Functional analysis of an alternatively spliced estrogen receptor lacking exon 4 isolated from MCF-7 breast cancer cells and meningioma tissue

Mokcular

and

ce llular End ocrinology

ELSEVIER

Molecular

and Cellular

Endocrinology

101 (1994) 237-245

Functional analysis of an alternatively spliced estrogen receptor lacking exon 4 isolated from MCF-7 breast cancer cells and meningioma tissue S.G.A. Koehorst *7a,J.J. Cox b, G.H. Donker a, S. Lopes da Silva b, J.P.H. Burbach b, J.H.H. Thijssen a, M.A. Blankenstein a p Department of Endocrinology, Academic Hospital Utrecht, Utrecht, Netherlands ‘Rudolf Magnus Institute. Utrecht Unklersity, Utrecht. Netherlands (Received

3 November

1993; accepted

7 June 1994)

Abstract An alternatively spliced mRNA coding for a variant estrogen receptor (ER) missing exon 4 (ERA41 was detected in the breast tumor cell line MCF7 and meningioma tissue by using the reversed transcriptase PCR technique. The trans-activational properties of this mutant ER were assessed in embryo carcinoma P19EC and human choriocarcinoma JEG3 cells by co-transfection of the ERA4 expression vector with an oxytocin promoter construct containing an estrogen-responsive element. ERA4 did not trans-activate the oxytocin promoter in either a hormone-dependent or -independent manner. Co-transfection of ERA4 together with the wtER did not show any interference of ERA4 on the stimulation of the oxytocin promoter by the wtER. ERA4 was translated in vitro. Its capacity to bind estradiol, and the binding of the variant to a synthetic estrogen-responsive element were compared to those of the wild-type receptor. ERA4 did not bind to a synthetic estrogen-responsive element, nor did it bind estradiol. Hence, ERA4 appears to be a silent variant and we speculate that it is without any role in tumor progression. Key words: Alternatively

spliced

estrogen

receptor;

Meningioma;

1. Introduction The ER and PR are members of a family of transcriptional regulatory factors that bind specific DNA sequences in response to binding of their cognate ligand (Carson-Jurica et al., 1988; Evans, 1988; Green and Chambon, 1988). Discrete functional domains for hormone binding, DNA binding, and trans-activation have been identified in the human ER (Kumar et al., 1987). For breast cancer several ER mRNA variants, especially many alternatively spliced ER mRNA variants, missing exon 2 (Wang and Miksicek, 19911, exon 3 (Wang and Miksicek, 1991; Fuqua et al., 19931, exon 5 (Fuqua et al., 1991) and exon 7 (Fuqua et al., 1992; Koehorst et al.. 1993a) were detected. The function of

* Corresponding author. S.G.A. Koehorst, Dept. of Endocrinology G02.625, Academic Hospital Utrecht, P.O. Box 85500, NL-3508 GA Utrecht, Netherlands. Phone: t + 31) 30507572; Fax: (+ 31) 30541750. 0303-7207/94/$07.00 0 1994 Elsevier SSDI 0303-7207(94)00019-6

Science

Ireland

Ltd. All rights

MCF7; T47D; Myometrium

many alternatively spliced ER mRNAs was established by transient transfection of the alternatively spliced variants. The ER mRNA variant missing exon 5 acted as a dominant positive ER variant in these experiments (Fuqua et al., 1991). In contrast to the ER mRNA variant missing exon 7 which acted in a dominant negative manner (Fuqua et al., 1992), the variant missing exon 3 behaved in a dominant negative manner in the experiments performed by Wang et al. (1991) or had no influence on transcription as stated by the experiments of Fuqua et al. (1993). A variant missing exon 2 had no influence on transcription (Wang and Miksicek, 1991). In the present paper the functional properties of the ER mRNA variant missing exon 4 (ERA41 are described. ERA4 mRNA was detected by three different groups (Koehorst et al., 1993a; Pfeffer et al., 1993; Skipper et al., 1993). A functional analysis of such a variant has not been reported yet. We isolated this variant from meningioma tissue and MCF7 breast cancer cell line (Koehorst et al., 1993a). reserved

