The Antiestrogen ICI 182780 Disrupts Estrogen Receptor Nucleocytoplasmic Shuttling

Journal of Cell Science 106, 1377-1388 (1993) 1377 Printed in Great Britain © The Company of Biologists Limited 1993

The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling

Sophie Dauvois, Roger White and Malcolm G. Parker* Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK *Author for correspondence

SUMMARY

The mouse estrogen receptor was shown to be constantly cytoplasm when cells were treated with the pure antie- shuttling between the nucleus and cytoplasm although strogen ICI 182780. The effect of the pure antiestrogen under steady-state conditions it is detected predomi- was to inhibit nucleocytoplasmic shuttling of the recep- nantly in the cell nucleus in both the absence and pres- tor by blocking its nuclear uptake. Thus although ligand ence of estradiol. Shuttling was demonstrated by moni- binding is not required by the estrogen receptor to toring the transfer of protein between nuclei in undergo nucleocytoplasmic shuttling, this process can be heterokaryons and by examining the subcellular distri- disrupted by the binding of a pure antiestrogen. bution of mutant receptors. In the presence of the partial antiestrogen 4-hydroxytamoxifen the receptor was Key words: antiestrogens, estrogen receptor, nucleocytoplasmic retained in the nucleus whereas it accumulated in the shuttling

