Transcriptional Interaction of an Estrogen Receptor Splice Variant and ErbB4 Suggests Convergence in Gene Susceptibility Pathways in Schizophrenia*

Mounting evidence from clinical and basic research suggests that estrogen signaling may be altered in the brains of people with schizophrenia. Previously, we found that DNA sequence variation in the estrogen receptor (ER) α gene, lower ERα mRNA levels, and/or blunted ERα signaling is associated with schizophrenia. In this study, we asked whether the naturally occurring truncated ERα isoform, Δ7, which acts as a dominant negative, can attenuate gene expression induced by the wild-type (WT) receptor in an estrogen-dependent manner in neuronal (SHSY5Y) and non-neuronal (CHOK1 and HeLa) cells. In addition, we determined the extent to which ERα interacts with NRG1-ErbB4, a leading schizophrenia susceptibility pathway. Reductions in the transcriptionally active form of ErbB4 comprising the intracytoplasmic domain (ErbB4-ICD) have been found in schizophrenia, and we hypothesized that ERα and ErbB4 may converge to control gene expression. In the present study, we show that truncated Δ7-ERα attenuates WT-ERα-driven gene expression across a wide range of estrogen concentrations in cells that express functional ERα at base line or upon co-transfection of full-length ERα. Furthermore, we find that ErbB4-ICD can potentiate the transcriptional activity of WT-ERα at EREs in two cell lines and that this potentiation effect is abolished by the presence of Δ7-ERα. Immunofluorescence microscopy revealed nuclear co-localization of WT-ERα, Δ7-ERα, and ErbB4-ICD, whereas immunoprecipitation assays showed direct interaction. Our findings demonstrate convergence between ERα and ErbB4-ICD in the transcriptional control of ERα-target gene expression and suggest that this may represent a convergent pathway that may be disrupted in schizophrenia.

Schizophrenia is a devastating mental illness of unknown cause, afflicting 1% of the population worldwide. At present, the molecular processes underlying the development and progression of schizophrenia have not been clearly identified; however, we have recently found evidence to support the hypothesis that schizophrenia arises from a derailment of normal brain maturation during adolescence where the brain fails to respond to the developmental increase in sex steroids (testosterone and estrogen) (1). Testosterone is commonly converted into estrogen by the action of the enzyme aromatase whereby the converted hormone serves as a ligand for estrogen receptors. Schizophrenia affects both men and women occurring at a ratio of ϳ3:2, respectively (2). Males typically have an earlier age of onset and more severe symptoms and tend to be less treatment responsive than females with schizophrenia (3). However, women with schizophrenia can have symptom exacerbation at times of low estrogen: during post-partum and menopause (4 -6), and schizophrenic symptoms can be ameliorated with estrogen treatment (7)(8)(9)(10)(11).
The regulatory effects of estrogen are mediated by two closely related receptors, estrogen receptor ␣ (ER␣ 3 (ESR1, NR3A1)) (36) and ER␤ (ESR2, NR3A2) (37). The ERs are ligandactivated transcription factors belonging to the steroid/thyroid hormone superfamily of nuclear receptors (38). In our current study, we focus our attention toward ER␣ because we have recently shown that sequence variation in the ER␣ gene, lower ER␣ mRNA, and/or decreased full-length or wild-type (WT) ER␣ mRNA is associated with the diagnosis of schizophrenia (1,39,40). The ER␣ gene contains eight coding exons, which trans-late into three independent but interacting functional domains including an activation domain, a DNA-binding domain, and an estrogen-binding domain (41). The estrogen-binding domain within the C-terminal region is necessary for liganddependent receptor dimerization. This region also contains the activation function 2, which promotes transcription by recruiting co-activators upon ligand binding (42)(43)(44). This estrogenbinding domain is coded for by exons 6 -8 of the ER␣ gene. We have previously detected as many as 18 distinct ER␣ splice variants in the human cortex with the exon-skipped ⌬7 being the most abundant naturally occurring splice transcript of the ER␣ expressed in the primate brain (1,39), in normal breast (45), and in ER-positive mammary carcinoma cell lines (46). We and others have previously shown that ⌬7-ER␣ encodes a truncated protein that can modulate WT-ER␣-mediated transcription in yeast and various non-neuronal cell lines by functioning as a dominant negative receptor with the ability to suppress transcriptional activation by WT-ER␣ (1,(47)(48)(49). However, no study to date has investigated the function of ⌬7-ER␣ in neuronal cells. As a primary aim, in this current study we determine whether the truncated ER␣, ⌬7, can act as a dominant negative receptor in neuronal cells to suppress WT-ER␣ mediated transcription in response to estrogen.
