The classical actions of estrogen are mediated via interaction

Transcription

1 Truncated Estrogen Receptor 46-kDa Isoform in Human Relationship to Acute Activation of Nitric Oxide Synthase Gemma A. Figtree, MD, DPhil; Denise McDonald, PhD; Hugh Watkins, MD, PhD, FRCP; Keith M. Channon, MD, MRCP Background Estrogen acutely activates endothelial nitric oxide synthase (enos). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results We detected an estrogen receptor (ER ) transcript in human endothelial cells that encodes a truncated 46-kDa ER ( 1a-hER -46). A corresponding 46-kDa ER protein was identified in endothelial cell lysates. Transfection of cdnas encoding the full-length ER (ER -66) and 1a-hER -46 resulted in appropriately sized recombinant proteins identified by anti-er antibodies. Confocal microscopy revealed that a proportion of both ER -66 and her -46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER isoforms colocalized with enos and mediated acute activation of enos in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by 1a-hER -46 was much less than with ER -66. Furthermore, 1a-hER -46 inhibited classical her -66 mediated transcriptional activation in a dominant-negative fashion. Conclusions These findings suggest that expression of an alternatively spliced, truncated ER isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen. (Circulation. 2003;107: ) Key Words: endothelium cells nitric oxide The classical actions of estrogen are mediated via interaction with nuclear steroid receptors leading to transcriptional regulation of various estrogen-responsive genes. However, recent evidence points to alternative pathways that mediate acute estrogen responses via cell-surface estrogen receptors (ERs). 1 4 An important example is the rapid activation of endothelial nitric oxide synthase (enos) 1,5 via a pathway involving mitogen-activated protein kinase and PI-3 kinase. 5,6 However, the exact identity of the receptors involved in these nonclassical estrogen responses remains unclear. Some evidence suggests that a subpopulation of ER is localized within caveolae and can mediate acute enos activation. 1 However, acute responses to estrogen in cell lines not expressing the classical ER (her -66) 7 and maintained vascular responses to estrogen in ER, 8 ER, 9 and double ER knockout mice 10 have led investigators to consider the possibility of other cell-surface ERs. Alternative ER proteins encoded by mrna splice variants are potential candidates for mediation of rapid estrogen responses, because there is no evidence for genes encoding ER homologues other than ER. Human ER mrna isoforms are generated by splicing of 5 alternative upstream exons (B through F) to a common acceptor site 70 nucleotides (at ,12 ) upstream of the translation start site in exon 1. This alternative splicing produces identical-length proteins but raises the potential for differential, and hence tissue-specific, transcriptional regulation. In addition, an exon-1 truncated transcript (designated 1a-hER -46) has recently been demonstrated in a breast carcinoma cell line, resulting from splicing of the 5 UTR variant exon E or exon F directly to exon In contrast to its full-length counterpart (her -66), recombinant 1ahER -46 has a predicted molecular weight of 46 kda and lacks a transactivation domain, AF-1. This truncated ER acts as a competitive inhibitor of her -66 mediated transactivation in some cell contexts. 13 Furthermore, an ER -like protein of uncertain molecular identity, but similar size (45 kda), was recently implicated in mediating acute estrogen responses in the EA.926 human endothelial cell line. 7 We and others 14 hypothesized that this protein could be translated from the 1a-hER -46 mrna, 13 suggesting a possible role for alternatively spliced ER transcripts in mediating acute estrogen responses. Accordingly, we sought to characterize the roles of the putative 1a-hER -46 and classical 66-kDa ER isoforms in Received June 21, 2002; revision received August 28, 2002; accepted September 13, From the Department of Cardiovascular Medicine, University of Oxford, UK. Correspondence to Prof Keith M. Channon, Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK. keith.channon@cardiov.ox.ac.uk 2003 American Heart Association, Inc. Circulation is available at DOI: /01.CIR F5 120