INTRODUCTION uptake of steroid receptors was initially thought to be mediated by a short cluster of basic residues conserved near the The estrogen receptor is a member of the nuclear receptor DNA-binding domain, similar to that identified in SV40 superfamily that acts as a ligand-dependent transcription large T antigen (Kalderon et al., 1984a,b; Lanford and factor (Beato, 1989; Evans, 1988; Ham and Parker, 1989). Butel, 1984). However, many nuclear targeting sequences Upon hormone binding the receptor binds to response ele- are more complex (Dingwall and Laskey, 1991), and con- ments in the vicinity of target genes and regulates their rates sistent with this is the demonstration that a number of basic of transcription. Since estrogens are mitogens in approxi- clusters appear to cooperate in the absence of hormone to mately one-third of breast tumors, specific estrogen antag- target estrogen and progesterone receptors into the cell onists have been developed for the treatment of hormone- nucleus (Guiochon-Mantel et al., 1989; Ylikomi et al., dependent breast cancer. The most widely used antiestrogen 1992). The observation that glucocorticoid receptors are to date is the non-steroidal compound tamoxifen (Jordan found in the cytoplasm in the absence of ligand and become and Murphy, 1990; Jordan, 1984), which promotes high nuclear upon hormone binding appears to be accounted for affinity DNA binding but inhibits transcriptional activation by masking of the nuclear targeting sequences by the hor- by the receptor (Berry et al., 1990). This antiestrogen, how- mone-binding domain in the absence of hormone (Picard ever, possesses partial agonist activity (Jordan, 1984) and and Yamamoto, 1987; Picard et al., 1990). for this reason compounds devoid of agonist activity have Recent work indicates that some nuclear proteins includ- been developed. The best known of these ‘pure’ antiestro- ing two nucleolar proteins (Borer et al., 1989) and several gens are ICI 164384 and ICI 182780, which are derivatives heat shock proteins (hsp) related to hsp70 shuttle continu- of estradiol but contain an alkylamide extension in the 7a ously between the nucleus and cytoplasm through the position (Bowler et al., 1989; Wakeling et al., 1991). One nuclear pore complex. Similar types of experiment indicate effect of these pure antiestrogens is to reduce steady-state that the progesterone receptor also undergoes this process levels of the estrogen receptor by increasing the turnover of nucleocytoplasmic shuttling (Guiochon-Mantel et al., of the protein (Dauvois et al., 1992; Gibson et al., 1991; 1989, 1991; Chandran and De Franco, 1992). The gluco- Reese and Katzenellenbogen, 1992). corticoid receptor has also been reported to shuttle since its Immunohistochemical studies have shown that the recep- re-entry into the nucleus was blocked by the protein phos- tors for estrogen and also progesterone and androgen are phatase inhibitor okadaic acid (De Franco et al., 1991). located predominantly in the cell nucleus even in the In view of our demonstration that the turnover of the absence of their respective hormone (King and Greene, estrogen receptor was increased by the binding of the pure 1984; Welshons et al., 1985; Perrot-Applanat et al., 1985; antiestrogen ICI 164384 (Dauvois et al., 1992) we have Jenster et al., 1992; Kemppainen et al., 1992). Nuclear investigated the subcellular distribution of the receptor. We 1378 S. Dauvois, R. White and M. G. Parker used a number of approaches to show that the estrogen 3% paraformaldehyde solution (3% (w/v) paraformaldehyde, 0.1 receptor shuttles from the nucleus to the cytoplasm and mM CaCl2 and 0.1 mM MgCl2, pH 7.4, in PBS) for 30 minutes. present evidence that pure antiestrogens interfere with The fixative was then quenched by the addition of 50 mM ammo- nucleocytoplasmic shuttling by blocking the nuclear uptake nium chloride in PBS for 10 minutes. The cells were washed with of receptor. PBS and permeabilised by incubation with 0.2% Triton X-100 in PBS for 3 minutes and washed with PBS, 10 mg/ml BSA. Two estrogen receptor antibodies were used, namely the rat monoclonal antibody H222 (Greene et al., 1984) whose epitope resides MATERIALS AND METHODS between residues 463-528 (Kumar et al., 1986) and the rabbit polyclonal antibody ER21 raised against a peptide corresponding Receptor expression vectors to residues 1-21. The antibodies were diluted in PBS, 10 mg/ml The mouse estrogen receptor (MOR) and mutant versions of the BSA, and 20 ml samples placed on parafilm in a box containing receptor are all expressed using the expression vector pJ3W (Mor- moist Whatman 3MM. The rat monoclonal antibody H222 (1 genstern and Land, 1990). The wild-type mouse estrogen recep- mg/ml) was used at 1/200 dilution while the rabbit polyclonal anti- tor (pJ3 MOR 1-599) and an N-terminal deletion mutant (pJ3 121- body ER21 (1 mg/ml) was used at a dilution of 1/50. Coverslips 599) have been described previously (Lees et al., 1989; White et were inverted onto the antibody and incubated for 1 hour at room al., 1987). Deletion mutants lacking nuclear localisation signals temperature. They were then washed three times in PBS, 10 mg/ml were constructed by PCR amplification using MOR cDNA as a BSA, and the above procedure repeated with an appropriate FITC- template and primers flanking the deleted region at amino acids conjugated second antibody. Cell hybrid DNA was stained by 205-211 and 344-349 and primers overlapping the deleted region addition of Hoechst 33258 (Sigma) dye at 0.5 mg/ml during the D259-275 (MOR D259-275) and D252-307 (MOR D252-307). The incubation with the second antibody. In all cases untransfected or deletions were then inserted in SP6MOR between BsmI and XbaI transfected cells incubated without first antibody were included as sites and transferred into the expression vector pJ3W at the EcoRI controls. After washing, the coverslips were mounted on slides site. The human estrogen receptor (HEGO) (Tora et al., 1989) was with a 90% glycerol solution in PBS. Slides were examined using expressed in pSG5. a Zeiss laser confocal microscope or a Zeiss Axioskop fluores- cence microscope and directly photographed using 400 ASA film Cell culture and transient transfection or images were captured with thermoelectrically cooled LC200 experiments CCD (Photometrics) and photographed from the screen after pro- The expression of wild-type and mutant estrogen receptors was cessing on a superMac computer. analysed by transient transfection experiments in COS-1 cells. COS-1 cells were grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf RESULTS serum (FCS) (Gibco, Paisley, Scotland) in a 37°C humidified atmosphere containing 10% CO2. After suspension in phosphate Subcellular localisation of estrogen receptor buffered saline (PBS), cells were transfected with 15 mg of either depends on the ligand pJ3 MOR 1-599, pJ3 MOR 121-599 or the nuclear localisation We first investigated the effect of different ligands on the mutants by electroporation at 450 V and 250 mF using a Bio-Rad subcellular localisation of the mouse estrogen receptor Gene Pulser. For immunofluorescence studies, transfected COS-1 cells were plated on 22 mm poly-L-lysine precoated coverslips expressed in COS-1 cells. The receptors were detected by placed in 6-well tissue culture plates in phenol red-free DMEM, immunofluorescence using the human estrogen receptor supplemented with 10% charcoal-stripped FCS. After 48 hours, antibody H222 and fluoroscein-conjugated anti-rat cells were treated with the different compounds tested and then immunoglobulins as the second antibody. No staining was processed for histological studies. In experiments using energy observed in untransfected COS-1 cells or when cells were synthesis inhibitors, the medium was replaced by DMEM with- stained only with the second antibody. For each treatment out glucose, supplemented with 6 mM deoxyglucose (Sigma), approximately 200 transfected cells were counted and the 10% dialysed charcoal-stripped FCS and 50 mM oligomycin receptor distribution was categorised as N, when the stain- (Sigma). The reversibility of this energy depletion was studied ing was 100% nuclear, N>C, when nuclear staining was after washing by leaving the cells for 4 hours in phenol red-free stronger than cytoplasmic staining, N=C when nuclear and DMEM, supplemented with 10% charcoal-stripped FCS in the cytoplasmic staining was of similar intensity, C>N when presence of 50 mg/ml cycloheximide. the cytoplasmic staining was stronger than the nuclear stain- Cell hybrid formation ing and C when the staining was exclusively cytoplasmic. COS-1 cells, transiently transfected with mouse estrogen recep- Receptor distribution was quantitated as presented in the tors, were plated on coverslips as described for transient trans- Tables and the most frequent distribution illustrated in the fection experiments. The following day the COS-1 cells were Figures. overlayed with 2´ 105 NIH 3T3 cells and fused 24 hours later as Estrogen receptors were predominantly nuclear in the previously described (Borer et al., 1989). Cells on coverslips were absence of hormone but nuclear staining was slightly inverted on prewarmed 50% polyethylene glycol 6000 (PEG) in increased by treatment with estradiol or the estrogen antag- PBS for 4 minutes, washed extensively in prewarmed PBS and onist 4-hydroxytamoxifen (Fig. 1 and Table 1). When cells then incubated in phenol red-free DMEM, supplemented with 10% were treated with the pure antiestrogens ICI 182780 (Fig. charcoal-stripped FCS containing 10 mM cytosine arabinoside (Sigma), 50 mg/ml cycloheximide (Sigma) and 10 nM estradiol or 1) or ICI 164384 (data not shown), however, the subcellu- 100 nM ICI 182780 for 3 hours. lar distribution of receptors was altered since they were detectable in the cytoplasm of approximately 80% cells. Indirect immunofluorescence The appearance of receptors in the cytoplasm was first For histological studies cells were washed with PBS and fixed in detectable within 15 minutes and continued to increase for Estrogen receptor shuttling 1379

