–PAGE sample buffer and loaded on SDS–polyacrylamide gels, followed by western blotting according to the standard procedures. A 4 µl aliquot of the input crude extract was used for detecting protein expression levels. The ER{alpha} proteins were detected using an anti-ER{alpha} polyclonal antibody (HC-20; Santa Cruz Biotechnology).

Gel shift assay
The double-stranded oligonucleotide (31), corresponding to the consensus ERE (5'-AGCTCTTTGATCAGGTCACTGTGACCTGACTTT-3') or mutant ERE (EREM; 5'-AGCTCTTTGATCAGTACACTGTGACCTGACTTT-3'), was 32P-labeled using T4 polynucleotide kinase (Promega). Binding reactions (20 µl) were performed in the presence or absence of ligands (1 µM) for 30 min using in vitro translated protein or the same amount of unprogrammed lysate (Promega) in 10 mM Tris pH 7.5, 50 mM NaCl, 1 mM MgCl2, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT and 0.1 µg/µl poly(dT–dC). The labeled ERE (0.5 ng) or EREM (0.5 ng) was then added, incubated for 20 min at room temperature, and analyzed on a 5% polyacrylamide gel. Protein–DNA binding was visualized by autoradiography.

RT–PCR analysis
Total RNA was extracted using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). First strand cDNA was reverse transcribed from 1.0 µg of total RNA with oligo(dT) primers using AMV reverse transcriptase as recommended by the supplier (Promega). A 1 µl aliquot of the first strand cDNA synthesis reaction mixture was used for PCR amplification in a total volume of 50 µl. The oligonucleotides 5'-TCTGCTGAGTCCGCAGCAG-3' and 5'-GAAAAGGGAGGCTGGTAAGGAAC-3' were used for amplification of a 233 bp fragment of XBP-1S, and 5'-TGGTTGCTGAAGAGGAGGCGGAAG-3' and 5'-GAGATGTTCTGGAGGGGTGACAACTG-3' for amplification of a 136 bp fragment of XBP-1U. PCR amplifications were performed for 35 cycles using the following cycling parameters: 94°C for 1 min, 68°C for 1 min and 72°C for 1 min. The RT–PCR products were purified and ligated to a T vector, and the resulting positive clones were sequenced. ß-Actin was amplified as described previously (32), and used as an internal control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Potentiation of ER{alpha} transcriptional activity by XBP-1S and XBP-1U
To investigate the effects of the XBP-1 proteins on ER{alpha} transcriptional activity, human breast cancer MDA-MB-435 cells, which lack the ER{alpha}, were co-transfected with the estrogen response element-containing reporter ERE-LUC, ER{alpha} and increasing amounts of XBP-1S or XBP-1U. As shown in Figure 1A, in the absence and presence of E2, 0.5 µg of XBP-1S enhanced the transcriptional activity of ER{alpha} 32- and 15-fold, respectively, whereas 0.5 µg of XBP-1U only enhanced ER{alpha} transcriptional activity 2.1- and 2.2-fold, respectively. This might be explained by the structural differences between the C-terminal transactivation domains of XBP-1S and XBP-1U. Both XBP-1S and XBP-1U increased ER{alpha} transcriptional activation in a dose-dependent manner (Fig. 1A). It is important to note that the magnitude of the ligand-independent activation of the ER{alpha} by XBP-1S (0.5 µg) was 5-fold higher than that observed with E2 (10 nM), and that XBP-1S and estrogen were synergistic in stimulating estrogen-regulated transcription. The activity of the ERE-LUC reporter activated by XBP-1S and XBP-1U decreased dramatically in the absence of exogeneous ER{alpha} (Fig. 1B), indicating that ER{alpha} itself was required for the maximal effects of XBP-1S and XBP-1U on ERE-LUC reporter transcription.



