Tamoxifen Aziridine Research Articles

Light-activated transcription and repression by using photocaged SERMs.

Recently developed methods to regulate the spatial and temporal patterning of genes in a light-directed manner hold promise as powerful tools for exploring the function of genes that act through their unique spatiotemporal patterning. To further explore the application of photocaged ligands of nuclear receptors to control gene expression patterning, the actions of photocaged analogues of selective estrogen-receptor modulators (SERMs) have been evaluated. Photocaged derivatives of hydroxytamoxifen (NB-Htam) and guanidine tamoxifen (NB-Gtam) have been synthesized that selectively antagonize ER alpha- and ER beta-mediated transcription at classic estrogen response elements (EREs) in response to light. When present only intracellularly, Htam and Gtam provide a similar transient repression response. When SERMs are allowed to diffuse out of the cell, transcription is recovered at a similar rate for Htam and Gtam (6.4 and 5.6 h(-1)), but is notably faster than is observed with the covalently binding SERM tamoxifen aziridine (Taz) (3.8 h(-1)). This suggests that the duration of agonist action is controlled by ligand off-rates/diffusion and not by receptor turnover. Gtam activates ER beta-mediated transcription at AP1 sites in a similar way to what has previously been reported for Htam. NB-Gtam and NB-Tam provide a light-activated transcription response at AP1-driven reporters, thus illustrating the unique ability of photocaged SERMs to simultaneously mediate light-activated transcription and repression.

Evidence of an estrogen receptor form devoid of estrogen binding ability in MCF-7 cells☆

In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [ 3H]estradiol (E 2). We show here that this property is not restricted to this antiestrogen. [ 3H]E 2 binding assays (whole cell assays, DCC assays on cell extracts) and enzyme immunoassays (Abbott) performed in parallel, establish the permanent presence of such unusual ERs in the absence of any exposure of the cells to a ligand. E 2 and the pure antiestrogen RU 58 668, which down-regulate ER, also decrease [ 3H]E 2 binding. In control cells, these ERs represent about the half of the whole receptor population; they also display a tendency to stabilize within the cell nucleus. Loss of E 2 binding ability appears irreversible, since we failed to label receptor accumulated under OH-Tam with [ 3H]E 2 or [ 3H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E 2-induced down regulation of ER, failed to stabilize [ 3H]E 2 binding (whole cell assay) after an [ 3H]E 2 pulse (1 h), confirming that regulation of E 2 binding and peptide level are related to different regulatory mechanisms. Loss of binding ability could not be ascribed to any ER cleavage as demonstrated by Western blotting with a panel of ER antibodies raised against its various domains (67 kDa ER solely detected). We propose that loss of E 2 binding ability is related to the aging process of the receptor, i.e. it is progressively converted to a form devoted to degradation after it has accomplished its physiological role. Ligands may favor (E 2, RU 58 668) or impede (OH-Tam) this elimination process.

Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF)

Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERα. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERα-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression.

Cysteine 530 of the human estrogen receptor alpha is the main covalent attachment site of 11beta-(aziridinylalkoxyphenyl)estradiols.

The efficiency of 11beta-[p(aziridinylethoxy)phenyl]estradiol 1 and 11beta-[p(aziridinylpentoxy)phenyl]estradiol 2 affinity labeling of the estrogen receptor alpha (ERalpha) was evaluated on the basis of their capacity to inhibit [(3)H]estradiol binding to lamb and human ERalphas. Relative to RU 39 411 (11beta-[p(dimethylaminoethoxy)phenyl]estradiol), the most closely related and chemically inert analogue of 1, the two electrophiles irreversibly inhibited [(3)H]estradiol binding to the lamb ERalpha. The fact that the compound effects were prevented (i) when the ERalpha hormone-binding site was occupied by estradiol and (ii) when the ERalpha-containing extracts were pretreated with methyl methanethiosulfonate (an SH-specific reagent) suggested that the compounds specifically alkylated ERalpha at cysteine residues. Wild-type human ERalpha was alkylated as efficiently as lamb ER, whereas the quadruple cysteine --> alanine mutant, in which all cysteines of the hormone-binding domain (residues 381, 417, 447, and 530) were changed to alanines, showed no significant electrophile labeling. The single C530A mutant was much less sensitive to the action of the electrophiles than the three other single mutants (C381A, C417A, and C447A). Moreover, analysis of the three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) showed that only the C381A/C530A mutant was less susceptible to electrophile labeling than the single C530A mutant. We concluded that in the hormone-binding pocket C530 was the main covalent attachment site of aziridines 1 and 2, whereas C381 could be a secondary site. These results agreed with the crystal structure of the hormone-binding domain of the human ERalpha bound to estrogen or antiestrogen, since C381 and C530 appeared to be (i) located in structural elements involved in delineating the hormone-binding pocket and (ii) in spatial proximity to each other, which was closer in the crystal structure of the ER:antiestrogen complex than in that of the ER:estrogen complex. Since C530 and C381 were also the main and secondary covalent attachment sites of tamoxifen aziridine (a nonsteroidal affinity-labeling agent), we propose a selective mode of superimposition of tamoxifen-class antiestrogens with RU 39 411-class antiestrogens, which could account for the relative positioning of the two types of ligands in the ERalpha hormone-binding pocket.