Fig. 1. Effect of ligand on the subcellular localisation of the mouse estrogen receptor. Transiently transfected COS-1 cells were treated with no hormone, 10 nM estradiol, 100 nM ICI 182780 or 100 nM OH-Tamoxifen for 1 hour. After ﬁxation of the cells, receptors were detected by indirect immunoﬂuorescence using the estrogen receptor monoclonal antibody H222. ´ 400.

Table 1. Effect of antiestrogens on the subcellar mone-binding domain and western blotting analysis failed localisation of mouse estrogen receptors to reveal degradation products with either of these anti- Nuclear Cytoplasmic bodies (data not shown). The effect of the antiestrogens on the subcellular locali- Treatment N N>C N=C C>N C sation of receptors was dose-dependent with cytoplasmic NH 83 17 receptors ﬁrst detected at a dose of 1 nM ICI182780 or 10 E2 95 5 ICI 182780 18 62 15.5 4.5 nM ICI164384 (Fig. 3), this difference is consistent with OH-TAM 96 4 their relative affinities for the estrogen receptor (Wakeling et al., 1991). We also examined the effect of the antiestro- Transiently transfected COS cells were treated for 1 hour with no gens on the human estrogen receptor since previous work hormone (NH), 10 nM estradiol (E2), 100 nM ICI 182780 (ICI 182780) or had failed to observe similar effects on their subcellular 100 nM 4-hydroxytamoxifen (OH-TAM). Results are expressed as percentage of cells falling into the following subcellular localisation localisation at 10 nM ICI 164384 (Ylikomi et al., 1992). categories: N, 100% nuclear staining; N>C, nuclear staining stronger than We conﬁrmed their results but found that human estrogen cytoplasmic staining; N=C, where nuclear and cytoplasmic staining have receptors were detected in the cytoplasm (Fig. 3) when cells the same intensity; C>N, where the cytoplasmic staining is stronger than were treated with 100 nM ICI 164384. Nevertheless, as the nuclear staining; C, where the staining is exclusively cytoplasmic. expected, given the enhanced turnover of receptor protein (Dauvois et al., 1992), there was a decrease in the inten- one hour (Fig. 2). We have previously demonstrated that sity of staining for both mouse and human receptors with the pure antiestrogens increase estrogen receptor turnover time and dose of antiestrogen. (Dauvois et al., 1992) and so the speckled staining suggested the possibility that the receptor was targeted to Pure antiestrogens block nuclear uptake of lysosomes. However we were unable to demonstrate coim- estrogen receptors muno-localisation of the cytoplasmic receptor with the lyso- We next investigated whether the cytoplasmic localisation somal enzyme cathepsin D (data not shown). Moreover the of receptors in the presence of pure antiestrogens was cytoplasmic form of the receptor does not seem to be caused by an inhibition in the nuclear uptake of estrogen degraded since staining was obtained with antibodies receptors. To analyse nuclear uptake we generated cyto- directed against the N-terminal domain (ER21) and the hor- plasmic receptors in two ways. First we used energy syn- 1380 S. Dauvois, R. White and M. G. Parker

Fig. 2. Time course of the effect of ICI 182780 on the subcellular localisation of the mouse estrogen receptor. Transiently transfected COS-1 cells were treated with no hormone (A) or 100 nM ICI 182780 for 15 minutes (B), 30 minutes (C) or 1 hour (D) and receptors were detected by indirect immunoﬂuorescence using the estrogen receptor monoclonal antibody H222.

Fig. 3. Effect of increasing doses of ICI 182780 or ICI 164384 on the subcellular localisation of the mouse (MOR) or human (HEGO) estrogen receptor. Transiently transfected COS-1 cells were treated for 1 hour with no hormone (NH), ICI 182780 at 10- 9 M (- 9), 10- 8M (- 8) and 10- 7M (- 7) or ICI 164384 at 10- 8M (- 8), 10- 7M (- 7) and 10- 6M (- 6) and receptors were detected by indirect immunoﬂuorescence using the estrogen receptor monoclonal antibody H222. Estrogen receptor shuttling 1381