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  		Figure 1. XBP-1S and XBP-1U enhance ER{alpha}-mediated transactivation in MDA-MB-435 cells. (A) Effects of XBP-1S and XBP-1U on ER{alpha}-mediated transactivation. Cells were co-transfected with 0.2 µg of ERE-LUC, 50 ng of the expression plasmid for ER{alpha} and increasing amounts of the expression vector for either XBP-1S or XBP-1U as indicated. Cells were then treated with control (0.1% ethanol) vehicle or 10 nM E2 for 24 h before luciferase assay. The LUC activity obtained on transfection of ERE-LUC and ER{alpha} without exogenous XBP-1S and XBP-1U in the absence of E2 was set as 1. Results are expressed as means ± SE for three independent experiments. (B) Effect of ER{alpha} on ERE-LUC reporter gene transcription by XBP-1S and XBP-1U. Cells were co-transfected with 0.2 µg of ERE-LUC and 0.5 µg of the expression vector for either XBP-1S or XBP-1U in both the absence and presence of the expression plasmid for ER{alpha}. Cells were then treated and analyzed as in (A). (C) Effects of antiestrogens on ERE-LUC reporter gene transcription by XBP-1S and XBP-1U. Cells were co-transfected with 0.2 µg of ERE-LUC, 50 ng of the expression plasmid for ER{alpha} and 0.5 µg of the expression vector for either XBP-1S or XBP-1U. Cells were then treated with control (0.1% ethanol) vehicle, 10 nM E2, 100 nM 4-OHT or 100 nM ICI 182,780 for 24 h before luciferase assay. Cells were analyzed as in (A).

 
To examine the effects of antiestrogens on ER{alpha} transactivation by XBP-1S and XBP-1U, MDA-MB-435 cells were co-transfected with the ERE-LUC reporter, ER{alpha} and XBP-1S or XBP-1U, and subsequently treated with antiestrogens, 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (Fig. 1C). Both ICI 182,780 and 4-OHT completely blocked the effects of XBP-1U on ER{alpha} transcriptional activity in the presence or absence of E2, whereas both ICI 182,780 and, to a lesser extent, 4-OHT reduced but did not abolish the ability of XBP-1S to transactivate ER{alpha}.

To determine whether the observed effects of XBP-1S and XBP-1U on ER{alpha} transactivation were specific to MDA-MB-435 cells, human breast cancer cell line MCF-7 and human embryonic kidney cell line 293T were used in co-transfection experiments. Although ER{alpha} transactivation by XBP-1S in MCF-7 and 293T cells was of a lower magnitude than in MDA-MB-435 cells, similar results were observed (Table 1). The transcriptional activity of ER{alpha} co-activated by XBP-1S was always greater than that co-activated by XBP-1U. Thus, XBP-1S and XBP-1U can act as positive regulators of ER{alpha}-dependent transcriptional activation in a variety of mammalian cell lines. To verify that this E2-independent enhanced transcriptional activity was not a result of increased ER{alpha} protein production, we examined protein expression in whole-cell extracts using western blotting analysis. Figure 2 shows that ER{alpha} levels were not increased by XBP-1S or XBP-1U expression. In addition, the greater effects of XBP-1S on ER{alpha} transcriptional activation were also not attributable to its high expression level. Conversely, the expression level of XBP-1S was lower than that of XBP-1U (Fig. 2). Together, our results suggest that XBP-1S and XBP-1U could function as a co-regulator to enhance ER{alpha} transcriptional activity in a ligand-independent manner.


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  		Table 1. XBP-1S and XBP-1U enhance ER{alpha}-mediated transactivation
 


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  		Figure 2. Western blotting showing the ER{alpha}, XBP-1S and XBP-1U protein levels in 293T cells. Cells were transfected as in Table 1. Whole-cell extracts were prepared, and equivalent amounts of each extract were probed with anti-ER{alpha} antibody (H-184; Santa Cruz Biotech) or anti-XBP-1 antibody (SC-7160; Santa Cruz Biotechnology).