In schizophrenia, estrogen signaling may also be altered or impacted through convergence with other disease causing pathways, such as neuregulin (NRG1)-ErbB4. The NRG1-ErbB4 pathway is a leading schizophrenia susceptibility pathway (50 -54), and the ErbB4 mRNA, protein, and functional coupling has been shown to be altered in the brains of patients with schizophrenia (55)(56)(57)(58). ErbB4 is a member of the type I or ErbB receptor tyrosine kinase subfamily that is activated by it's ligand NRG1. Binding of NRG1 stimulates sequential cleavage of the full-length ErbB-4 receptor by tumor neurosis factor ␣ converting enzyme and ␥-secretase, which releases the transcriptionally active intracytoplasmic domain (ErbB4-ICD).
Evidence suggests that alterations in cortical ErbB4-ICD and WT-ER␣ may be involved in the molecular pathology of schizophrenia. Cross-talk between WT-ER␣ and ErbB4-ICD has been implicated in the development and progression of endrocrine-related cancers (59). However, in neurons, interaction between WT-ER␣ and ErbB4 has yet to be described. We hypothesize that ER␣ and ErbB4 may converge to regulate ER␣-mediated gene transcription at EREs where ErbB4-ICD may potentiate estrogen signaling by acting as a co-activator. Thus, as a secondary aim, we also determined whether ErbB4-ICD can potentiate transcriptional activity of WT-ER␣ at EREs in both neuronal and non-neuronal cells.
Although reductions in cortical WT-ER␣ mRNA and ErbB4-ICD protein have been linked to schizophrenia susceptibility, we have also found possible increases in non-WT ER␣ transcripts (splice variants) in mental illness (1,39,58). Considering that ⌬7-ER␣ is the most abundant naturally occurring ER␣ splice transcript, as a final aim, we determined whether ⌬7-ER␣ can impact WT-ER␣ and ErbB4-ICD transcriptional interaction at EREs.
Cell Culture-All of the cell lines were obtained from the American Type Culture Collection and were grown at 37°C in a 5% CO 2 atmosphere. CHOK1 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DF12 1:1 mixture) containing 10% (v/v) FBS supplemented with 100 units/100 g/ml penicillin/streptomycin and 2 mM glutamax. HEK293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) FBS supplemented with 100 units/100 g/ml penicillin/streptomycin and 2 mM glutamax. HeLa and SHSY5Y cells were cultured in RPMI containing 10% (v/v) FBS supplemented with 100 units/100 g/ml penicillin/ streptomycin and 2 mM glutamax. To differentiate SHSY5Y cells, all-trans-retinoic acid (10 M) was added to the culture medium and incubated for 9 days. Prior to transfection the cells were washed twice in PBS and incubated for 24 h in phenol red-free medium containing 10% (v/v) charcoal-stripped FBS supplemented with glutamax (2 mM). Transfections and treatments were conducted for 24 h in the same medium. All of the treatments were added to cells either in absolute ethanol or dimethyl sulfoxide and compared with vehicle-only controls. No cell toxicity was observed for any treatment at the concentrations employed.
Luciferase Reporter Assay-On day 1, the cells were seeded in 48-well plates (density was 2 ϫ 10 4 for CHO K1, 3 ϫ 10 4 for HeLa, and 2 ϫ 10 5 for SHSY5Y). For SHSY5Y, the cells were differentiated 9 days prior. On day 2, the cells were washed and incubated in phenol red-free medium containing 10% (v/v) charcoal-stripped FBS supplemented with 2 mM glutamax. On day 3, 3ϫERE-luc reporter plasmids (a kind gift from Professor Rakesh Kumar, M. D. Anderson Cancer Center, Houston, TX)) (125 ng/well), pZsGreen-ErbB4-ICD (50 ng/well), pDsRed-CMV-wtER␣ (12.5 ng/well), and/or pDsRed-CMV-⌬7ER␣ (37.5 ng/well) were transfected into cells for 24 h using Lipofectamine 2000 (0.5 l/well) or Lipofectamine 2000 combined with magnetofection (0.2 l/well; 20 min on magnet) (Chemicell) for SHSY5Y. The pRL-TK Renilla internal control plasmid (Promega) (12.5 ng/well) was co-transfected for normalization of transfection efficiency. On day 4, the cells were treated with 0.01 nM or the indicated concentrations of 17␤-estradiol for 24 h. On day 5, the cells were washed and resuspended in 50 l of 1ϫ passive lysis buffer. The luciferase assays were performed using a dual luciferase assay reporter system (Promega) according to the manufacturer's instructions in a FluorStar OPTIMA (BMG). The results were normalized to the Renilla control and expressed as relative fold change in luciferase activity relative to the empty vector vehicle-treated control condition.