Fig. 4. Energy depletion of COS-1 cells expressing MOR. Cells were first treated with 50 mM oligomycin for 2 hours (left panel) in the absence of hormone (NH) or in the presence of 10 nM estradiol (E2) or 100 nM ICI 182780 (ICI). Oligomycin was then removed and cells incubated with no hormone (NH), 10 nM estradiol (E2), 100 nM ICI 182780 (ICI) or with 1 mM estradiol (E2) (after ICI 182780 treatment) for 4 hours in the presence of 50 mg/ml of cycloheximide (right panel). Receptors were detected by indirect immunofluorescence using the estrogen receptor monoclonal antibody H222. thesis inhibitors as previously described (Guiochon-Mantel tor MORD259-275 was detected in the cytoplasm of cells et al., 1991) and secondly we used mutant receptors that in the absence of hormone (Fig. 6). The nuclear staining lacked nuclear localisation signals. We then compared the observed could be due either to passive diffusion of the effect of different ligands on the nuclear uptake of these deletion mutant or to the presence of residual estrogen in cytoplasmic receptors. the charcoal-stripped serum. However this cytoplasmic When transfected COS-1 cells were treated with receptor could be translocated into the cell nucleus in the oligomycin to deplete them of energy there was an efflux presence of estradiol or 4-hydroxytamoxifen (Fig. 6 and of receptors from nuclei into the cytoplasm (Fig. 4). Irre- Table 2), demonstrating ligand-inducible nuclear uptake of spective of the ligand bound the cytoplasmic receptors stained with a speckled appearance similar to that previ- Table 2. Subcellular localisation of receptors deficient in ously observed when cells were treated with ICI 182780 nuclear localisation alone. To investigate the reversibility of the process, oligomycin was removed and cells were incubated with dif- Time Nuclear Cytoplasmic ferent ligands for 4 hours in the presence of cycloheximide Treatment (min) N N>C N=C C>N C to inhibit de novo protein synthesis. After pretreatment with NH 19 69 12 no hormone or estradiol there was quantitative re-entry of E2 15 74 26 receptors into the cell nucleus when oligomycin was 30 81 19 removed and cells maintained in the absence or presence 60 15 78 7 of estradiol but not if ICI 182780 was added. After ICI 120 63 37 182780 pretreatment nuclear uptake was only achieved OH-TAM 15 34 47 19 when the antiestrogen was replaced with estradiol. If ICI 30 59.5 34.5 6 182780 treatment was maintained when the oligomycin was 60 6 74 19 1 120 48 45 7 removed receptors were no longer detectable in the major- ity of cells. We conclude therefore that the accumulation ICI 182780 15 9 18 71 2 30 10 18 69 3 of estrogen receptors in the cell nucleus is energy-depen- 60 5 24 52 19 dent and that, in contrast to estradiol, this process is inhib- 120 3 3 8 86 ited by the antiestrogen ICI 182780. We have also generated cytoplasmic receptors by delet- The mutant receptor (MOR D259-275) was transiently transfected in COS cells and treated for increasing periods of time with no hormone ing lysine/arginine rich motifs that have been shown to con- (NH), 10 nM estradiol (E2), 100 nM 4-hydroxy-tamoxifen (OH-TAM) or tribute to nuclear localisation of the estrogen receptor 100 nM ICI 182780 (ICI 182780). Results are expressed as percentage of (Ylikomi et al., 1992; Picard et al., 1990). When two of cells falling into each of the subcellular localisation categories as defined these motifs, I and II, are deleted (Fig. 5) the mutant recep- for Table 1. 1382 S. Dauvois, R. White and M. G. Parker

Fig. 5. Structure of the wild-type and nuclear localisation-defective forms of the mouse estrogen receptor. Different regions of mouse estrogen receptor are schematically represented at the bottom of the Figure. The amino acid sequence of the wild-type region D is represented with basic amino acids in bold and the K/R rich stretches of amino acids called I, II and III. The two deletions made in this region are represented by a plain line between the mentioned amino acid number. Filled circles indicate part of the zinc ﬁnger motif. this mutant form of receptor. When the cells were treated MOR121-599, which retains the nuclear localisation signals with ICI 182780 there was no nuclear uptake of receptor but lacks the ER21 epitope. In the absence of hormone but rather an increase in the proportion of cells containing MORD252-307 is predominantly cytoplasmic but treatment cytoplasmic receptors over the two hour treatment period with estradiol or 4-hydroxytamoxifen results in nuclear (Fig. 6 and Table 2). Thus, as with the energy-depletion staining in 42% and 24% cells respectively (Fig. 7A and experiments, we conclude that nuclear uptake of estrogen Table 3). Nuclear accumulation of MORD252-307 could be receptors is inhibited by the antiestrogen ICI 182780. further increased to approximately 80% by coexpressing it with MOR121-599 in the presence of estradiol or 4-hydrox- Estradiol and hydroxytamoxifen promote estrogen ytamoxifen; this increase was particularly evident in the receptor dimerisation presence of 4-hydroxytamoxifen when the proportion of Interactions between receptor monomers can be studied by cells expressing solely nuclear receptors increased from 1% coexpressing mutant forms of receptors that can be distin- to 26% (Fig. 7B and Table3). This result indicates that the guished with speciﬁc antibodies. For this purpose we con- nuclear receptor MOR121-599 must shuttle from the structed the deletion mutant MORD252-307, which contains nucleus to the cytoplasm in order to dimerise and carry the epitope for the antibody ER21 but lacks the basic MORD252-307 from the cytoplasm into the cell nucleus. nuclear localisation motifs I, II and III (Fig. 5) and In contrast, MORD252-307 fails to accumulate in the