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Meningiomas are mostly benign brain tumors, of which 80% of the cases have an ER-negative/PR-positive phenotype at the protein level (Blaauw et al., 1986). Analogous to the ER-negative/PR-positive breast tumor in which the ERA5 variant is supposed to act in a dominant positive way (Fuqua et al., 19911, ERA4 may act in a similar way. Exon 4, which codes for amino acid 255-366, encodes the last part of the DNA binding domain (DBD), the hinge region and the first forty-eight amino acids of the ligand binding domain (LBD). The two zinc fingers of the DBD are intact and probably no estradiol will be bound by this variant because a great part of the LBD is missing. Also the highly positively charged region situated between amino acids 251 and 271 is missing. This sequence is very important for the formation of the non-DNA binding 8-9 S ER complexes, which bind heat shock protein 90 (hsp90) (Chambraud et al., 1990). A variant missing the amino acids 251-271 such as ERA4 may not be able to bind hsp90 and may therefore be always in the 4-5 S DNA binding form. These characteristics of ERA4 suggest a putative role as an estrogen independent trans-activator. The present report describes the functional properties of ERA4. Also the putative tumor specific occurrence was investigated by reversed-transcriptase PCR in solid breast tumors, meningioma tissue, and the breast tumor cell lines MCF7 and T47D. As non-tumor control normal human myometrium was used.

2. Materials

and methods

2.1. Tissue collection and cell lines

The MCF7 and T47D breast cancer cell lines were routinely grown in RPMI-1640 with 10% fetal calf serum (Gibco BRL, Paisley, UK) with phenol red. Human meningioma tissue, solid breast tumors and myometrium were placed on ice immediately after removal from the patient. Representative specimens were frozen at -80°C until they were used for RNA extraction. 2.2. RNA preparation and cDNA synthesis Total RNA was isolated from cells and tissue using the acid-guanidinium thiocyanate extraction method (Chomczynski and Sacchi, 1987). cDNA was synthesized using random hexamers as previously described (Koehorst et al., 1992). 2.3. PCR amplification Primer ATCT-3’)

pair Pl (5’-GGAAGTATGGCTATGGAand P2 (5’-GATCTTCGAACATGCT-

GCTGG-3’1 were used to amplify exon 2 - exon 6 of the human ER cDNA synthesised from total RNA from meningiomas and MCF7 breast cancer cells. PCR reactions were carried out in 50 ~1 10 mM Tris-HCl (pH 8.3) containing: 50 mM KCl, 1.5 mM MgCl,, 100 ng primers Pl and P2, and 1 U Ampli-Taq (Perkin Elmer Cetus, Norwak, CT, USA). Each cycle of amplification consisted of 30 s denaturation (95°C) and 60 s annealing (55”C), followed by 90 s extension (72°C). Each PCR reaction consisted of 35 cycles. PCR products were loaded on a 2% agarose gel, electrophoresed, and transferred to Hybond N membrane (Amersham, Amersham, UK). The membrane was hybridized over night at 50°C with different ‘“P-end labelled internal probes, one in exon 4, P3 (5’GGAGACATGAGAGCTGCCAAC-3’) and one in exon 5, P4 (5’-GAATGTGCCTGGCTAGAGATCCTGATG-3’). Hybridization was performed in the buffer recommended by the membrane manufacturer. 2.4. Construction of the expression piasmid HEGAI The pUc-19 clone containing the exon 4 variant PCR fragment, which was generated by cloning specific PCR products in the, Smal digested, blunt-ends pUc-19 plasmid (Koehorst et al., 1993a), was digested with Apa-Ll and XbaI. The Apa-Ll-XbaIA4 fragment was exchanged for the Apa-Ll-XbaI fragment of HEGO. HEGO is an expression plasmid containing the wtER coding region cloned in the eukaryotic expression vector pSG5 (HEGO was a kind gift of Prof. Dr. P. Chambon, Strasbourg, France). The expression plasmid containing the ER which is missing exon 4 is called HEGA4. The integrity of HEGA4 was confirmed by sequence analysis. 2.5. Transfection and luciferase assay Human choriocarcinoma cell line JEG3 or mouse embryocarcinoma cell line P19EC were cultured in Dulbecco’s modified Eagle’s medium with 7.5% fetal calf serum (FCS) for P19EC cells and 10% FCS for JEG3 cells. On the day of transfection the medium was replaced with Dulbecco’s modified Eagle’s medium without phenol red and dextran coated charcoal treated FCS resulting in a basal 17P-estradiol concentration of 3 X lo-” M in the medium. The cells were transfected overnight by means of the calcium phosphate precipitation method. 6.5 pg oxytocin COT) promoter luciferase plasmid (p-172ROLUC) (Adan et al., 1991) was cotransfected with 1 pg HEO, HEGO or HEGA4 (HEO, which is a gift of Prof. Dr. P. Chambon, is an expression vector coding for an ER that is less sensitive to estrogen). The expression in JEG3 cells of the expression plasmids HEGO and HEGA4 were verified by immunocytochemistry using the monoclonal antibody