 
To determine the specificity of XBP-1S and XBP-1U in ER{alpha}-mediated transactivation, we tested the effects of XBP-1S and XBP-1U on AR-mediated transactivation using the ARE-containing reporter construct PSA-LUC. As expected, transcription of the reporter was induced by an androgen, R1881, in MDA-MB-435, MCF-7 and 293T cells (Table 2). XBP-1S and XBP-1U failed, however, to enhance AR-mediated transcription in both the presence and absence of R1881. Conversely, in the presence and absence of R1881, XBP-1S decreased AR transcriptional activity ~2- and 4-fold, respectively, in MCF-7 and 293T cells (Table 2). This result suggests that XBP-1S and XBP-1U may not be general co-activators for steroid receptors.


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  		Table 2. XBP-1S and XBP-1U do not enhance AR-mediated transactivation
 
Both the N- and C-terminal domains of ER{alpha} contribute to ER{alpha} transactivation by XBP-1S and XBP-1U
To determine which domain of ER{alpha} is involved in the co-activation of ER{alpha} by XBP-1S and XBP-1U, ER{alpha} constructs containing N-terminal AF-1 and DNA-binding domain (DBD) (ABC domain) or C-terminal AF-2 and DBD (CDEF domain) were co-expressed with XBP-1S or XBP-1U in MDA-MB-435 cells. XBP-1S and, to a lesser extent, XBP-1U enhanced both the constitutive transactivation activity of AF-1 and ligand-dependent transcriptional activity of AF-2 (Fig. 3). However, co-expression of XBP-1S or XBP-1U with full-length ER{alpha} resulted in a more efficient enhancement of the reporter transcriptional activity (2- to 5-fold), compared with ABC and CDEF domains expressed separately (Fig. 3). Thus, both N- and C-terminal domains of ER{alpha} contribute to ER{alpha} transcriptional activity by XBP-1S and XBP-1U.



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  		Figure 3. Both N- and C-terminal domains contribute to ER{alpha} transcriptional activity regulated by XBP-1S and XBP-1U. MDA-MB-435 cells were co-transfected with 50 ng of the expression vector for ER{alpha}, ER{alpha} ABC domain or ER{alpha} CDEF domain, 0.2 µg of ERE-LUC and 0.5 µg of the expression vector for either XBP-1S or XBP-1U as indicated. The LUC activity obtained on transfection of ERE-LUC without exogenous ER{alpha}, ER{alpha} ABC domain, ER{alpha} CDEF domain, XBP-1S and XBP-1U in the absence of E2 was set as 1. Results are expressed as means ± SE for three independent experiments.

 
Cooperative co-activation of ER{alpha} by XBP-1 and SRC-1
SRC-1 is a member of the p160 steroid receptor coactivator (SRC) family (33). SRC-1 interacts with ER{alpha} and stimulates E2-mediated gene transcription. SRC-1 enhances the interaction between the N-terminal AF-1-containing and C-terminal AF-2-containing regions of the ER{alpha}, allowing for full ER{alpha} activation. To determine whether XBP-1 plays a role in SRC-1-mediated co-activation of the ER{alpha}, MDA-MB-435 cells were co-transfected with the ERE-LUC reporter construct and expression vectors for XBP-1 and SRC-1 (Table 3). As expected, in the absence of E2, transfection of XBP-1S or XBP-1U alone co-activated ER{alpha} transcriptional activity, whereas transfection of SRC-1 did not have a significant effect. Co-transfection with XBP-1S or XBP-1U, plus SRC-1 expression vectors gave greater than additive effects of XBP-1S or XBP-1U and SRC-1 measured independently. In the presence of E2, XBP-1S, XBP-1U and SRC-1 all enhanced ER{alpha} transcriptional activity to different levels. When SRC-1 was co-expressed with either XBP-1S or XBP-1U, however, the ER{alpha} transcriptional activity was cooperatively enhanced. Therefore, the synergistic enhancement of ER{alpha} activity by co-expression of SRC-1 and XBP-1S or XBP-1U is ligand independent.