Table 3. Effect of ligands on the dimerisation and nuclear uptake of a nuclear localisation deﬁcient mutant (MOR D252-307) and an N-terminal deletion mutant (MOR 121-599) Nuclear Cytoplasmic Receptor Treatment N N>C N=C C>N C MORD252-307 NH 7 59 34 E2 1 41 48 10 ICI 182780 2 8 90 OH-TAM 1 23 62 14 MORD252-307 NH 12 71 17 +MOR121-599 E2 11 68 20 1 ICI 182780 2 10 88 OH-TAM 26 57 17

Cells were transfected with MOR D252-307 in the absence or presence of MOR 121-599 and treated for 2 hours with no hormone (NH), 10 nM estradiol (E2), 100 nM ICI 182780 (ICI 182780) or 100 nM 4-hydroxytamoxifen (OH-TAM). The subcellular localisation of MOR D252-307 was detected with the receptor antibody ER21, which does not recognise MOR 121-599. Results are expressed as percentage of cells falling into each of the subcellular localisation categories as deﬁned for Table 1. Estrogen receptor shuttling 1383

Fig. 6. Effect of ligands on the subcellular localisation of estrogen receptors defective in nuclear localisation. The mutant receptor (MOR D259-275) was transiently transfected in COS-1 cells. Cells were treated in (A) with no hormone, 10 nM estradiol, 100 nM OH- Tamoxifen or 100 nM ICI 182780 for 1 hour and in (B) with 10 nM estradiol or 100 nM ICI 182780 for different periods of time. Receptors were detected by indirect immunoﬂuorescence using the estrogen receptor monoclonal antibody H222. 1384 S. Dauvois, R. White and M. G. Parker

Fig. 7. Effect of ligands on dimerisation and nuclear uptake of the mouse estrogen receptor. The nuclear localisation-deﬁcient mutant (MOR D252-307) was expressed alone (A) or with the N-terminal deletion mutant MOR 121-599 (B) in transiently transfected COS-1 cells treated with no hormone, 10 nM estradiol, 100 nM ICI 182780 or 100 nM OH-Tamoxifen for 3 hours. The subcellular localisation of MOR D252-307 was monitored with the estrogen receptor polyclonal antibody ER21 that recognises an epitope absent in MOR 121-599. The structures of the mouse estrogen receptor deletion mutants used to study oligomerization are represented at the top of the Figure. nucleus in the presence of ICI 182780 even when it is coex- receptor-expressing and non-expressing cells and then pressed with MOR121-599, but this is not suprising given examine the transfer of receptors between their nuclei. For that the receptor bound with the antiestrogen is retained in these experiments, we fused transiently transfected COS-1 the cytoplasm (Figs 1-3). These results suggest that estra- cells, of which 30-40% were expressing receptor, with diol or 4-hydroxytamoxifen induce the interaction of mouse NIH-3T3 cells that were devoid of receptor. Their MORD252-307 with MOR121-599. nuclei were distinguished with Hoechst dye, the COS-1 nuclei being uniformally stained whereas NIH 3T3 nuclei Nucleocytoplasmic shuttling of estrogen were smaller and showed punctate staining (Fig. 8). After receptors occurs in the presence of estradiol but fusion, the cell hybrids were treated with cycloheximide to not pure antiestrogens inhibit de novo protein synthesis and the distribution of A more direct approach to monitor nucleocytoplasmic shut- receptors with no hormone, estradiol or ICI 182780 was tling of receptors is to generate heterokaryons between determined after 3 hours. In the absence of hormone or in Estrogen receptor shuttling 1385