S.G.A. Koehorst et al. /Molecular

and Cellular Endocrinology

ERlD5 (Immunotech, Marseille, France). As an internal control 0.5 pg (P19EC) or 0.1 pg (JEG3) pRSVGAL was included in the co-transfection assay. The next day the cells received increasing amounts of 17/3estradiol (10-9-10-7 M) with ethanol as vehicle. Control cells received ethanol only. After 24 h the cells were harvested in 420 ~1 lysis buffer (1% Triton X-100, 0.1 M KPO, (pH 7.6-7.8) 15% glycerol, 2 mM DTT) and luciferase activity was measured in 100 ~1 of extract according to the protocol of De Wet et al. (1987) using a Lumac/3 m biocounter M2010A luminometer. Galactosidase activity was determined according to Sambrook et al. (1989). 2.6. In vitro transcription and translation In vitro performed tem, the (Promega, performed tions.

transcription and translation reactions were in a coupled transcription/translation sysTnT Coupled Reticulocyte Lysate System Madison, WI, USA). The reactions were according to the manufacturer’s instruc-

2.7. Western blotting 5 ~1 of in vitro translated wtER and ERA4 were separated onto 12.5% SDS-polyacrylamide gel and transferred to a PDVF-membrane. After blotting the membrane was blocked in 5% (wt/vol) nonfat milk in Tris-buffered saline containing 0.5% Tween 20. The blots were incubated overnight at room temperature with the anti-ER antibodies H222 (a gift from Abbott Laboratories, Abbott Park, USA) and H226 (a gift from Prof. Dr. G.L. Greene, Univ. of Chicago, Chicago, IL, USA). After washing in Tris-buffered saline containing 0.5% Tween 20 the membranes were incubated with 0.5 pug/ml goat-antirat immunoglobulin G conjugated to alkaline phosphatase. Immunoreactive bands were visualized using nitroblu tetrazolium 5-bromo-4chloro-3-inodylphosphate.

2.8. Biochemical assay of steroid hormone binding The ER and PR content of the tumors and the in vitro translated ER were measured as described previously (Blankenstein et al., 1983) by the ligand binding assay according to the guidelines of the EORTC, Breast Cancer Cooperative Group (EORTC, 1980). 2.9. Gel mobility shift assay for in LjitroDNA binding As probe for the gel mobility shift assay the ERE of the Xenopus vitellogenin A2 was used (Klein-Hitpass et al., 1986). Both strands corresponding to the ERE were synthesized as 31 (5’-GATCCGTC AGGTCACAGTGACCTGATGGATC-3’; palindrome

101 (1994) 237-245

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is underlined) base oligonucleotides using a DNA synthesizer (Applied Biosystems, San Jose, CA, USA). Equimolar amounts of the two strands were annealed in buffer (10 mM Tris (pH S.O>, 1 mM EDTA) by heating to 95°C and cooling to room temperature during a period of 2 h. The double-stranded ERE oligomer was end-labelled using [ y- 32PIATP (Amersham, Amersham, UK) and T4 polynucleotide kinase (Boehringer Mannheim, Germany). A 5 ~1 aliquot of in vitro translated receptor was preincubated for 15 min at room temperature in 20 ~1 binding buffer (100 mM KCl, 100 mM Tris (pH 8.0), 1 mM EDTA, 10 mM monothioglycerol, 10% glycerol (v/v), 2 pg poly (dI.dC). 1 ng 32P-labelled double-stranded ERE oligomer was added and the reaction mixture incubated for 20 min at 20°C. The ER-ERE complexes were separated by electrophoresis on a 4% acrylamide (37.5 : 1, acrylamide-his) gel using a buffer consisting of 50 mM Tris, 50 mM boric acid, 1 mM EDTA (pH 8.0). The gels were run at 180 V for 3 h, vacuum dried and autoradiographed. To verify the presence of ER in the shifted complex, anti-ER monoclonal antibodies ER P31 (Medac, Hamburg, Germany), H222 and D547 (gifts from Abbott Laboratories, Chicago, IL, USA) were included in the binding reactions. 1 ~1 of the translation reaction mixture was added to 17 ~1 antibody solution in a total of 20 ~1 binding buffer.