Immunofluorescence Staining and Fluorescence Microscopy-On day 1, the cells were seeded in 24-well plates containing sterile 19 ϫ 19-mm glass coverslips (density was 4 ϫ 10 4 for CHOK1, 6 ϫ 10 4 for HeLa, and 4 ϫ 10 5 for SHSY5Y). For SHSY5Y, the cells were differentiated 9 days prior. On day 2, the cells were washed and incubated in phenol red-free medium containing 10% (v/v) charcoal-stripped FBS supplemented with 2 mM glutamax. On day 3, the cells were transfected with 100 ng/well overexpression plasmids for 24 h using Lipofectamine 2000 (1 l/well) or Lipofectamine 2000 combined with magnetofection (0.2 l/well; 20 min on magnet) (Chemicell) for SHSY5Y. On day 4, the cells were treated with 0.01 nM 17␤estradiol for 24 h. On day 5, the cells were washed twice with PBS and fixed with 3% (v/v) formaldehyde/PBS for 15 min at room temperature or 37°C for SHSY5Y. For fluorescence microscopy without immunostaining, the cells were then rinsed twice with PBS and mounted on glass slides using mounting medium containing anti-fading reagent ProLong Gold antifade reagent with DAPI (Invitrogen). For immunofluorescence microscopy, the cells were rinsed with PBS (3 ϫ 5 min) and then permeabilized with 0.1% (v/v) Triton X-100 in PBS (5 min). After washing with PBS (3 ϫ 5 min), the cells were incubated with 10% FBS (v/v) in PBS for 1 h at room temperature. The cells on the coverslips were then incubated with primary antibody against ER␣ (Santa Cruz catalog number sc-8005) 1:50 dilution in 10% FBS (v/v) in PBS plus 0.1% (w/v) of saponin) for 16 h at 4°C. After washing with 10% FBS (v/v) in PBS (3 ϫ 5 min), the cells were incubated with 5 g/ml of Alexa Fluor 594-conjugated secondary antibody (Molecular Probes catalog number A21203) for 1 h at room temperature. The cells were washed with 10% FBS (v/v) in PBS (3 ϫ 10 min) and then mounted on glass slides using mounting medium containing anti-fading reagent ProLong Gold antifade reagent with DAPI (Invitrogen). The images were obtained using a Nikon D-Eclipse C1 confocal microscope (Nikon).
Immunoprecipitation and Western Blotting-On day 1, all of the cells were seeded at a density of 2 ϫ 10 6 cells/dish. HEK293 cells were seeded in poly-L-lysine-coated 60-mm dishes. HeLa and CHOK1 cells were seeded in 60-mm dishes. On day 2, the cells were transfected for 48 h with pcDNA3.1V5His-wtER␣ or pcDNA3.1V5His-⌬7ER␣ (2 g) using Lipofectamine LTX transfection reagent (Invitrogen). On day 4, the cells were washed twice with PBS and then scraped into 1 ml of ice-cold PBS. The cells were pelleted and lysed in 1ϫ Tris-buffered saline with 0.01% Triton X-100 (TBST) containing protease inhibitor mixture (Sigma). The cell lysates were then precleared with 20 l of protein A/G-agarose plus beads (Santa Cruz) by rotating for 30 min at 4°C. After centrifugation at 5000 ϫ g for 5 min, precleared lysates (supernatants) were rotated overnight at 4°C with protein A/G-agarose plus beads prebound with 2 g of ErbB4 polyclonal antibody (Santa Cruz catalog number sc-283). The beads were pelleted by centrifugation at 2500 ϫ g and washed with 1ϫ TBST ϩ protease inhibitor mixture for 10 min at 4°C with rotation. This was repeated three times. The beads were then mixed with 5ϫ SDS loading buffer and boiled for 5 min at 95°C. For SHSY5Y and MCF-7 lysates, 40 and 10 g, respectively, were mixed with 5ϫ SDS loading buffer and boiled for 5 min at 95°C. All of the samples were subjected to SDS-PAGE on 12% Bis-Tris gels (Bio-Rad). The proteins were transferred onto nitrocellulose membranes and incubated with blocking solution (5% w/v nonfat milk, 0.1% (v/v) Tween 20 in PBS (PBST) at room temperature for 1 h). The membranes were incubated with a primary antibody for V5 (Invitrogen catalog number R960-25) diluted 1:10000 in blocking solution at 4°C overnight. For detection of endogenous ER␣, the membranes were incubated with a primary antibody for ER␣ (Novocastra catalog number NCL-ER-6F11) diluted 1:500 in 5% bovine serum albumin-PBST; 4°C; overnight. The membranes were washed three times for 10 min with PBST and incubated with peroxidase-conjugated affinity-purified secondary antibodies (anti-mouse (for V5 antibody, diluted 1:5000 in blocking solution; for ER␣ antibody, diluted 1:1000 in 5% bovine serum albumin-PBST) at 4°C for 1 h) (Chemicon International). After further washing, the bound antibodies were incubated with enhanced chemiluminescence reagent (Millipore) and visualized by autoradiography or by chemiluminescence on a Chemidoc Imaging System (Bio-Rad).