Fig. 8. Effect of ligands on the ability of estrogen receptors to shuttle between nuclei in heterokaryons. COS-1 cells expressing the mouse estrogen receptor were fused with NIH-3T3 cells devoid of estrogen receptor and treated for 3 hours with no hormone (NH), 10 nM estradiol (E2) or 100 nM ICI 182780 (ICI 182780). The subcellular localisation of the estrogen receptor was observed in cell hybrids by indirect immunofluorescence using the monoclonal antibody H222 (left panel). Nuclei from the two cell types were distinguished using Hoechst 33258 dye (right panel), open arrows indicate COS-1 cell nuclei while solid arrows indicate NIH-3T3 nuclei. 1386 S. Dauvois, R. White and M. G. Parker the presence of 10 nM estradiol receptors accumulated in similar to that obtained when cells were treated with NIH3T3 nuclei showing that they were able to shuttle from oligomycin to deplete them of energy. A similar speckled COS-1 cell nuclei (Fig. 8). In the presence of 100 nM ICI appearance has been observed for a number of other nuclear 182780, however, estrogen receptors were not detected in receptors (Jenster et al., 1992; Guiochon-Mantel et al., NIH-3T3 nuclei (Fig. 8) suggesting that the antiestrogen 1991; Barsony et al., 1990; Lee and Mahdavi, 1993) but interferes with the process of nucleocytoplasmic shuttling their nature is unknown. From our coimmunolocalisation in heterokaryons. studies they are unlikely to represent lysosomes since they do not coimmunolocalise with the lysosomal enzyme cathepsin D. DISCUSSION In spite of the cytoplasmic accumulation of receptors in the presence of pure antiestrogen it was apparent that cells Immunochemical analysis indicates that the receptors for varied in the distribution of receptor between the cytoplasm estrogens, progestins and androgens are located predomi- and nucleus. This might be a consequence of cells being in nantly in the cell nucleus in the absence as well as the pres- different phases of the cell cycle since this has been shown ence of hormonal ligand (King and Greene, 1984; Welshons by the group of DeFranco to affect the subcellular location et al., 1985; Perrot-Applanat et al., 1985; Jenster et al., of the glucocorticoid receptor. By using synchronised cells 1992; Kemppainen et al., 1992) whereas the glucocorticoid nuclear retention was found to be impaired during the G2 and mineralocorticoid receptors initially accumulate in the phase of the cell cycle and this may be related to the state cytoplasm and are transported into the cell nucleus upon of phosphorylation of the receptor (De Franco et al., 1991; hormone binding (Picard and Yamamoto, 1987; Hsu et al., Hsu et al., 1992). 1992). Such immunochemical analysis, however, fails to The pure antiestrogens ICI 164384 and ICI 182780 reveal the fact that proteins can be predominantly nuclear reduce the steady-state levels of the estrogen receptor by and yet are not necessarily stationary but are in dynamic reducing the half-life of the protein (Dauvois et al., 1992; equilibrium. Gibson et al., 1991; Reese and Katzenellenbogen, 1992) Two approaches have been used to demonstrate the abil- and so it is possible that the increased receptor turnover ity of certain nuclear proteins to shuttle between the nucleus results from a disruption in nucleocytoplasmic shuttling. and the cytoplasm. The most direct approach is to monitor We demonstrated previously that a number of residues transfer of the protein between nuclei in heterokaryons and important for receptor dimerisation were essential for estro- the second way is to show that the nuclear protein can trans- gen binding suggesting that these two functions overlap one port cytoplasmic forms of the protein into the nucleus when another (Fawell et al., 1990a). In view of this we proposed it undergoes shuttling. Both these approaches have been that the pure antiestrogens might sterically interfere with used to demonstrate that progesterone receptors (Guiochon- dimerisation by means of their 7a -alkylamide extensions Mantel et al., 1989, 1991; Chandran and De Franco, 1992) (Fawell et al., 1990b; Arbuckle et al., 1992) and suggested and, as described in this paper, estrogen receptors, con- that the increased turnover might result from impaired stantly shuttle between the nucleus and the cytoplasm. The dimerisation (Dauvois et al., 1992). This is consistent with ability of oligomycin to inhibit the re-entry of estrogen our observation that the nuclear uptake of the nuclear-local- receptors into the cell nucleus confirms the results of Mil- isation-defective mutant by its interaction with MOR121- grom and coworkers suggesting that nuclear uptake is an 599 is promoted by estradiol and 4-hydroxytamoxifen but energy-dependent step (Guiochon-Mantel et al., 1991). It not by the pure antiestrogens. has been reported that the glucocorticoid receptor also shut- Another possible explanation for the effects of ICI tles (De Franco et al., 1991) and so it seems likely that 164384 and ICI 182780, however, is suggested by the nucleocytoplasmic shuttling is a general feature of all observation that the protein phosphatase inhibitor, okadaic steroid receptors and possibly other nuclear receptors. acid, inhibits nuclear retention of the glucocorticoid recep- We demonstrate that, while the initial nuclear uptake and tor (De Franco et al., 1991). Thus the pure antiestrogens the subsequent nucleocytoplasmic shuttling of the mouse might interfere with dephosphorlylation of the estrogen estrogen receptor occur in the absence or presence of hor- receptor and thereby inhibit nuclear uptake. Finally, since mone, the binding of the pure antiestrogen ICI 182780 dis- hsp70-related proteins appear to be involved in targeting rupts the shuttling process. It appears that ICI 182780 proteins into the cell nucleus and given the observation that reversibly inhibits nuclear uptake of the receptor but it is hsp70 is associated with the estrogen receptor (Scherrer et also conceivable that the antiestrogens may preferentially al., 1993) it is conceivable that pure antiestrogens disrupt target the receptor to another cellular compartment, thereby the interaction of the receptor with hsp70 which in turn interfering with nuclear re-entry. The cytoplasmic location leads to inhibition of nuclear uptake and an increase in pro- of receptors suggests that receptor efflux from the nucleus tein degradation. is not affected by the antiestrogen. Human estrogen recep- The ability of proteins to shuttle between the nucleus and tors also accumulated in the cytoplasm in the presence of cytoplasm implies that they may have a cytoplasmic as well 100 nM ICI 164384. This concentration is approximately as a nuclear role or that they may be involved in trans- 10-fold higher than that required by the mouse receptor, porting proteins from one compartment to another. This which probably accounts for the absence of cytoplasmic seems to be the case for two nucleolar proteins, nucleolin receptor at 10 nM ICI 164384 observed by Chambon and and B23/No38, that shuttle and yet, like the steroid recep- coworkers (Ylikomi et al., 1992). The speckled appearance tors, are not significantly detected in the cytoplasm under of receptors observed in the presence of antiestrogen was steady-state conditions (Borer et al., 1989). Nucleolin and Estrogen receptor shuttling 1387