3. Results 3.1. Selective amplification of ER cDNA from meningioma tissue and MCF7 breast cancer cells reL)eals the presence of an alternatively spliced receptor variant lacking exon 4

The amplification of exon 2-6 of the human ER from cDNA derived from myometrium tissue, MCF7-, T47D breast cancer cells, solid breast tumor tissue and meningioma tissue revealed, in addition to the 770 bp wt transcript, an extra band of 450 bp. Also a minor band of approximately 650 bp is seen. The sequence of this extra band was not determined. However the 450 bp fragment was detected upon hybridization with an exon 5 probe (Fig. 1A) but was not visible when using an exon 4 probe (Fig. 1B). Sequence analysis showed that this variant was an alternatively spliced product missing exon 4 (Koehorst et al., 1993a; Ponglikitmongkol et al., 1988)). It contains an in frame deletion and codes for an ER missing amino acids 255-366 (see Fig. 10 An expression plasmid HEGA4 was constructed that codes for the ERA4. In vitro translation experiments with [ 3sS]methionine were first performed to prove that the ERA4 protein is produced in rabbit reticulocyte lysates. Fig. 2A shows an autoradiograph of the translation products analyzed

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and Cellular Endocrinology

on a sodium dodecyl sulfate (SDS)-polyacrylamide gel. As expected, wtER has a molecular weight of 65.5 kDA (Greene et al., 1986). The translation product of ERA4 has a mobility of 53.7 kDa as was predicted from the cDNA sequence (Greene et al., 1986). This product was further tested for recognition by anti-ER

A

Myometr. -l---In-n

+ +

ER PR

Breast tumors

MCF7 T47D

++

+ +

+ +

+ +

+ +

@

C

IO1 (1994) 237-245

wtER

ERA4

AZ.. 1% .,, -97 kD -66 kD

C

-45 kD

+ -

1.03 kb 0.65 kb 0.45 kb -

EC II ER PR 1.03 kb 0.65 kb 0.45 kb -

+

--+

+

-InIn + +

+

’ wtER

ERA4’

‘wtER

ERA4’

M

-

MCF7

T47D

+ +

+ +

+ +

Breast tumors + -

+ +

+ +

1.03 kb 0.65 kb 0.45 kb -

1+1_ +

C

Meningiomas

T47D ER PR

M

In

-..

Myometr.

B ER PR

+_+

C

Meningiomas

T47D

H226

H222

I +

-

-

-

-

+

+

+

-

C

Fig. 2. (A) SDS - polyacrylamide gel analysis. Aliquots of translation reactions labeled with [“‘SlMethionine from reticulocyte lysates lacking DNA (lane 0, containing HEGO (lane 2) and HEGA4 (lane 3) were separated onto 12.5% SDS-polyacrylamide gel. (B) Western blot analysis of the in vitro translated wtER and ERA4. 5 ~1 of in vitro translated wtER and ERA4 were separated onto 12.5% SDSpolyacrylamide gel and transferred to a membrane. After blotting the membrane was incubated with the anti-ER antibodies H222 and H226. The anti-ER monoclonal antibodies H222, which has its epitope at the C-terminal part of domain E, and H226, which has its epitope in the A/B domain of the human ER. Both antibodies recognize the 67 kDa wtER and 54 kDa ERA4. (Prestained molecular weight markers on the outer lanes were used).