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  		Table 3. Synergistic enhancement of ER{alpha} activity by SRC-1 and XBP-1
 
Interaction of XBP-1S and XBP-1U with ER{alpha} in vitro and in vivo
Our observation that XBP-1S and XBP-1U could function as a co-regulator to enhance ligand-independent ER{alpha} transactivation suggested that XBP-1S and XBP-1U might physically interact with ER{alpha}. To test this possibility, GST pull-down experiments were performed in which in vitro translated [35S]methionine-labeled XBP-1S or XBP-1U was incubated with full-length GST–ER{alpha}. The binding of XBP-1S and XBP-1U to GST–ER{alpha}, but not to GST, was observed in both the absence and presence of E2 (Fig. 4A). E2 did not increase the interaction of XBP-1S and XBP-1U with GST–ER{alpha}. Notably, the binding of XBP-1S to ER{alpha} was stronger than that of XBP-1U to ER{alpha}.




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  		Figure 4. XBP-1S and XBP-1U bind to ER{alpha} in vitro and in vivo. (A) Interaction of XBP-1S and XBP-1U with ER{alpha} in vitro. GST pull-down assay was performed as described in Materials and Methods. Full-length GST–ER{alpha} fusion proteins, immobilized on beads, were mixed with in vitro translation reaction mixtures of XBP-1S or XBP-1U in the absence or presence of E2 (10–6 M) as indicated. The bound proteins were subjected to SDS–PAGE followed by autoradiography. (B) Interactions between either XBP-1S or XBP-1U and ER{alpha} in vivo. 293T cells were co-transfected with the expression vectors for either the FLAG-tagged XBP-1 or the FLAG-tagged XBP-1U and ER{alpha} as indicated. Lysates from the transfected cells were immunoprecipitated (IP) using anti-FLAG antibody (Sigma-Aldrich), and the immunoprecipitates were probed with an anti-ER{alpha} antibody (HC-20; Santa Cruz Biotech). (C) Mapping of interaction regions of XBP-1S and XBP-1U in ER{alpha}. GST pull-down assay was performed using 35S-labeled XBP-1S or XBP-1U and fusion proteins between GST and three different ER{alpha} fragments. (D) Mapping of the ER{alpha} interaction region in XBP-1S and XBP-1U. GST pull-down assay was performed using 35S-labeled ER{alpha} and fusion proteins between GST and six different XBP-1 fragments.

 
To determine whether XBP-1S and XBP-1U interact with ER{alpha} in vivo, 293T cells were transfected with ER{alpha} and FLAG-tagged XBP-1S or XBP-1U and cultured in both the absence (Fig. 4B) and presence of 10 nM E2 (data not shown). FLAG-XBP-1S or XBP-1U was immunoprecipitated from cell lysates by an anti-FLAG antibody and analyzed for ER{alpha} binding by western blotting analysis. The results showed that ER{alpha} could be co-immunoprecipitated in a ligand-independent manner in the presence, but not in the absence, of FLAG-XBP-1S or FLAG-XBP-1U (Fig. 4B, and data not shown). Consistent with the co-activation results, the binding of XBP-1S to ER{alpha} was stronger than that of XBP-1U to ER{alpha}. The in vivo interaction of XBP-1S and XBP-1U with ER{alpha} was unlikely to be mediated by nucleic acids, as it was not affected by the treatment with ethidium bromide that disrupts DNA–protein interaction (data not shown).

Mapping of the ER{alpha} and XBP-1 interaction domains
To determine which region of ER{alpha} binds to XBP-1S or XBP-1U, GST pull-down experiments were performed. As shown in Figure 4C, the GST–ER{alpha}(180–282) containing the DBD bound specifically to in vitro translated [35S]methionine-labeled XBP-1S or XBP-1U, but the GST-ER{alpha}(1–185) containing the AF-1 and the GST–ER{alpha}(282–595) containing the AF-2 did not. Consistent with the in vivo binding results, XBP-1S interacted with GST–ER{alpha}(180–282) in vitro more strongly than XBP-1U.