2 Figtree et al Truncated ER Activates enos 121 mediating acute estrogen responses in human endothelial cells. In particular, we aimed to investigate the relative expression of these transcripts, their cellular localization, and their role in the acute activation of enos by estrogen in human endothelial cells. Methods Cell Culture Cell lines EA.926, hmec, COS-1, and MCF-7 were propagated according to standard protocols. Human umbilical vein endothelial cells (HUVECs) were isolated from single donors and used at passages 2 through 4. For estrogen starvation, cells were incubated in medium without phenol red or serum. Reverse Transcriptase Polymerase Chain Reaction RNA was obtained from human endothelial-derived cell lines and HUVECs with the use of Trizol reagent (Life Technologies). MCF-7 cells were used as a positive control. Reverse transcriptase (RT) was carried out by random priming with 1 g of DNAase-treated RNA (Promega). Polymerase chain reaction (PCR) was performed with the use of forward primers based in 5 exons, as follows: 1A-F: 5 -GGAGCTGGCGGGGGGCGTTG-3 ; 1BF: 5 - CGCGTTTAT- TTTAAGCCCAG- 3 ; 1CF: 5 - CGGCCCTTGACTTCTACAAG- 3 ; 1DF: 5 -CTTCTTCACCTGAGAGAGCC-3 ; 1EF: 5 CAG- AGAAATAATCGCAGAGC-3 ; 1FF: 5 -CCAAAACTGA- AAATGCAGGC-3. The reverse primer, located in exon 2, as follows, was common to all reactions: 2R: 5 -CCTTGCAG- CCCTCACAGGAC-3. Construct Preparation The 1a-hER -46 psg5 expression plasmid was generously provided by the Gannon laboratory (European Molecular Biology Laboratory, Heidelberg, Germany). 13 her -66 psg5 expression plasmid was created by subcloning the 5 coding sequence of ER (exons 1 through 4) from pegfp-c1 her (a gift from the Mancini laboratory, Baylor College of Medicine, Houston, Tex 15 ) into psg5- ER46 with the use of EcoRI and the HindIII site in exon 4. The fusion constructs 1a-hER -46-GFP and ER -66-GFP were created by cloning from psg5 into pegfpn1 (Clontech Laboratories, Inc), with PCR used to generate an in-frame GFP fusion cdna. The luciferase reporter gene (ERE) 2 -tk-luc was a gift from the Scanlan laboratory (University of California, San Francisco). 16 Plasmids were transfected into cells with Fugene 6 (Roche). Immunoblotting and Fluorescent Confocal Microscopy of ER -Related Proteins Immunoblotting and immunofluorescence studies of ER -related protein were performed with monoclonal antibodies to ER C-terminal (F-10, Santa Cruz Biotechnology). enos immunostaining (Transduction laboratories) was performed on COS cells cotransfected with an enos expression plasmid and GFP-tagged ER isoforms to examine the spatial relationships between enos and ER. Bound primary antibodies were visualized with TRITCconjugated goat anti-mouse secondary antibodies (Sigma). Cells were imaged with a Biorad MRC 1024 confocal laser-scanning microscope. Determination of E2-Surface Binding With E2-Conjugated BSA-FITC Cells were transiently transfected with 1a-hER -46, her -66, or -gal control plasmid. After 48 hours, cells were fixed and incubated in E2-conjugated BSA-FITC (Sigma) equating to 30 nmol/l of 17 -estradiol. Unconjugated BSA-FITC was used to examine for nonspecific binding. Cells were mounted in Vectorshield (Vector Laboratories) containing DAPI and TO-PRO-3 and visualized by confocal microscopy. Determination of Estrogen-Induced enos Activation To visualize NO production, we loaded cells with diacetylated DAF-2 (DAF-2DA: Calbiochem). This membrane-permeable dye is hydrolyzed intracellularly by cytosolic esterases releasing DAF-2, which is converted in the presence of NO into a fluorescent product, DAF-2 triazole. 17 Forty-eight hours after cotransfection with 1ahER -46; ER -66 or control; and enos or control plasmids, COS cells were incubated in 1 mol/l DAF-2DA solution for 30 minutes at 37 C in the dark and then washed. Cells were stimulated for 15 minutes in 17 -estradiol (30 nmol/l) in the presence or absence of L-NMMA and then washed and fixed before examination under the confocal microscope. Estrogen-Dependent Transactivation in the Presence of 1a-hER -46 The (ERE) 2 -tk-luc reporter gene construct was used to assess the effect of 1a-hER -46 expression on the transcriptional activity of her -66 in the human endothelial cell derived hmec and EA.926 cell lines and in COS cells. Cells were grown in 24-well plates and transfected at 24 hours with a total of 1 g of DNA per well (0.25 g of (ERE) 2 -tk-luc; 0.25 g of 1a-hER -46 expression vector or control expression vector; 0.25 g ofher -66 expression vector or control expression vector; 0.25 g of internal control -galactosidase expression vector). Cells were stimulated in 30 nmol/l 17 -estradiol for 24 hours and then assayed for