In

antibodies (see Fig. 2B). The antibodies used were: antibody H226 (a gift of Prof. Dr. G.L. Greene, Chicago, IL, USA) which is directed to A/B domain

0.45 kb -

256

365

u7EmunYu.Y

*PLlCED

GLY

PHE

366

317

Fig. 1. PCR amplification of exon 2-6 of the human estrogen receptor using cDNA from different tissues and cell lines. 1 pg of mRNA of myometrium (Myometr.), solid breast tumors (Breast tumors), meningiomas and the breast cancer cell lines MCF7 and T47D were reverse transcribed. The cDNA was used for the PCR amplification using two oligonucleotides spanning exon 2-6 of the human ER. The PCR products were loaded on a 2% agarose gel, electrophoresed, and transferred to a membrane. The membrane was hybridized with two 3zP end-labelled internal probes one in exon 5 (A) and one in exon 4 (B). The expected size of the wt ER transcript is 770 bp. Note the variant at 450 bp (A), which is not detected by the internal primer of exon 4 (B). (Tumors and cell lines were stated negative when ER and PR contents were < 3 fmol/mg protein and positive when the content was > 10 fmol/mg protein) (0 Schematic diagram of the sequencing result of the 450 bp variant. Note the in frame deletion of exon 4 of the 450 bp variant (ERA4).

S.G.A. Koehorst et al. /Molecular

HE0

HEGO

and Cellular Endocrinology

HEGd4 HEGO+HEGd4

Fig. 3. Stimulation of the rat oxytocin COT) promoter by wtER and mutant ERA4 at various 17P-estradiol concentrations. JEG3 cells were co-transfected with p172ROLUC (a construct containing the - 172/ + 16 upstream region of the rat OT gene in front of the luciferase gene) together with a plasmid that expressed the human wtER (HEGO), or the mutant ERA4 (HEGdO, or both (HEGO+ HEGd4) and treated with various 17/Sestradiol concentrations (IO-‘, 10-s* 10e7 M). As control, medium with DCC treated FCS was used. However, this still contained 3 x 10 -‘* M 17&estradiol. Even at this concentration the transfected wtER shows trans-activation on p-172ROLUC. Therefore p-172ROLUC was also co-transfected with a plasmid that expressed an ER that is less sensitive to 17/Sestradiol treatment (HEO). This results confirm that the trans-activation is 17p-estradiol specific. ERA4 did not trans-activate the ERE containing oxytocin promoter nor did it interfere the wtER trans-activation. The average luciferase activity f standard deviation is shown.

and H222 (a gift of Abbott Laboratories, Chicago, IL, USA) which is directed to the C-terminal part of domain E. HEGA4 is recognized by both antibodies. This confirms the integrity of the in vitro synthesized product. Immunocytochemistry with the anti-ER monoclonal antibody revealed that after transfection with the HEGO or HEGA4 in JEG3 cells a comparable percentage of cells stained positively, indicating successful transfection of the expression plasmids. 3.2. The tram-activational properties of ERA4 The trans-activational properties of ERA4 on the estrogen-responsive oxytocin COT) promoter p172ROLUC (Adan et al., 1991) were compared to those of the wtER at different p-estradiol concentrations by means of transient transfection. To this end, expression vectors coding for ERA4 (HEGA4) and for the wtER (HEGO) were co-transfected with p172ROLUC in Pl9EC and in JEG3 cell lines. The activity of the OT promoter measured in JEG3 cells is given in Fig. 3.