XBP-1S and XBP-1U are proteins of 261 and 376 amino acids, respectively, with an identical N-terminus (amino acids 1–164). To delineate the domains in the XBP-1S and XBP-1U that mediate the protein–protein interaction with ER{alpha}, a series of mutant GST–XBP-1 fusion proteins were used in GST pull-down experiments (Fig. 4D). Deletion of the XBP-1 N-terminal amino acids (1–82) reduced but did not abolish the ability of the XBP-1 proteins to bind to the ER{alpha}. Either region (amino acids 1–101, containing the bZIP domain, or either amino acids 148–376 or amino acids 148–261, containing the transactivation domain) of XBP-1S or XBP-1U was sufficient for ER{alpha} binding. However, the full-length GST–XBP-1 interacted with ER{alpha} more strongly than any GST–XBP-1 fragments. In addition, GST–XBP-1S associated with ER{alpha} more strongly than GST–XBP-1U.

The N-terminal portion of XBP-1 is required for ER{alpha} transactivation function
To test the possibility that the maximal interaction of XBP-1S and XBP-1U with ER{alpha} is required for the enhancement of ER{alpha} transcriptional activation, mutants of XBP-1S and XBP-1U ({Delta}XBP-1S and {Delta}XBP-1U) were made in which the N-terminal region from amino acids 1 to 82 was deleted. MDA-MB-435 cells were co-transfected with the ERE-LUC reporter, ER{alpha} and either FLAG-tagged XBP-1S, XBP-1U, {Delta}XBP-1S or {Delta}XBP-1U. As shown in Figure 5A, the mutations lacking some of the ER{alpha}-binding sites completely abolished the ER{alpha} transcriptional activation in a ligand-independent manner. This is not attributable to decreased expression of the {Delta}XBP-1S and the {Delta}XBP-1U deletion mutants. In contrast, {Delta}XBP-1S and {Delta}XBP-1U were expressed at higher levels than XBP-1S and XBP-1U (Fig. 5B). Taken together, these findings suggest that the XBP-1S and XBP-1U action of ER{alpha} by their maximal binding contributes to the transactivation function of ER{alpha}.



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  		Figure 5. The deletion mutants of XBP-1S and XBP-1U abolished the ER{alpha} transactivation. (A) MDA-MB-435 cells were co-transfected with 0.2 µg of ERE-LUC, 50 ng of the expression plasmid for ER{alpha} and 0.5 µg of the expression vector for FLAG-tagged XBP-1S, XBP-1S{Delta}82, XBP-1U or XBP-1U{Delta}82 as indicated. Cells were then treated with control (0.1% ethanol) vehicle or 10 nM E2 for 24 h before luciferase assay. The LUC activity obtained on transfection of ERE-LUC and ER{alpha} without exogenous XBP-1 and its derivatives in the absence of E2 was set as 1. Results are expressed as means ± SE for three independent experiments. (B) Western blotting showing expression of FLAG-tagged XBP-1S, XBP-1S{Delta}82, XBP-1U and XBP-1U{Delta}82. Cells were transfected as in (A). Whole-cell extracts were prepared, and equivalent amounts of each extract were probed with anti-FLAG antibody (Sigma-Aldrich).

 
Neither XBP-1S nor XBP-1U binds to ERE
The transcription factor XBP-1 was found to bind preferably to the cAMP responsive element (CRE)-like element GATGACGTG(T/G)nnn(A/T)T (34). ER{alpha} binds to the ERE GGTCAnnnTGACC. Since both XBP-1 and ER{alpha} target sequences have the sequence TGAC, we tested whether XBP-1S and XBP-1U bind to the consensus ERE, using a gel shift assay. As expected, the 32P-labeled ERE, but not mutant ERE (EREM), bound to in vitro-translated ER{alpha} in the absence or presence of E2 (Fig. 6 and data not shown). The binding was specifically inhibited by a 100-fold molar excess of a cold ERE oligonucleotide. Moreover, neither XBP-1S nor XBP-1U bound to the ERE.