Statistical Analysis-All of the experiments are representative of at least two independent experiments. For luciferase, the data are presented as the means Ϯ S.E. from three replicate cultures representative of two independent experiments. The statistical analyses were conducted using Statistica 7 (StatSoft Inc., 2000, STATISTICA for Windows). One-way analysis of variance (ANOVA) was used to assess significance of increasing 17␤-estradiol concentration. The distinct concentration or the type(s) of transfected vectors were used as the independent variable, and relative luciferase activity was used as the dependent variable. ANOVAs were followed up with the Fisher LSD post-hoc analysis to assess the significance among groups. A p value less than 0.05 was considered statistically significant.

Endogenous ER␣ Expression in Cell
Lines-In the present study we used three cell types to assess ER␣-driven gene expression. The neuronal cell line SHSY5Y and the non-neuronal cell lines HeLa and CHOK1. By immunofluorescence staining, we observed nuclear localization of ER␣ in SHSY5Y (Fig. 1, A-C). HeLa cells are reported to be ER-negative (60) and were used as a negative control. We confirmed that no expression of ER␣ was observed in HeLa cells (Fig. 1, D-F). Conversely, CHO K1 cells are ER␣-positive and were used as a positive control. We observed robust staining of ER␣ in CHO K1 cells, most of which is localized in the nucleus (Fig. 1, G-I).
We determined whether this lack of response was due to the type of ER␣ expressed in SHSY5Y cells at base line. We found that SHSY5Y cells express both WT-ER␣ and ⌬7-ER␣ mRNA and protein (supplemental Fig. S1). Endogenous ER␣ protein was detected by Western blotting using an ER␣ antibody targeted to an epitope at the N-terminus of the ER␣ protein between amino acids 1 and 184. We have previously characterized this antibody extensively, demonstrating that it detects WT-ER␣ and smaller ER␣ isoforms in both cell lysates and brain tissue (61). Indeed, multiple bands were detected in SHSY5Y and the MCF-7 positive control. Compared with MCF-7, expression of WT-ER␣ in SHSY5Y is low to negligible. The prominent bands migrating below WT-ER␣ are putative truncated ER␣ proteins. Aside from ⌬7-ER␣, which migrates at ϳ45 kDa, we predict that the band migrating at ϳ42 kDa to be a possible ⌬5-ER␣ based on the predicted molecular masses as previously reported (60). Although both cell types express similar banding pattern, expression levels between the two differed, consistent with our previous suggestion that expression of ER␣ isoforms may be cell type-specific (61).