B23/No38 may be involved in the translocation of riboso- Fawell, S. E., White, R., Hoare, S., Sydenham, M., Page, M. and Parker, mal components across the nuclear envelope as well as in M. G. (1990b). Inhibition of estrogen receptor-DNA binding by the rRNA synthesis or processing. Several hsp70-related pro- ‘pure’ antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization. Proc. Nat. Acad. Sci. USA 87, 6883-6887. teins that also shuttle across the nuclear envelope (Mandell Gibson, M. K., Nemmers, L. A., Beckman, W. C. Jr, Davis, V. L., Curtis, and Feldherr, 1990) appear to have a role in transporting S. W. and Korach, K. S. (1991). The mechanism of ICI 164,384 proteins into the cell nucleus (Shi and Thomas, 1992). The antiestrogenicity involves rapid loss of estrogen receptor in uterine tissue. functional significance of shuttling of the steroid receptors Endocrinology 129, 2000-2010. Greene, G. L., Sobel, N. B., King, W. J. and Jensen, E. V. (1984). is unclear but it might present a means for coupling steroid Immunochemical studies of estrogen receptors. J. Steroid Biochem. 20, receptors with other signalling pathways. For example, it 51-6. has been found that dopamine can stimulate the transcrip- Guiochon-Mantel, A., Loosfelt, H. Lescop, P., Sar, S., Atger, M., Perrot- tional activity of a number of nuclear receptors (Power et Applanat, M. and Milgrom, E. (1989). Mechanisms of nuclear al., 1991) and epidermal growth factor can mimic some of localization of the progesterone receptor, evidence for interaction between monomers. Cell 57, 1147-1154. the effects of estrogen on the estrogen receptor in the uterus Guiochon-Mantel, A., Lescop, P., Christin-Maitre, S., Loosfelt, H., (Ignar-Trowbridge et al., 1992). Perrot-Applanat, M. and Milgrom, E. (1991). Nucleocytoplasmic We conclude that the estrogen receptor is constantly shut- shuttling of the progesterone receptor. EMBO J. 10, 3851-3859. tling between the nucleus and the cytoplasm in the pres- Ham, J. and Parker, M. G. (1989). Regulation of gene expression by ence of the estrogen antagonist 4-hydoxytamoxifen as well nuclear hormone receptors. Curr. Opin. Cell Biol. 1, 503-511. Hsu, S.-C., Qi, M. and De Franco, D. B. (1992). Cell cycle regulation of as estradiol. In contrast the pure antiestrogens ICI 182780 glucocorticoid receptor function. EMBO J. 11, 3457-3468. and ICI 164384 disrupt shuttling and therefore the result- Ignar-Trowbridge, D. M., Nelson, K. G., Bidwell, M. C., Curtis, S. W., ing nuclear exclusion of the estrogen receptor must con- Washburn, T. F., McLachlan, J. A. and Korach, K. S. (1992). tribute to its inactivation. Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc. Nat. Acad. Sci. USA 89, 4658-4662. Jenster, G., Van der Korput, A. G. M., Trapman, J. and Brinkmann, A. We thank A. Wakeling (ICI Pharmaceuticals) for supplying ICI O. (1992). Functional domains of the human androgen receptor. J. Steroid 164384, ICI 182780 and OH-tamoxifen; C. Nolan (Abbot Labo- Biochem. Molec. Biol. 41, 671-675. ratories) for the H222 antibody; G. L. Greene for ER21 antibody; Jordan, V. C. (1984). Biochemical pharmacology of antiestrogen action. and P. Chambon for the human estrogen receptor cDNA clone. Pharmacol. Rev. 36, 245-76. We also thank the members of the Molecular Endocrinology lab- Jordan, V. C. and Murphy, C. S. (1990). Endocrine pharmacology of oratory, C. Dickson, R. Laski and G. Warren, for advice and com- antiestrogens as antitumor agents. Endocrine Rev. 11, 578-610. ments on the manuscript. Kalderon, D., Richardson, W. D., Markham, A. T. and Smith, A. E. (1984a). Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311, 33-38. Kalderon, D., Roberts, B. L., Richardson, W. D. and Smith, A. E. REFERENCES (1984b). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509. Arbuckle, N. D., Dauvois S. and Parker, M. G. (1992). Effects of Kemppainen, J. A., Lane, M. V., Sar, M. and Wilson, E. M. (1992). antioestrogens on the DNA binding activity of oestrogen receptors in Androgen receptor phosphorylation, turnover, nuclear transport, and vitro. Nucl. Acids Res. 20, 3839-3844. transcriptional activation. J. Biol. Chem. 267, 968-974. Barsony, J., Pike, J. W., De Luca, H. F. and Marx, S. J. (1990). King, W. J. and Greene, G. L. (1984). Monoclonal antibodies localize Immunocytology with microwave-fixed fibroblasts shows 1a,25- oestrogen receptor in the nuclei of target cells. Nature 307, 745-7. dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow Kumar, V., Green, S., Staub, A. and Chambon, P. (1986). Localisation of reorganization of vitamin D receptors. J. Cell Biol.11, 2385-2395. the oestradiol-binding and putative DNA-binding domains of the human Beato, M. (1989). Gene regulation by steroid hormones. Cell 56, 335- oestrogen receptor. EMBO J. 5, 2231-2236. 344. Lanford, R. E. and Butel, J. S. (1984). Construction and characterization Berry, M., Metzger, D. and Chambon, P. (1990). Role of the two of an SV40 mutant defective in nuclear transport of T antigen. Cell 37, activating domains of the oestrogen receptor in the cell-type and 801-813. promoter-context dependent agonistic activity of the anti-oestrogen 4- Lee, Y. and Mahdavi, V. (1993). The D domain of the thyroid hormone hydroxytamoxifen. EMBO J. 9, 2811-2818. receptor alpha1 specifies positive and negative transcriptional regulation Borer, R. A., Lehner, C. F., Eppenberger, H. M. and Nigg, E. A. (1989). functions. J. Biol. Chem. 268, 2021-2028. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56, Lees, J. A., Fawell, S. E. and Parker, M. G. (1989). Identification of two 379-390. transactivation domains in the mouse oestrogen receptor. Nucl. Acids Res. Bowler, J., Lilley, T. J., Pittam J. D. and Wakeling, A. E. (1989). Novel 17, 5477-5488. steroidal pure antiestrogens. Steroids 54, 71-99. Mandell, R. B. and Feldherr, C. M. (1990). Identification of two HSP70- Chandran, U. R. and De Franco, D. B. (1992). Internuclear migration of related Xenopus oocyte proteins that are capable of recycling across the chicken progesterone receptor, but not simian virus-40 large tumor nuclear envelope. J. Cell Biol. 111, 1775-1783. antigen, in transient heterokaryons. Mol. Endocrinol. 6, 837-844. Morgenstern, J. P. and Land, H. (1990). A series of mammalian Dauvois, S., Danielian, P. S., White, R. and Parker, M. G. (1992). expression vectors and characterisation of their expression of a reporter Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by gene in stably and transiently transfected cells. Nucl. Acids Res. 18, increasing its turnover. Proc. Nat. Acad. Sci. USA 89, 4037-4041. 1068. De Franco, D. B., Qi, M., Borror, K. C., Garabedian, M. J. and Perrot-Applanat, M., Logeat, F., Groyer-Picard, M. T. and Milgrom, E. Brautigan, D. L. (1991). Protein phosphatase types 1 and/or 2A regulate (1985). Immunocytochemical study of mammalian progesterone receptor nucleocytoplasmic shuttling of glucocorticoid receptors. Mol. using monoclonal antibodies. Endocrinol. 116, 1473-84. Endocrinol. 5, 1215-1228. Picard, D. and Yamamoto, K. R. (1987). Two signals mediate hormone- Dingwall, C. and Laskey, R. A. (1991). Nuclear targeting sequences - a dependent nuclear localization of the glucocorticoid receptor. EMBO J. 6, consensus? Trends Biochem. Sci. 16, 478-481. 3333-40. Evans, R. M. (1988). The steroid and thyroid hormone receptor Picard, D., Kumar, V., Chambon, P. and Yamamoto, K. R. (1990). superfamily. Science 240, 889-895. Signal transduction by steroid hormones: nuclear localization is Fawell, S. E., Lees, J. A., White, R. and Parker, M. G. (1990a). differentially regulated in estrogen and glucocorticoid receptors. Cell Characterization and colocalization of steroid binding and dimerization Regul. 1, 291-299. activities in the mouse estrogen receptor. Cell 60, 953-962. Power, R. F., Mani, S. K., Codina, J., Conneely, O. M. and O’Malley, B. 1388 S. Dauvois, R. White and M. G. Parker