Implication of proteasome in estrogen receptor degradation

In MCF-7 breast cancer cells, estradiol (E 2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E 2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH 4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E 2 and RU 58668; this selective effect was also recorded in cells prelabeled with [ 3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.

Inhibition of estradiol binding to its receptor by the cupric ion

Treatment of human recombinant estrogen receptor (hER) expressed in yeast with very low concentrations of the cupric ion decreased its ability to bind [3H]estradiol ([3H]E2) and [125I]tamoxifen aziridine (minimal Cu2+ concentration: 1 nM). This decrease was reflected in a loss of immunoreactivity for monoclonal antibodies raised against the hormone binding domain (HBD). An ER recombinant expressing solely the HBD confirmed that the ion operated at this level. Cysteines located within this domain contributed to the inhibitory action of the Cu2+ in view of a partial restoration of the [3H]E2 binding activity with β-mercaptoethanol. Histidines were also implicated since the influence of Cu2+ on the [3H]E2 binding parameters (Scatchard plot analysis) was maintained after oxidation of thiol groups by methyl methanethiosulfonate, and partly reversed by imidazole.

Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements

The estrogen receptor (ER) is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements (EREs). Estradiol-liganded ER (E 2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally-occurring AT-rich sequences with a stoichiometry of one E 2-ER dimer per ERE. When ER is bound by 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen (TAM), the receptor binds to EREs with high affinity. However, one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA, yielding a stoichiometry of one [ 3H]4-OHT molecule per ERE. To determine whether DNA-binding induced ligand dissociation is a general property of type I antiestrogens that are not covalently attached to the ER, we examined the interaction of ER liganded by tamoxifen (TAM) with EREs. We demonstrate that TAM-ER binds EREs with lower affinity than E 2-ER, 4-OHT-ER, or ER liganded by the covalent antiestrogen tamoxifen aziridine. Unlike E 2-ER, both TAM and 4-OHT-ER bind EREs non-cooperatively. Like 4-OHT, TAM appears to dissociate from the liganded ER as the receptor binds EREs. Additionally, partial proteolysis of ERE-bound ER by trypsin revealed different cleavage patterns for E 2 versus 4-OHT and TAM. These findings indicate that the behavior of the ER liganded by TAM is generally similar to that of the antiestrogen 4-OHT.

Tamoxifen aziridine binding to cytosolic proteins from human breast specimens is negatively associated with estrogen receptors, progesterone receptors, pS2, and cathepsin-D.

[3H]Tamoxifen Aziridine ([3H]TAZ) is a derivative of the antiestrogen tamoxifen that covalently labels the Estrogen Receptor (ER), and perhaps other uncharacterized proteins. In a previous article we described that [3H]TAZ binds to a cytosolic protein from human uterine tissues that shares some, but not all, the ER properties. Here we have extended these studies to [3H]TAZ binding to cytosol proteins from human breast cancer specimens, and studied its quantitative association with other molecular markers and clinico-pathological variables. Cytosols were obtained in hypotonic buffer containing 20 mM molybdate and protease inhibitors, incubated with [3H]TAZ, and subjected to Sucrose Gradient Analysis (SGA). A [3H]TAZ labeled peak that consistently migrated with the 4S fractions was found in most of the assayed cytosols (range of 0 to 1278 fmol/ mg p.). The 4S peak of [3H]TAZ was partially inhibited by both estrogens and antiestrogens. When [3H]E2 was used instead of [3H]TAZ, only an 8S peak was detected. [3H]TAZ was covalently bound to a protein with an apparent MW of 65 kDa, as determined by SDS-PAGE and fluorography. The mean of [3H]TAZ binding was significantly higher in the subgroups of samples classified as ER-, PR-, pS2- or cathepsin D-, than in the respective positive subgroups (P < 0.01 in all the cases). [3H]TAZ binding was not associated with clinico-pathological variables, except that its mean was significantly larger in tumors larger than 5 cm than in smaller tumors. These results, and those previously reported, suggest that: 1) [3H]TAZ labels a cytosolic protein present in human breast cancers and uterine tissues that does not share all the ER properties, and 2) the [3H]TAZ binding by breast cancer cytosols is negatively associated with markers of estrogenic dependency, and its quantification may provide valuable information on antiestrogen responsiveness of a given tumor.