ErbB4-ICD Potentiation of Transcription at EREs-Previous studies
have shown that ErbB4-ICD could stimulate ER␣ transcription at EREs in non-neuronal cell types (59).  However, the effect of the full-length ErbB4 receptor has yet to be shown. In our next approach, we determined whether ErbB4 potentiation of ER␣ could be incited by the membrane-bound form of ErbB4 or whether it was dependent on cleavage of the full-length ErbB4. Transfection of the full-length ErbB4 receptor into CHOK1 cells did not stimulate transcription at EREs beyond the increase that was seen with 17␤-estradiol alone (p Ͼ 0.1) (Fig. 3A). Analysis of subcellular localization of the GFPtagged full-length ErbB4 receptor showed only plasma membrane and cytoplasmic localization with no apparent localization to the nucleus (Fig. 3B). Together, this implies that cleavage of the ICD (nuclear form) of ErbB4 is necessary to increase transcription at EREs. Because the ICD domain of ErbB4 can exist as 2 isoforms: one that is PI3K binding (CYT1) and one that lacks the PI3K binding domain (CYT2), we next determined whether the ErbB4-ICD PI3K-binding site influences ER␣-mediated transcription. Using CHO K1 cells, we found that both the CYT1-ErbB4-ICD and CYT2-ErbB4-ICD potentiated transcription at EREs to a similar extent. No difference in ERE-driven gene transcription was observed between the two ErbB4-ICD isoforms (p Ͼ 0.1) (Fig. 3C). Both CYT1 and CYT2 ErbB4-ICDs were capable of stimulating transcriptional activity at EREs in the absence of estrogen, although this effect was greater in the presence of estrogen. Considering that no difference was observed between the two ErbB4-ICD isoforms, CYT1 ErbB4-ICD was used in subsequent experiments.
⌬7-ER␣ Disrupts Synergy in Transcriptional Activation by WT-ER␣ and ErbB4-ICD at EREs-In our next approach, we overexpressed both WT-ER␣ and ⌬7-ER␣. As we previously found, overexpression of WT-ER␣ increased transcriptional activity at EREs in all three cell types (all p Ͻ 0.0001), whereas overexpression of ⌬7-ER␣ did not stimulate transcription (all p Ͻ 0.00001) (Fig. 5, second and third columns). Co-expression of WT-ER␣ together with ⌬7-ER␣ significantly attenuated transcriptional activity at EREs in all three cell types (all p Ͻ 0.0001) (Fig. 5, fourth column). These findings confirm what we show in Fig. 2.
When ErbB4-ICD was co-expressed with ⌬7-ER␣ in the absence of WT-ER␣ overexpression, we observed no significant change in transcriptional activity from the control condition in all three cell types (Fig. 5, seventh column), suggesting that ErbB4-ICD potentiation cannot be mediated through ⌬7-ER␣.
ErbB4-ICD Interacts with WT-ER␣ and ⌬7-ER␣-To determine whether ErbB4-ICD physically interacts with WT-ER␣ or ⌬7-ER␣, HEK293, HeLa and CHOK1 cells transfected with either V5-tagged WT-ER␣ or ⌬7-ER␣ (Fig. 7, A, C, and E) were immunoprecipitated for endogenous ErbB4 and immunoprobed via Western blotting for the V5-tagged ER␣. In all three cell types, both WT-ER␣ and ⌬7-ER␣ were found to co-immunoprecipitate with endogenous ErbB4 (Fig. 7, B, D, and F). Con-  sistent with our co-localization data (Fig. 6, D, H, L, P, T, and X), we show that a direct protein-protein interaction between WT-ER␣ and ErbB4-ICD as well as ⌬7-ER␣ and ErbB4-ICD within three different cell types is possible.

DISCUSSION
In this current study, we present several major findings. 1) We showed that the truncated ER␣ receptor, ⌬7-ER␣, can abolish or attenuate estrogen-dependent WT-ER␣-mediated transcription in neuronal and non-neuronal cell lines. 2) In addition, we showed that ErbB4-ICD, but not full-length ErbB4, can potentiate the transcriptional activity of WT-ER␣ at EREs. 3) The positive effect of ErbB4-ICD on WT-ER␣-mediated transcription at EREs is not dependent on the CYT form of ErbB4-ICD. 4) Moreover, we show that ErbB4-ICD potentiation of ER␣-driven gene expression at EREs can occur in the CHO ovary and SHSY5Y neuronal cell line but not in HeLa cells, suggesting that only certain cell types provide a permissive ER␣-responsive environment. 5) Finally, we present two lines of evidence showing direct interaction between ER␣ and ErbB4-ICD. Both WT-ER␣ and ⌬7-ER␣ were found to co-localize with ErbB4-ICD in the nucleus of neuronal and nonneuronal cells. Moreover, immunoprecipitation revealed direct protein-protein interaction between ErbB4-ICD and WT-ER␣ and between ErbB4-ICD and ⌬7-ER␣.
Interestingly, although treatment with estrogen and/or overexpression of ErbB4-ICD can potentiate ER␣-mediated transcription in neuronal cells, this effect was only observed in the presence of exogenous WT-ER␣ expression but not at base line despite positive ER␣ immunoreactivity. In SHSY5Y cells, although we detect ER␣ by immunoprobing, analysis of the predicted sizes for ER␣ splice variants from Western blotting showed low to negligible levels of the full-length WT-ER␣ but with relatively higher levels of smaller ER␣ reactive bands and ⌬7-ER␣ mRNA (supplemental Fig. S1) at base line. This would suggest that SHSY5Y cells may naturally express increased truncated forms of ER␣ and would thus normally be in a repressed state with regards to ER␣-mediated transcription. Previous studies have reported that the relative levels of WT-ER␣ to ⌬7-ER␣ are critical in determining the effectiveness of WT-ER␣ transcriptional activity (47,49). Because ⌬7-ER␣ is naturally expressed with WT-ER␣, it has been suggested that the dominant negative function of the ER␣ splice variants may act to protect tissues from excessive estrogenic signals. This notion is based on a 30-fold higher expression of the ⌬3-ER␣ variant in normal breast cells rather than in breast cancer tissues (62,63). Moreover, estradiol treatment of human endometrial adenocarcinomas grown in nude mice show decreased WT-ER␣ and increased ⌬7-ER␣ protein expression (64). Our finding that SHSY5Y cells are capable of robust induction of estrogen-stimulated luciferase reporter activity by exogenous WT-ER␣ overexpression is consistent with this notion. In the presence of exogenously expressed ⌬7-ER␣, WT-ER␣-mediated transcription at EREs was severely attenuated in the SHSY5Y neuronal cell line.
ErbB4-ICD Potentiation of Transcription at EREs-ErbB4 is thought to be unique among the receptor ErbB tyrosine kinase family members because proteolytic processing of the fulllength ErbB4 receptor releases a bioactive ICD. The biological significance of the ErbB4-ICD is not fully understood. In this current study, we showed that it is the ErbB4-ICD but not fulllength ErbB4 receptor that is responsible for the stimulation in transcriptional activity. By transient transfection of the fulllength ErbB4 receptor, we observed no change in transcriptional activity at EREs or targeting of the full-length receptor to the nucleus of the cell, suggesting that potentiation of ER␣mediated transcription may depend upon cleavage and nuclear localization of ErbB4-ICD. Moreover, this potentiation is not dependent on the availability of the PI3K-binding site, suggesting that coupling to the PI3K/AKT signaling pathway does not affect the ability of the ErbB4-ICD to transclocate to the nucleus and stimulate transcription because both CYT1 and CYT2 ErbB4-ICDs stimulated transcription at EREs to the same extent. This is in contrast to the work of Sundvall et al. (65), who showed that CYT2-ErbB4-ICD was more efficient than CYT1-ErbB4-ICD at stimulating ␤-casein promoter activity. These differences in transcriptional activity may depend on the cell type and gene promoter to which the ErbB4-ICDs are targeted to. This is likely because ErbB4-ICD has been shown to affect a variety of cellular processes in different settings (66,67).
ErbB4-ICD and ER␣-Although the dominant negative activity of ⌬7-ER␣ has been described (1,(47)(48)(49), the mechanism by which this occurs is debatable. Although ⌬7-ER␣ possesses a nuclear localization signal, previous studies have reported nuclear exclusion and primarily cytoplasmic localization of ⌬7-ER␣ (60), suggesting that ⌬7-ER␣ interferes with WT-ER␣ entering the nucleus. In contrast, we showed nuclear localization of ⌬7-ER␣, suggesting that interference with WT-ER␣ occurs within the nucleus. In contrast, ErbB4-ICD is likely to be acting as a co-activator for WT-ER␣-mediated transcription in both a ligand-dependent and independent manner because ErbB4-ICD was able to slightly stimulate transcriptional activity in CHOK1 cells in the absence of estrogen stimulation. However, it is possible that there is endogenous production of estrogen by CHOK1 cells, although ErbB4-ICD stimulated transcriptional activity even further in the presence of exogenous estrogen. The potentiation of ErbB4-ICD-WT-ER␣-mediated transcription was blunted in the presence of ⌬7-ER␣. Other studies have shown by in vitro protein-protein interaction that ⌬7-ER␣ is unable to induce co-activator recruitment and binding in the presence of estrogen (49). Thus, our finding that WT-ER␣ and ⌬7-ER␣ can co-immunoprecipi-  HEK293 (A and B), HeLa (C and D), and CHOK1 cells (E and F) were transfected with V5-tagged WT-ER␣ or ⌬7-ER␣ for 48 h. Protein from cells was then harvested and immunoprecipitated with ErbB4 antibody. The immunoprecipitants were separated by SDS-PAGE and immunoprobed with V5 antibody. Immunoblotting for V5 in whole cell lysates (A, C, and E) and immunoblotting for V5 after pulldown with ErbB4 c-terminal antibody (B, D, and F) are shown. Whole cell lysates and immunoprecipitated bands (V5-WT-ER␣, ϳ69 kDa; V5-⌬7-ER␣, ϳ48 kDa) are shown. NS, nonspecific.
tate with ErbB4 suggests that ErbB4-ICD can directly interact with WT-ER␣-⌬7 heterodimers or WT-WT and ⌬7-⌬7 homodimers. Our finding of ⌬7-ER␣ repression of WT-ER␣-ICD mediated transcription at EREs suggests that the presence of the dominant negative ER␣ as a dimerization partner suppresses a large transcription complex and renders it less estrogen-responsive.
ER␣ and ErbB4-ICD in Human Disease-Although the focus of our laboratory is to determine the relationship between ER␣ and ErbB4 in schizophrenia, the mechanistic data we present in this study suggest that WT-ER␣, ⌬7-ER␣, and ErbB4-ICD may regulate a wide variety of cellular processes that are essential for normal cell function. Indeed, dysregulation of ER␣ and ErbB4 is implicated in various human diseases. In schizophrenia, we have previously reported decreased frequency of WT-ER␣ mRNA and increases in splice variant mRNA expression (1). In various cancers including breast, meningiomas, endometrial hyperplasias, and moderate to well differentiated endometrial adenocarcinomas, changed frequencies of WT-ER␣ and ⌬7-ER␣ mRNA have been reported (68,69). Similarly, with Alzheimer disease, expression of the both WT-ER␣ mRNA and ⌬7 are substantially increased (70). Collectively our finding that ⌬7-ER␣ can function as a dominant negative receptor to suppress estrogen-dependent ER␣-mediated transcription in neuronal and non-neuronal cells suggests that increases in non-wild-type ER␣ mRNA expression may represent a predisposition to disease.
Using postmortem tissue from the human prefrontal cortex, we and others found elevations in the JMA/CYT1 form of the ErbB4 receptor in schizophrenia (56 -58) and decreases in ErbB4-ICD protein (58). Considering that full-length ErbB4 was unable to potentiate WT-ER␣-mediated transcription at EREs, this would suggest that increases in full-length ErbB4 and reductions in ErbB4-ICD may alter the appropriate balance of transcriptional versus nontranscriptional ErbB4 signaling in neurons in schizophrenia.
Summary-To date, only one other study in ER␣-positive breast cancer cells has shown a convergence in function between WT-ER␣ and ErbB4-ICD (59). In our current work, we confirm their findings using another ER␣-positive cell line, CHOK1, and extend it by showing that convergence between WT-ER␣ and ErbB4-ICD can be detected in neuronal cells. Moreover, we found that the ability of ErbB4-ICD to potentiate ER␣-mediated transcription may be dependent on cell type. Additionally, we show for the first time that ⌬7-ER␣ can not only disrupt the transcriptional effects of WT-ER␣ but also disrupt the potentiation mediated by ErbB4-ICD in neuronal and non-neuronal cells. The impact of ErbB4-ICD on ER␣ is likely to be mediated by direct nuclear interaction.
Collectively, our current findings suggest that gene regulation by ER␣-ErbB4-ICD may function as a general mechanism of ER␣-mediated transcription in various WT-ER␣-expressing cell types. In addition, disruption of this interaction by ⌬7-ER␣ may act as an additional transcriptional regulatory mechanism in controlling ER␣ target gene expression. Thus, our findings demonstrate that molecular alterations of ER␣ and ErbB4 expression in ER␣ expressing neuronal and non-neuronal could lead to blunted transcriptional control of DNA promot-ers regulated by estrogen. In regards to schizophrenia, we suggest that the lack of appropriate transcriptional control of steroid-driven maturation in the brain may represent a faulty regulatory pathway where two potential schizophrenia susceptibility genes, ER␣ and ErbB4, may converge to cause alterations in neuronal functioning.