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  		Figure 6. Neither XBP-1S nor XBP-1U binds to ERE. Gel shift assay was performed as described in Materials and Methods. The 32P-labeled ERE probe was incubated with the in vitro-translated ER{alpha}, XBP-1S and XBP-1U proteins as indicated, in the presence of 1 µM E2. For competition experiments, a 100-fold molar excess of unlabeled ERE was mixed with the radioactive probe. The 32P-labeled mutant ERE (EREM) probe was used as a negative control.

 
XBP-1S and XBP-1U mRNAs are expressed in breast cancer cell lines
Human XBP-1 (XBP-1U) was originally isolated as a transcription factor which binds to the X2 box present in the promoter region of human major histocompatibility complex (MHC) class II genes (14). More recently, XBP-1U mRNA was found to be spliced in response to the endoplasmic reticulum stress, resulting in XBP-1S (18,19). To determine whether XBP-1S mRNA is expressed in breast cancer cells, we prepared mRNA from five ER-positive (MCF10A, T47D, MCF7, ZR75-1 and BT474) and five ER-negative (MDA-MB-436, MDA-MB-435, MDA-MB-231, SKBR3 and MDA-MB-453) breast cancer cell lines and performed RT–PCR using primers specific for the unique XBP-1S region. As shown in Figure 7, in addition to XBP-1U mRNA, we could also detect XBP-1S mRNA in all of the breast cancer cell lines tested. The identity of XBP-1S detected was further confirmed by DNA sequencing. XBP-1S was also expressed in the immortalized normal breast epithelial cell line MCF-10A, although at a lower level. Since a limited number of breast cancer cell lines were examined, there is no significant correlation between XBP-1 and ER{alpha} expression. However, both XBP-1S and XBP-1U were widely expressed in breast cancer cells. Interestingly, XBP-1S was also expressed in mammary and ovary two-hybrid cDNA libraries, which were prepared from the corresponding tissues of individuals who died from trauma, indicating that XBP-1S is more widely expressed than previously thought. After searching expressed sequence tags (ESTs) for XBP-1S in GenBank (www.ncbi.nlm.nih.gov/blast), we also found that human XBP-1S mRNA is expressed in breast adenocarcinoma, lung tumor, stomach, brain, skin and pancreas, and mouse XBP-1S is expressed in mammary tumor, lung tumor metastatic to mammary, and neural retina. Taken together, these data suggest that, like XBP-1U, XBP-1S may play a critical role under physiological and pathological conditions.



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  		Figure 7. XBP-1 mRNA expression in breast cancer cell lines. RT–PCR from the selected cell lines and cDNA libraries was performed as described in Materials and Methods. ß-Actin was used as an internal control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several recent studies using cDNA microarray analysis have shown that XBP-1 mRNA expression is associated with ER{alpha} status in breast tumors (20–25), although the forms of XBP-1 are unknown. This raises at least two possibilities: (i) XBP-1 participates in regulating the ER{alpha} promoter or ER{alpha} in regulating the XBP-1 promoter; and (ii) XBP-1 interacts with ER{alpha}. In this manuscript, we report for the first time that XBP-1 physically and functionally interacts with ER{alpha} in a ligand-independent manner. Since XBP-1S and XBP-1U do not bind to the ERE, it is possible that ER{alpha} recruits XBP-1 to the ERE-containing promoter to stimulate gene transcription.