The two native estrogen receptor forms of 8S and 4S present in cytosol from human uterine tissues display opposite reactivities with the antiestrogen tamoxifen aziridine and the estrogen responsive element

We have investigated the capability of the different native ER forms, present in cytosols from human uterine tissues, of reacting with the antiestrogen [ 3H]Tamoxifen aziridine ([ 3H]TA) and with the Estrogen Responsive Element (ERE). Cytosols from uterine leiomyoma (myoma) prepared in buffer containing 40 mM molybdate and protease inhibitors, labelled with [ 3H]estradiol and analyzed in low-salt sucrose gradient showed 8S and 4S ER forms. The same cytosols labelled with [ 3H]TA only showed a 4S ER form, whereas the ERE only reacted with fractions from the 8S peak. The band of ERE reaction in the EMSA assay showed a lower relative mobility than the band labelled with [ 3H]TA, but both bands contained immunoreactive ER of 65 kDa. Electrophoretic mobility of the [ 3H]TA-labelled band in that system was not affected by cytosol treatment with cross-linkers or SDS, which suggests that it is a monomeric protein. The [ 3H]TA-binding 4S ER form was found in all studied myoma samples, as well as in human endometrium or myometrium, but not in rat tissues. These results suggest that the 8S and 4S ER form were already present before cytosol from human uterine tissues comes into contact with the molybdate buffer. They both contain the same ER molecule of 65 kDa, either in the free form or as an oligomer. Only the ER dimers, which have been described both in the cytosolic 8S form and in the nuclear 4-5S form, react with the ERE. [ 3H]TA only binds to the 4S ER monomer probably because its binding site is concealed in the 8S form under these experimental conditions. The opposite reactivity of the 8S and 4S ER forms with [ 3H]TA and the ERE support the hypothesis that they may constitute separate entities with a different physiological role.

Protein synthesis is not implicated in the ligand-dependent activation of the estrogen receptor in MCF-7 cells

In MCF-7 cells, estrogen receptor (ER) elimination occurs rapidly under stimulation with estradiol (E 2) at 1 nM (‘ER processing’); cycloheximide (CHX) at 50 μM impedes this phenomenon. ER processing is also observed when E 2 is removed after the first hour of incubation, indicating that the role of the hormone would be limited to the initiation of this process. When CHX is removed at the same time, receptor processing and, later, the induction of progesterone receptor (PgR) both proceed. The initial estrogenic signal which activates ER is therefore not influenced by CHX. In support of this conclusion, no effect of the drug on E 2 binding affinity of residual ER was detected. A similar result was recorded for a series of estrogens and antiestrogens, indicating that CHX exerts no influence on the potential agonistic/antagonistic potency of any ligand. Size-exclusion chromatography (FPLC) revealed that [ 3H]E 2-induced ER activation leads to the cleavage of the native receptor (67 kDa) into low molecular weight isoforms which subsequently become less detectable over time (proteolysis). In the presence of CHX, such ER isoforms persist, confirming the absence of interference of the drug with the activation step. When the cells were prelabelled with [ 3H]tamoxifen aziridine ([ 3H]TAZ) before their exposure to E 2, ER cleavage could not be detected due to the lack of activation potency of the antiestrogenic ligand. However, the [ 3H]TAZ-ER complexes were subjected to E 2-induced processing; CHX blocked this phenomenon, which is associated with the maintenance of ER synthesis and activation.

17 alpha (haloacetamidoalkyl) estradiols alkylate the human estrogen receptor at cysteine residues 417 and 530.