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Transfection with HEGA4 did not show transactivation on p-172ROLUC. The wtER (HEGO) stimulated the OT promoter at the used P-estradiol concentrations (3 x lo-‘*, lo-‘, lo-‘, 10e7 M). The wtER, especially when transfected and therefore overexpressed, is very sensitive to P-estradiol in the cells. Although the FCS was DCC treated, it still contained 3 x 10 -‘* M P-estradiol. Even at this concentration the transfected wtER showed trans-activation on p172ROLUC. To confirm that this activation is pestradiol specific and not due to an other factor, transfection with HE0 was performed. The resulting ER of this plasmid is less sensitive to estradiol (Tora et al., 1989) and showed no induction of the OT-promoter at 3 x 10 - ‘* M /3-estradiol. This result confirms that the trans-activation by the wtER (HEGO) on p-172ROLUC is 17p-estradiol specific and not due to another factor. Co-transfection of HEGA4 together with HEGO did not show any interference of HEGA4 on the stimulation of the OT promoter by HEGO. 3.3. L&and binding of ERA4 The in vitro translated wtER and ERA4 were subjected to a ligand binding assay. The wtER showed binding of estradiol up to 177 fmol per 50 ~1 in vitro translation reaction with a K, of 0.138 nmol/l. HEGA4, however, showed no estradiol binding. 3.4. Gel mobility shift assay The gel mobility shift assay was performed with the perfect ERE of the Xenopus vitellogenin gene A2 as probe. The specificity of this element for ER has been proven both by other groups (Klein-Hitpass et al., 1986, 1989) and by us (Koehorst et al., 1993b). The in vitro translated wtER was found to bind to its responsive element in the presence and absence of estradiol (Fig. 4). wtER binding to the ERE resulted in two retarded bands. This is due to the presence of rabbit reticulocyte lysate in the reaction. Only one retarded band is present when the in vitro translated ER is 20-fold diluted as seen in Fig. 5, where it is diluted with antibody solution; no extra retarded band is seen in this figure. This ERE was also used in other experiments where no rabbit reticulocyte lysate was used in the binding reaction and only one retarded band was seen (Koehorst et al., 1993bl. We therefore conclude that the two retarded bands in Fig. 4 and 6 are the result of the presence of rabbit reticulocyte lysate in the binding reaction. The HEGA4 in vitro translated product showed no ERE binding (Fig. 4). The monoclonal-anti-ER antibodies ER P31 (Medac, Hamburg, Germany), H222 and D547 (Abbott Laboratories, Chicago, IL) were included in the binding reactions in order to establish

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the presence of ER in the shifted complex (Fig. 5). The antibodies H222 and D547 supershifted the ER-ERE complex. Antibody ER-P31, in contrast, failed to supershift the ER-ERE complex. From earlier experiments (Koehorst et al., 1993b) we know, however, that antibody ER-P31 inhibits ER-ERE binding and has to be pre-incubated prior to the incubation with the ERE, which was not done in the present experiment. When HEGO and HEGA4 were simultaneously translated an extra band, that would demonstrate heterodimerization between wtER and ERA4, did not occur (Fig. 4). The translation reaction was performed simultaneously

E2

C

wtER

+

+

ERA4 -

+

wtER + ERA4

WtER ER_P31 H222 0547

I-+--I -

n + -

+

-

-

+ -

+

wtER

-

ERA4 + -

ER - ERE Complex l

F

Fig. 4. Binding of the in vitro translated wtER and ERA4 to the perfect ERE of the vitellogenin gene A2. 5 ~1 lysate containing either wtER (1 gg of HEGO was used in the translation reaction, lanes 2,3), ERA4 (1 pg HEGA4 was used in the translation reaction, lanes 4,5), or simultaneously translated wtER and ERA4 (0.5 pg of each plasmid was used in the translation reaction, lanes 6.7) were incubated for 15 min. with 1 ng 32P-labeled ERE in the presence (+) or absence (-) of 10-s M 17-p estradiol (E,). The retarded bands in lanes 6-7 are weaker compared to those in the other lanes. This is due to different amounts of plasmid used in the translation reactions. Lane one contains a control lysate incubated with the ERE probe. The asterisk indicates a non-specific complex formed between the ERE and a protein present in the rabbit reticulocyte lysate (F = free probe).

Fig. 5. Detection of ER in the ER-ERE complex by the anti-ER monoclonal antibodies ER-P31, H222 and D547. In addition to the 1 ~1 lysate with the in vitro translated wtER (lanes l-4) or simultaneously translated wtER and ERA4 (lanes 5-8) binding reactions were performed in the absence (-) or presence (+) of anti-ER antibodies. The antibodies H222 and D547 supershifted the ER-ERE complex, which proves the presence of ER in this complex. No extra supershifted band is seen in lanes 5-8 which would indicate heterodimer complex forming between wtER and ERA4 (F = free probe).