ER{alpha} has been shown to interact with a number of cofactors, including co-activators and co-repressors (1). Almost all of the co-activators enhance ER{alpha} transcriptional activity in a ligand-dependent fashion. They include the SRC-1 family (35), CBP/p300 (36,37), p68 (38), ARA70 (39) and many others (1). In addition to the conventional ligand-dependent regulation of the activity of ER{alpha}, ER{alpha} has been shown to be activated by non-steroidal agents including dopamine and growth factors (40). This ligand-independent activation is possibly due to the ER{alpha} phosphorylation. However, by definition (1), none of the agents are co-activators, as they do not interact with ER{alpha}. Cyclin D1 is the first co-activator to have been shown to enhance ER{alpha} transactivation in a ligand-independent manner (41–43). Cyclin D1 acts through physical association to activate ER{alpha}. To the best of our knowledge, XBP-1 is the second co-activator to enhance ligand-independent ER{alpha} transcriptional activation. XBP-1S, the spliced form of XBP-1, is more potent in ER{alpha} activation than XBP-1U, the unspliced form of XBP-1. This is consistent with the stronger binding affinity of XBP-1S for ER{alpha} both in vitro and in vivo. Effects of antiestrogens on the cyclin D1-induced activation of the ER{alpha} are controversial. Neuman et al. have shown that ICI 182,780 and, to a lesser extent, 4-OHT significantly inhibit cyclin D1-mediated activation of the ER{alpha}, which is in contrast to the findings of Zwijsen et al. (41,42). In our study, both ICI 182,780 and 4-OHT inhibited the effects of XBP-1 on ER{alpha} activity in a similar manner to that reported by Neuman et al.

Co-activators that interact with the DBD of nuclear receptors are less characterized or defined. Our study showed that XBP-1S and XBP-1U interact with the ER{alpha} region containing the DBD. Recently, several co-activators that interact with the DBDs of nuclear receptors have been reported, including GT198 (44) and SNURF (45). GT198 interacted with the DBD of glucocorticoid receptor (GR) and potently stimulated transcription mediated by GR. SNURF is a small RING finger protein that co-activates AR-mediated transcription through interaction with the DBD of AR. Since the DBDs of nuclear receptors are highly conserved, we determined the effects of XBP-1U and XBP-1S on AR-mediated transcription. In contrast to the co-activation of ER{alpha} by XBP-1S and XBP-1U, XBP-1S decreased transcriptional activity of AR in a cell type-dependent manner, whereas XBP-1U had little effect on AR transcriptional activation (Table 2). This may be exemplified by the cofactor RIP140 (46–50). This cofactor interacts with ER{alpha} and increases ER{alpha} transcriptional activity in the presence of E2 (46,51). RIP140 has also been shown to act as a co-activator for the rat AR (47). However, recent studies have indicated that RIP140 represses the transcriptional activation of the GR and the orphan nuclear receptor TR2 by interaction with the receptors (48,49). It will be interesting to examine the effects of XBP-1S and XBP-1U on the transcriptional activities of other nuclear receptors.

Although XBP-1 mRNA expression levels have been shown to be elevated during cancer development and progression (20–26), the forms of XBP-1 are unclear. Initially, XBP-1U was thought to be the only form of XBP-1 mRNA expression (14). Recently, it was shown that XBP-1S was expressed in response to endoplasmic reticulum stress (18,19). XBP-1S was also found to be induced during the differentiation of antibody-secreting B cells (50). We report here that XBP-1S mRNA is constitutively expressed in breast cancer cell lines as well as an immortalized human mammary epithelial cell line. Future experiments are required to test whether XBP-1S is expressed in clinical samples. Since the XBP-1 mRNA expression pattern is highly correlated with ER{alpha} and highly upregulated in a subset of breast cancers (20–26), XBP-1, especially XBP-1S, may play an important role in breast cancer growth and represent a new target for therapeutic intervention.


   ACKNOWLEDGEMENTS
 
We thank Rong Li for sharing reagents, Philip Leder and Fen Zhou for kindly providing the MCF10A, MDA-MB-435, MDA-MB-436, MDA-MB-453, ZR75-1, SKBR3 and BT474 cell lines, and Hsiang-Fu Kung for critical reading of the manuscript. This work was supported by Innovative Grant from AMMS and SRF for ROCS, and Fujian Qian Bai Wan Outstanding Scientist Plan Grant.


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