luciferase and -galactosidase activity. Reporter gene activity was normalized for transfection efficiency according to the activity of the cotransfected reference control. Results 1a-hER -46 Is Expressed in Human We first used RT-PCR to investigate the expression of ER transcripts in human endothelial cells. Both truncated ( 1ahER -46) and full-length ER transcripts were present in human endothelial cells, including the EA.926 cell line, the hmec microvascular endothelial cell line, and primary HUVEC cultures (Figure 1). In these cells, her transcripts were derived exclusively from exons 1E and 1F. The smaller RT-PCR product observed in human endothelial cells was confirmed by sequencing to result from alternative mrna splicing from 5 untranslated exons 1E or 1F directly to exon 2 (Figure 1) in a manner previously described for the truncated 1a-hER -46 transcript in MCF-7 cells. 13 Next, we sought to investigate whether appropriately sized ER proteins corresponding to these her transcripts were present in endothelial cells. Western blots with an ER C-terminal antibody revealed only a 46-kDa ER protein in EA.926 cells in contrast to both 46- and 66-kDa ER proteins in MCF-7 cells (Figure 2). This 46-kDa protein appeared of identical molecular weight to recombinant 1a-hER -46 expressed in COS cells after transient transfection of the respective cdna (Figure 2). Although some her -66 expression was detected by RT-PCR, repeated Western blots did not detect any ER protein corresponding to the 66-kDa full-length isoform, consistent with observations by the Bender laboratory. 7 Subcellular Localization of her 46- and ER 66-GFP Fusion Proteins We used ER-GFP constructs to investigate the subcellular localization of the 2 ER isoforms in transfected COS cells.

3 122 Circulation January 7/14, 2003 Figure 1. A, RT-PCR demonstrating the expression of 1a-hER -46 transcript (lower bands) in human endothelialderived cell lines EA.926, hmecs, HUVECs, and the positive control MCF-7 cells. The larger molecular weight bands seen are her -66. The lower molecular weight products were confirmed by sequencing to result from a splice event from the 5 exons directly to exon 2 (B). 1a-hER -46 transcripts in the endothelial cells are derived from the 5 exons 1E and 1F. Forward primers for RT-PCR were based on sequence in 5 exons 1A through 1F, and the reverse primer based on sequence in exon 2 (common to all reactions) (B). The arrow points to the donor/acceptor site of 5 exon E/F and exon 2, respectively. The GFP was tagged to the C terminal, ensuring a similar position relative to both ER isoforms. Furthermore, the functional ability of the ER 66-GFP fusion protein to mediate estrogen-dependent transactivation of a target sequence was confirmed before additional investigation. Most her 46- and her -66-GFP fluorescence was observed Figure 2. Western blot demonstrating a 46-kDA ER immunoreactive band in lysates of both MCF cells and EA.926 cells. This was the same molecular weight as that of the recombinant 1ahER -46 isolated from transfected COS cells. The upper band seen in MCF-7 cells and COS cells transfected with ER -66 is the full-length ER isoform. No bands were identified in untransfected COS cells or in COS cells transfected with control gal plasmid. within the nucleus. However, significant proportions of both recombinant her -46 and her -66 were observed outside the nucleus and in association with the plasma membrane (Figure 3). Similar findings were observed in EA.926 cell and hmecs (data not shown). Nonpermeabilized, transfected cells immunostained for ER with C terminal antibody suggested cell-surface localization of a proportion of the receptor, as well as C terminal integrity of the ER component of the fusion proteins. Nontransfected COS cells or cells transfected with GFP-control vector were not recognized by the ER antibody. We determined whether ER -GFP fusion proteins localized in the same way as ER itself. The distribution of fluorescence was similar to that observed in nontransfected human endothelial cell line EA.926 when stained with primary anti-er antibody (data not shown), arguing against the observed distribution being a consequence of transfection or overexpression. The complete nuclear localization of her -66 after prolonged exposure to physiological levels of estrogen additionally suggested that the observed extranuclear localization of the ER isoforms was not a result of the GFP disrupting nuclear localization signals (data not shown).