since wtER easily forms stable homodimers which could prevent the formation of heterodimers in posttranslational mixing experiments of wtER and ERA4. The retarded bands in the lanes in which wtER and ERA4 were simultaneously translated is weaker as compared to the separated translated products. This due to the fact that in the reaction in which wtER and ERA4 were simultaneously translated 0.5 pg HEGO and 0.5 pg HEGA4 was used. In contrast 1 pg of each expression plasmid was used in the separate translation of HEGO and HEGA4. Even with increasing amounts of HEGA4 in the translation reactions no extra band was discovered (data not shown). Similarly, no heterodimerisation was observed when the wtER and ERA4 were translated separately in vitro and both added in the binding reaction of the band shift assay (Fig. 6). Adding increasing amounts of ERA4 protein to the wtER in the

S.G.A. Koehorst et al. /Molecular

plwtER d ERA4 g lysate

0 0 5

1 1 0 1 543210

1 2

1 3

1 4

and Cellular Endocrinology

1 5

ER - ERE Complex l

F

Fig. 6. ERA4 does not inhibit or stimulate the binding of wtER to the estrogen-responsive element. 1 ~1 in vitro translated wtER were post-translationally mixed with increasing amounts of ERA4 as indicated on top of each lane. Adding increasing amounts of ERA4 to the wtER in the binding reaction did not change the binding of wtER to the ERE (F = free probe).

ERE binding reaction did not change the binding of wtER to the ERE (Fig. 6).

4. Discussion ER mRNA variants were detected in solid breast tumors or breast tumor cell lines. The ERA4 mRNA variant described here was isolated from meningioma tissue and the MCF7 breast cancer cell line. Two different groups also detected this variant mRNA. Pfeffer et al. (1993) detected this variant also in the human breast cancer cell line, Skipper et al. (1993) detected ERA4 mRNA in the brain and uteri of lizards and rats. This last group predicted that the corresponding protein encoded by ERA4 will not bind estradiol and may therefore belong to the growing subclass of the steroid/thyroid/vitamin D receptor superfamily known as orphan receptors. In their opinion ERA4 protein may function as a ligand-independent transcription factor that acts on the same DNA responsive element as the wtER (Skipper et al., 1993). Our group predicted the same features for ERA4 in order to explain the ER-negative/PR-positive phenotype of 80% of human meningiomas (Koehorst et al., 1993a). Pfeffer et al. suggested another role for ERA4. They speculated that ERA4 is responsible for the type II low affinity estrogen binding activity (Pfeffer et al., 1993). Therefore in this paper the functional analysis

101 (1994) 237-245

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of ERA4 is described in order to confirm or to reject the predicted functions of ERA4. We first determined whether the occurrence of ERA4 was tumor specific. Therefore solid breast tumors, meningioma tissues and the breast tumor cell lines MCF7 and T47D were tested. As non-tumor material myometrium was used. The presence of ERA4 mRNA was investigated by means of the reversed PCR-technique. ERA4 was detected in all the tissues and cells tested (Fig. 1A and 1B). This confirms the results of Skipper et al. (19931, who detected ERA4 mRNA in uteri and normal brain of lizards and rats. Therefore we conclude that the presence of ERA4 is not tumor-specific. A minor band of approximately 650 bp was seen in these experiments (Fig. 1A and 1B). One can speculate that this reflects, a variant missing exon 3, but the sequence of this extra band was not determined. Besides the variant bands also wtER mRNA in meningioma tissues which, were negative for ER proteins as determined by the ligand binding assay, were detected. This is probably due to the sensitivity of the reversed PCR-technique used. Fuqua et al. detected also wtER mRNA besides ERA5 mRNA in apparently ER negative breast tumors as it was determined by ligand binding (Fuqua et al., 1991). The transcriptional properties of ERA4 were assessed by co-transfection of the ERA4 expression vector with an oxytocin promoter construct containing an estrogen-responsive element. The wtER was used as control in these transfection experiments. The transfected wtER expression plasmid HEGO was able to trans-activate on p-172ROLUC (Fig. 3). The wtER, especially transfected and therefore overexpressed, in the cells is very sensitive for estradiol. Although the FCS was DCC treated, it still contained 3 x 10 -‘* M estradiol. Even at this concentration the transfected wtER showed trans-activation on p172ROLUC. Transfection with HE0 as control revealed that the trans-activation by the wtER (HEGO) on p-172ROLUC is estradiol specific and not due to another factor. HEGA4 did not trans-activate the ERE containing oxytocin COT) promoter p-172ROLUC. Since ERA4 mRNA was always detected together with the wtER mRNA we were interested in the influence of HEGA4 on the trans-activation of HEGO. HEGA4 showed no influence at all on the trans-activation properties of HEGO. Thus, ERA4 did not compete for the binding of wtER to p-172ROLUC and heterodimers of ERA4 and wtER which could influence the wtER transactivation properties are not formed. To further characterize the properties of ERA4, the variant and wtER were synthesized in vitro. The in vitro translated wtER was found to bind to its responsive element in the presence and absence of estradiol