Results obtained in a previous study suggested that cysteine residues in the estrogen receptor were covalent attachment sites for four 17 alpha-(haloacetamidoalkyl) estradiols (halo, bromo or iodo; alkyl, methyl, ethyl, or propyl). To identify the putative concerned cysteines, we expressed wild-type and various cysteine --> alanine mutants of the human estrogen receptor in COS cells and determined their ability to be alkylated by the four electrophiles. The quadruple mutant, in which all the cysteines (residues 381, 417, 447, and 530) of the hormone-binding site were changed to alanines, showed very little electrophile labeling, whereas the four single mutants (C381A, C417A, C447A, and C530A) were alkylated as efficiently as the wild-type receptor. These results (i) demonstrate that cysteine residues were covalent attachment sites of electrophiles and (ii) indicate that more than one cysteine residue could be alkylated. Analysis of three double mutants (C381A/C530A, C417A/C530A, and C447A/C530A) provided strong evidence that only C417 and C530 were sites for electrophile covalent attachment. Since C530 was also alkylated by tamoxifen aziridine, a nonsteroidal affinity-labeling agent, we propose a selective mode of superimposition of tamoxifen-class antiestrogens with estradiol, which could account for the relative positioning of the two types of ligands in the receptor hormone-binding pocket. According to the structure of the hormone-binding pocket of nuclear receptors, as inferred from crystallographic studies and general sequence alignment of hormone-binding domains, C417 and C530 appear to be (1) located at the extreme border or in structural elements involved in delineation of the hormone-binding pocket, (2) spatially in close proximity to each other, and (3) in positions highly homologous to those of glucocorticoid receptor sites alkylated by affinity- and photoaffinity-labeling agents, respectively.

Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line.

We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogen-response element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A18 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ER77. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways.

Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

We have adressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.Ca++ (0.1–10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca++- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.

Calmodulin Decreases the Estrogen Binding Capacity of the Estrogen Receptor

We previously demonstrated the ability of calmodulin (CaM) to decrease the binding affinity of estradiol (E2) to the rat uterine estrogen receptor (ER). We show now that CaM induces a loss of E2binding capacity especially when ER molecules exhibit a lower binding affinity for the hormone. By Western blotting and [125I]tamoxifen aziridine covalent labeling we found that this CaM-induced loss is not associated with a disappearance of the ER protein. In addition, we were able to demonstrate a CaM-mediated decrease in E2binding of a human recombinant ER expressing solely its hormone binding domain (HBD, aa 282-595). Hence, CaM can modulate the structure of the HBD of the ER without any involvement of a degradative process, this conformational change is not mediated by other domains of the receptor and / or components of the native ER heterocomplex.

Footprint analysis of estrogen receptor binding to adjacent estrogen response elements.

Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, Kd of approximately 0.25 nM, for estradiol-liganded ER (E2-ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E2, 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.

Estrogenic and antiestrogenic regulation of the half-life of covalently labeled estrogen receptor in MCF-7 breast cancer cells

Effect of estrogens and antiestrogens (AEs) on estrogen receptor (ER) half-life was analyzed in MCF-7 cells by assessing its progressive disappearance after covalent labeling in situ with [ 3H]tamoxifen aziridine ([ 3H]TAZ). Cells were incubated for 1 h with 20 nM [ 3H]TAZ either in the absence or presence of a 500-fold excess of unlabeled estradiol (E 2) (non-specific binding). The entire ER population was labeled by this method as established by subsequent incubation of the cells with [ 125I]E 2. [ 3H]TAZ labeled cells were maintained in culture for additional 5 h in the absence (control) or presence of increasing amounts (0.1 nM-1 μM) of either a given estrogen (E 2, estrone, diethylstilbestrol, bisphenol), a pure AE (RU 58 668, ICI 164 384) or an AE with residual estrogenic activity (RU 39 411, 4-hydroxytamoxifen, keoxifene). The progressive disappearance of nuclear and cytosolic [ 3H]TAZ-ER complex during 5 h incubation were assessed by their immunoprecipitation with anti-ER monoclonal antibody (H 222) followed by scintillation counting or SDS-PAGE and fluorography. Fading of labeled receptors was extremely slow (∼ 10% loss after 6 h) in absence of any hormone/antihormone indicating a long half-life of the [ 3H]TAZ-ER complex. Addition of estrogens as well as pure AEs led to a dramatic reduction of the half-life while AEs with residual estrogenic activity were extremely less efficient in this regard providing an explanation for the ability of latter compounds to up-regulate the receptor since they do not affect ER mRNA synthesis and stability. Receptor disappearance induced by estrogens was closely related to their binding affinity for ER. Newly synthesized ER emerged during the treatment with hormones or antihormones seems to be implicated in the phenomenon since [ 3H]TAZ was covalently bound and could, therefore, not be displaced by these compounds. Induction of synthesis of a short half-life peptide(s) with degradative activity was demonstrated by addition of cycloheximide or puromycine (both at 50 μM) which completely blocked ER disappearance. The fact that no cleavage products of ER were detected by SDS-PAGE suggested a lysosomial hydrolysis. Hence, hormonal modulation of only a part of ERs may down-regulate their total population until it reaches the steady-state level.