4 Figtree et al Truncated ER Activates enos 123 enos proteins with the use of fluorescent confocal microscopy. In cells incubated without estrogen, a proportion of both her -46 and her -66 was observed to colocalize with enos (Figure 4). Colocalization with enos remained after 10 minutes of stimulation with 30 nmol/l 17 -estradiol, but was lost after 24 hours of stimulation because of redistribution of ER to the nucleus (data not shown). These results demonstrate that her -46 associates with enos in human endothelial cells in a similar manner to her -66. Figure 3. Cells transfected with GFP-tagged ER isoforms. Most of both conjugate proteins were located in the nucleus compared with the empty vector control (GFP). However, a significant proportion of both ER 46- and ER 66-GFP was extranuclear and associated with plasma membranes, as supported by the ER immunostaining (red, top panel) seen in nonpermeabilized COS cells transfected with ER 46-GFP and ER 66-GFP. ER -immunostaining was not observed in cells transfected with GFP-control plasmid. Secondary antibody controls were performed (bottom panel). her -46 Associates With enos in Human To investigate the potential role of her -46 in enos activation, we next investigated the association between her -46 and Cells Expressing ER -46 and ER -66 Bind Estrogen at the Cell Surface Because we observed recombinant 1a-hER -46 at the cell membrane, we sought to investigate the ability of this protein to function as an estrogen receptor at this site. The cellimpermeable BSA-FITC tagged estradiol (E2coBSA-FITC: Sigma) was used to investigate estrogen binding at the surface of nonpermeabilized cells transfected with 1ahER -46, her -66, or control ( -gal) expression vectors. When transfected with the -gal control vector, nonpermeabilized cells did not bind the cell-impermeable E2coBSA- FITC compound. However, cells transfected with 1ahER -46 or her -66 expression vectors bound E2coBSA- FITC at the cell surface at concentrations equivalent to 30 nmol/l 17 -estradiol (Figure 5). Preincubation with unla- Figure 4. enos immunostaining on COS cells cotransfected with enos and GFP-tagged ER isoforms. The spatial relationship of these molecules was examined by fluorescent confocal microscopy. The confocal microscopy images demonstrate that a proportion of both 1a-hER -46 (ER 46) and her -66 (ER 66) colocalizes with enos (yellow color indicated by white arrow) in the absence of estrogen stimulation.

5 124 Circulation January 7/14, 2003 Figure 5. Nonpermeabilized COS cells transfected with 1ahER -46 (ER 46) or ER 66 expression vectors contain surface binding sites for 17 -estradiol (E2) detectable by cell-impermeant ligand binding (E2coBSA covalently linked to E2-FITC). Cells transfected with control plasmid ( gal), or cells preincubated in unlabeled 17 -estradiol (E2-FITC E2) demonstrate no binding, as do cells exposed to BSA-FITC control compound. No background fluorescence was observed in control cells. beled estradiol (3 mol/l) abolished E2coBSA-binding, and the FITC-BSA control compound did not bind, showing ligand specificity. These results suggest that her -46 functions as a specific estrogen-binding protein at the cell surface. ER -46 Mediates Estrogen-Induced enos Activation Having observed estrogen binding by her -46 at the cell membrane and colocalization with enos, we next investigated its role in enos activation. Estrogen-induced enos activation was assessed in COS cells loaded with the NO-sensitive dye DAF-2DA. Confocal microscopy revealed that expression of 1a-hER -46 or her -66 and enos resulted in estrogeninduced NO production not observed in the presence of either the ER isoforms or enos alone (Figure 6). NO production was antagonized by the enos inhibitor L-NMMA. The NOsensitive DAF-fluorescence was observed in a punctate pattern both in association with the membrane and in the cytoplasm. These data indicate that estrogen-induced NO production in endothelial cells is mediated by functional associations between her -46 or her -66 and enos. ER -46 and ER -66 Mediate Differential Transcriptional Responses to Estrogen in Human Because both her -46 and her -66 mediate acute enos activation, we next compared their role in mediating classical, transcriptional responses after longer estrogen administration. Prolonged estradiol treatment at physiological levels (30 nmol/l for 24 hours) resulted in loss of membrane-associated GFP fluorescence and loss of GFP-eNOS colocalization (data not shown). In transient transfection experiments with the (ERE) 2 -tk-luc reporter gene construct, her -66 mediated a Figure 6. Estrogen-induced NO production in DAF-2DA loaded, transfected COS cells. In the presence of NO, DAF-2 is converted into a fluorescent product, DAF-2 triazole, which can be visualized. Cells transfected with enos alone did not respond to acute estrogen stimulation. However, cells expressing both enos and either 1ahER -46 (ER 46) or ER 66 demonstrated estrogen-induced enos activation (green fluorescence) that was inhibited by L-NMMA.

6 Figtree et al Truncated ER Activates enos 125 Figure 7. Transient transfection studies using the estrogen response element reporter plasmid (ERE) 2 -tk-luc to examine the relative estrogen-dependent transcriptional activation mediated by 1a-hER -46 (ER 46) compared with ER 66. 1a-hER -46 has much less ability for estrogen-dependent transcriptional activity and inhibits that of ER 66 in human endothelial cells. HMEC, EA.926, and COS cell lines were transfected with different combinations of 1a-hER-46 (her 46), her 66, or control expression vectors, as indicated. Cells were treated with or without estradiol (E2: 30 nmol/l) for 24 hours before being assayed for luciferase activity. Results are expressed as a percentage of the reporter gene activity measured from her 66 transfected cells stimulated with estrogen. Luciferase activities were normalized with the use of the internal reference control -galactosidase. represents 0.25 g of DNA used per 4 wells. In each case, the total quantity of DNA transfected was equalized with a control plasmid. Values correspond to the average SEM of at least 3 separate transfection experiments, each performed in quadruplicate. *P 0.05; **P 0.01; ***P compared with E2-induced transactivation mediated by her 66. Luciferase activity of control plasmids pgl3basic (no promoter) and pgl3control (containing SV40 promoter and enhancer) is provided for comparison. marked transcriptional response to estrogen in hmecs, EA.926, and COS cell lines (Figure 7). In contrast, 1ahER -46 mediated only a fraction of this reporter gene expression in these cell lines (P 0.001; n 3). Furthermore, coexpression of 1a-hER -46 and her -66 reduced estrogeninduced transactivation compared with that mediated by her -66 alone (P 0.01; n 3). These observations demonstrate that her -46 is a weak transcriptional activator compared with her -66 and acts as a competitive inhibitor of her -66 mediated transcriptional responses in these cells. Discussion This study demonstrates expression of an exon 1 truncated her isoform, her -46, in human endothelial cells. This truncated ER localizes to the cell membrane, specifically binds E2, and mediates acute estrogen-induced enos activation. Because her -46 mediates minimal estrogen-induced transcriptional activation and acts to competitively inhibit transcriptional activation by her -66, these findings provide new evidence for differential receptor-mediated mechanisms underlying acute and transcriptional estrogen signaling in human endothelial cells. Despite the importance of estrogen-mediated effects in the vasculature, the identity of estrogen receptors that mediate acute effects of estrogen in endothelial cells has remained unclear. The classical ER -66 copurifies with and activates enos in isolated caveolae from ovine pulmonary arterial endothelial cells. 1 However, our findings now suggest an important role for a novel splice variant of ER as an additional mediator of acute estrogen responses in human endothelial cells. Immunoidentification confirmed the predominance of the 46-kDa protein in EA.926 cells, the identical molecular weight of the recombinant 1a-hER -46. Primary HUVECs also express a 46-kDa ER variant. 7 Use of her -46 and her -66 GFP fusion proteins enabled us to determine the localization of these her isoforms in living cells in relation to enos. The peripheral and membrane-associated localization of her -46 and the partial colocalization with enos demonstrated by the her 46-GFP fusion protein was indistinguishable from that of her -66, previously shown by ultracentrifugation and immunofluorescence studies to localize to plasma membrane caveolae. 1 We used a non GFP tagged 1a-hER -46 expression vector to examine for the role of this truncated ER isoform in acute and transcriptional responses to estrogen. Nontransfected COS cells posses no ER immunoreactivity and no transcriptional or acute response to estrogen. However, COS cells expressing 1a-hER -46 bound estrogen at the cell surface and demonstrated acute enos activation in response to estrogen in a similar manner to those expressing ER -66. Furthermore, these findings clarify previous observations in EA.926 cells, in which only a 45-kDa ER -like protein was detected, despite intact acute responses to estrogen. 7 Our demonstration of 1a-hER -46 expression in several other human endothelial cell types now suggests that the exon 1 truncated ER isoform plays important roles in endothelial estrogen signaling. Specific binding of BSA-estradiol conjugates to the surface of cells expressing ER -66 or 1a-hER -46 is additional evidence supporting the ability of both isoforms to function as estrogen receptors in the mediation of acute, surfacerelated effects. A concern with the use of BSA-estradiol compounds is the possibility of incomplete conjugation that allows binding of free estradiol. 18 However, this is only a

7 126 Circulation January 7/14, 2003 limitation in studies addressing the acute physiological effects of BSA-estradiol administration. In our binding studies, any unconjugated estrogen would decrease the FITC signal seen at the cell surface by direct competition, as shown by addition of excess unconjugated estradiol. We used multiple additional controls, including BSA-FITC, together suggesting that membrane binding in cells expressing ER isoforms is estrogen-specific. The expression of her in endothelial cells has important implications for acute versus transcriptional effects of estrogen signaling. Because her -46 is only a weak mediator of transcriptional responses to estrogen and acts to inhibit the strong transcriptional activation mediated by her 66, the predominant expression of her 46 in endothelial cells would shift the balance of estrogen signaling toward acute rather than transcriptional responses. Differential expression of alternatively spliced ER variants may therefore provide a novel mechanism for modulation of estrogen responses in the vascular endothelium. Our identification of ER 46 as an important mediator of acute estrogen effects in human endothelial cells highlights the importance of recent findings in ER knockout mouse models. Alternative ER transcripts in the mouse, corresponding to human 1ahER -46, may explain residual estrogen responsiveness in ER knockout mice ( ERKO), in which only the first coding exon was targeted for disruption. 19 In these mice, a short ER transcript is expressed that encodes an N-terminal truncated, mutant ER that binds estrogen but possesses significantly reduced estrogendependent transcriptional activity compared with that of the wildtype ER, 20 resulting in the same functional domain changes as our findings with ER -46 in human endothelial cells. Indeed, the vascular responses to estrogen that are preserved in these ERKO mice 8 are abolished in a new knockout mouse with total deletion of ER gene Specifically, expression of alternatively spliced transcripts lacking the AF-1 domain in ERKO mice seems to mediate estrogen-induced endothelial NO production that is absent in the complete knockout. 24 These insights from mouse models suggest that mouse homologs of 1a-hER -46 are sufficient to mediate the acute endothelial effects of estrogen and in principle support our observations of the functional effects of her -46 in human endothelial cells, although the 46-kDa truncated ER protein was not readily detected in mouse aortic lysates. 24 Additional studies are required to investigate possible roles for truncated ERs in mediating acute and transcriptional effects of estrogen in human pathophysiology. In conclusion, this study identifies a novel, exon 1 spliced ER in human endothelial cells. The exon 1 truncated ER is similar to ER -66 in its subcellular localization and ability to mediate estrogen binding at the surface of the membrane in a ligand-specific fashion, leading to estrogen-induced enos activation. However, its inability to mediate transcriptional responses in endothelial cells suggests a unique role of this truncated ER in mediating differential acute versus transcriptional responses to estrogen in the vascular endothelium. Acknowledgments This work was supported by grants from the British Heart Foundation and from the Wellcome Trust. Dr Figtree was supported by the Rhodes Trust and by Trinity College, University of Oxford. References 1. Chambliss KL, Yuhanna IS, Mineo C, et al. Estrogen receptor alpha and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res. 2000;87:E44 E Chen Z, Yuhanna IS, Galcheva-Gargova Z, et al. 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