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(Fig. 4). wtER binding to the ERE resulted in two retarded bands. This is due the presence of rabbit reticulocyte lysate in the binding reaction. The in vitro translated wtER binds to the ERE under our conditions (Fig. 4) and therefore we conclude that, if ERA4 is a DNA binding variant it would also bind DNA under these conditions. However, ERA4 did not bind the ERE (Fig. 4). It seems likely that the changes in amino acid composition near the last zinc finger of the DBD which results from the truncation of exon 4 destabilizes the zinc fingers in such a way that DNA binding can not occur. Mader et al. (1993) recently defined a minimal estrogen receptor DBD. Their study revealed that amino acids 250-262 are essential for DNA binding and any mutation in this region abolishes DNA binding. Therefore, the truncation at 254 in ERA4 probably destabilizes the DNA binding domain in such a way that binding to the ERE cannot take place. Another mechanism of action of the ERA4 on an ERE could be through heterodimerisation with the wtER resulting in a change in trans-activation by the heterodimer as compared to the action of wtER alone. In the band shift assay no extra band was detected which would confirm the presence of a DNA-bindingheterodimer (Fig. 4). The retarded bands in the lanes in which wtER and ERA4 were simultaneously translated is weaker as compared to the separated translated products. This is due a fact that in the reaction in which wtER and ERA4 were simultaneously translated 0.5 pg HEGO and 0.5 pg HEGA4 were used. In contrast 1 pg of each expression plasmid was used in the separate translation of wtER and ERA4. Adding increasing amounts of ERA4 protein to the wtER in the ERE binding reaction did not change the binding of wtER to the ERE (Fig. 6). Therefore it is unlikely that a non-DNA-binding-heterodimer between wtER and ERA4 is formed. This was confirmed by the transfection experiments in which ERA4 had no influence on the trans-activation properties of the wtER. From our experiments, the alternatively spliced variant ERA4 appears to be a silent variant without any role in tumor progression. The role of alternatively spliced variants in hormone independent growth is questionable. Variant mRNAs of the human ER missing exon 5 (Fuqua et al., 19911, exon 7 (Fuqua et al., 1992; Koehorst et al., 1993a), exon 2 (Wang and Miksicek, 19911, exon 3 (Wang and Miksicek, 1991; Fuqua et al., 19931, and exon 4 (Koehorst et al., 1993a; Pfeffer et al., 1993; Skipper et al., 1993) have now been described. All these variant mRNAs were detected along with wtER mRNA. The occurrence of the variant mRNA only in combination with i the wtER mRNA, even in ER negative tissue (Fuqua et al., 1991; Koehorst et al., 1993a) leads us to conclude that these variants are by-products made by

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the splicing machinery. The assays used, reversed trancriptase PCR (Fuqua et al., 1991; Koehorst et al., 1993a; Pfeffer et al., 1993) and RNA protection (Fuqua et al., 19921, are probably sensitive enough to detect mistakes in the splicing machinery. This conclusion is strengthened by the fact that thus far no mutations at the intron exon boundaries of the ER are detected by breast tumors which contained ER alternatively spliced mRNA (Fuqua et al., 1993). The role of ERA4 as ligand-independent transcription factor as was proposed earlier (Koehorst et al., 1993a; Skipper et al., 1993) can not be maintained after the present experiments.

5. Acknowledgements The authors gratefully acknowledge Prof. Dr. P. Chambon, Institut de Chimie Biologique, Strasbourg, France, for the gift of plasmids HE0 and HEGO, Abbott Laboratories, Abbott Park, USA, for the gift of the antibodies H222 and D547 and Prof. Dr. G.L. Greene, University of Chicago, Chicago, USA, for the gift of antibody H226.

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