W. (1991). Dopaminergic and ligand-independent activation of steroid mutation which alters its hormone binding properties. EMBO J. 8, 1981- hormone receptors. Science 254, 1636-1639. 1986. Reese, J. C. and Katzenellenbogen, B. S. (1992). Examination of the Wakeling, A. E., Dukes, M. and Bowler, J. (1991). A potent speciﬁc pure DNA-binding ability of estrogen receptor in whole cells: implications for antiestrogen with clinical potential. Cancer Res. 51, 3867-3873. hormone-dependent transactivation and the actions of antiestrogens. Mol. Welshons, W. V., Krummel, B. M. and Gorski, J. (1985). Nuclear Cell. Biol. 12, 4531-4538. localization of unoccupied receptors for glucocorticoids, estrogens, and Scherrer, L. C., Picard, D., Massa, E., Harmon, J. M., Simons, S. S. Jr, progesterone in GH3 cells. Endocrinology 117, 2140-7. Yamamoto, K. R. and Pratt, W. B. (1993). Evidence that the hormone White, R., Lees, J. A., Needham, M., Ham, J. and Parker, M. (1987). binding domain of steroid receptors confers hormonal control on Structural organization and expression of the mouse estrogen receptor. chimaeric proteins by determining their hormone-regulated binding to Mol. Endocrinol. 1, 735-744. hsp90. Biochemistry (in press). Ylikomi, T., Bocquel, M. T., Berry, M., Gronemeyer, H. and Chambon, Shi, Y. and Thomas, J. O. (1992). The transport of proteins into the nucleus P. (1992). Cooperation of proto-signals for nuclear accumulation of requires the 70-kilodalton heat shock protein or its cytosolic cognate. estrogen and progesterone receptors. EMBO J. 11, 1-14. Mol. Cell. Biol.12, 2186-2192. Tora, L., Mullick, A., Metzger, D., Ponglikitmongkol, M., Park, I. and Chambon, P. (1989). The cloned human oestrogen receptor contains a (Received 8 September 1993 - Accepted 25 October 1993).