Dissociation of 4-hydroxytamoxifen, but not estradiol or tamoxifen aziridine, from the estrogen receptor as the receptor binds estrogen response element DNA

Estradiol-liganded estrogen receptor (E 2-ER) binds EREs with a stoichiometry of one E 2-ER dimer per estrogen response element (ERE). In contrast, although 4-hydroxytamoxifen (4-OHT)-liganded ER (4-OHT-ER) binds EREs with high affinity, its saturation ERE binding capacity is consistently half that of E 2-ER, giving an apparent stoichiometry of one 4-OHT-ER monomer per ERE. Here we show that one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA. Under equilibrium conditions, the type I antiestrogen tamoxifen aziridine (TAz), covalently attached to ER (TAz-ER), binds a single ERE with high affinity ( K d = 0.27 nM), comparable to that of E 2-ER and 4-OHT-ER. In contrast to 4-OHT-ER, the ERE binding stoichiometry of TAz-ER was identical to that of E 2-ER: one dimeric receptor per ERE. By measuring [ 3H]ligand that was initially bound to ER, a significant loss of [ 3H]4-OHT from ER was detected after ERE binding, whereas all [ 3H]E 2 or [ 3H]TAz remained ER-bound. These results confirm that one molecule of 4-OHT ligand dissociates from the ER dimer as a consequence of ERE binding. Binding of 4-OHT and TAz are likely to induce a conformation in ER dimers that alters their capacity for gene activation. Upon ER binding to DNA, this conformation reveals itself by allowing 4-OHT dissociation, and predictably would allow TAz dissociation were it not bound covalently.

Functional Estrogen Receptors in a Human Preosteoclastic Cell Line

Abstract The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.

Molecular characterization by mass spectrometry of the human estrogen receptor ligand-binding domain expressed in Escherichia coli.

The ligand binding domain of the human estrogen receptor (hER-LBD), encompassing the sequence MDPS282AG...V595, has been expressed at high levels in Escherichia coli from a pET-23d vector, and a purified preparation has been characterized both by mass spectrometry and biochemical methods. Inclusion bodies from the bacterial expression were solubilized by sonication and the hER-LBD was purified to near homogeneity by affinity chromatography over an estradiol-Sepharose column in urea-containing buffer. This material ran as a single peak on reversed-phase HPLC, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band with apparent molecular mass of 31-32 kilodaltons (kDa), somewhat smaller than that expected from the construct (35.6 kDa). Edman degradation revealed a single sequence of MDPSAGDMRA, consistent with an intact N terminus. Further characterization of this material using low resolution matrix-assisted laser desorption ionization mass spectrometry indicated an apparent single protein species of average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray ionization mass spectrometry, however, revealed that this purified hER-LBD preparation was actually composed of a number of both modified and unmodified LBD protein fragments between 32,900-33,400 Da. The bulk of this 33-kDa protein mixture consisted of three LBD protein fragments with C termini at Ala571 (70%), Ala569 (23%), and Ser575 (4%), with only a trace amount (3%) of the full length expressed LBD (MDPS282...V595; 35, 602 Da). These four protein species also appear to be partially chemically modified by carbamylation. The functional integrity of this hER-LBD preparation was investigated by a ligand-exchange assay, which showed that the hER-LBD retained full estradiol-binding capacity; specific, covalent labeling was also observed using either the electrophilic affinity-labeling ligand tamoxifen aziridine (TAZ) or the photoaffinity-labeling ligand hexestrol diazirine. Thus, this expressed hER-LBD preparation, while appearing nominally pure by conventional biochemical techniques and having the expected ligand-binding capacity, was shown by sensitive high resolution electrospray ionization mass spectrometry techniques to be largely truncated 20-26 amino acids from the expected C terminus and to have a substantial level of covalent modification arising from the urea.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional estrogen receptors in a human preosteoclastic cell line.

The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis.