Rev-erb beta 2, a novel isoform of the Rev-erb family of orphan nuclear receptors.

We have isolated a rat complementary DNA clone corresponding to a novel isoform of the hormone nuclear receptor superfamily. This clone encodes a 383 amino acid residue protein designated as Rev-erb beta 2. This protein is identical until residue 382 to the Rev-erb beta 1 protein, which is 195 amino acids longer. Several arguments pointed out that the Rev-erb beta 2 cDNA may originate from the same gene as Rev-erb beta 1 by alternative splicing and using a different polyadenylation site. Our results indicate that Rev-erb beta 2 is a new isoform of the Rev-erb family of orphan nuclear receptors.

Studies on the activation of the oestrogen receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384 by using three monoclonal antibodies.

Three monoclonal antibodies, H222, H226 and D547, which provided evidence of the structural transformation and change in exposure of the functional domains of the oestrogen receptor from fetal guinea-pig uterus upon activation, were used to study the receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384. No differences in the structure of non-activated 4-hydroxytamoxifen- and ICI 164,384-receptor complexes, as compared with the oestradiol-receptor complex, were detected by the three monoclonal antibodies. When heated at 28 degrees C, both anti-oestrogen-receptor complexes became capable of binding the D547 antibody, which reacts selectively with the activated receptor; however, this binding was lower than that of the oestradiol-receptor complex. The interaction with the H226 antibody showed that anti-oestrogens can induce receptor dimerization, but to a lesser extent than oestradiol. In addition, both anti-oestrogen-receptor complexes can bind to DNA-cellulose and are retained in nuclei from intact cells at 28 degrees C, but less efficiently than the oestradiol-receptor complex. On the other hand, the nuclear receptor seems to have a similar dimeric structure when bound to either anti-oestrogens or oestradiol, as detected by the three monoclonal antibodies. The data suggest that 4-hydroxytamoxifen and ICI 164,384 induce and impaired activation of the oestrogen receptor; this difference, although quantitative rather than qualitative, might be related to the partial agonistic action of these anti-oestrogens in the fetal guinea-pig uterus.

Antiestrogen action in mammary cancer and in fetal cells.

The present data confirm the very complicity of the response of antiestrogen when this compound is studied in different experimental conditions. The new and potent antiestrogen ICI 164,384, which is considered as a full antagonist in most models studied, concerning the progesterone receptor in the isolated cells of the uterus and vagina of guinea-pig acts as a real agonist. However, this compound antagonizes cell proliferation, progesterone receptor, and decreases the concentration of estradiol in different hormone-dependent mammary cancer cell lines. Another interesting aspect is the response of the antiestrogen 4-hydroxytamoxifen which in isolated cells of very close tissues such as the uterus and vagina is an antagonist for the former and agonist for the latter concerning the progesterone receptor. In conclusion, the present data added new information in the complicity of the mechanism of action of antiestrogens, but using new models interesting possibilities are opened to understand their responses and their mechanism.

[Characterization of activated and non-activated estrogen receptor using monoclonal antibodies]. / Caractérisation des formes activées et non-activées du récepteur oestrogénique en utilisant des anticorps monoclonaux.

Three monoclonal antibodies (D547, H222 and H226) prepared against the human estrogen receptor interact with the estrogen receptor from fetal guinea pig uterus depending on its activation state. The D547 antibody, whose epitope is located between the hormone-binding and DNA-binding domains, recognizes only the activated receptor. The H226 antibody, whose epitope is in the N-terminal region of the receptor ("A/B" region), recognizes both the activated and non-activated receptor. However, the H226 epitope seems to be partially masked in the non-activated receptor (oligomeric form 9 S) but completely exposed following activation. This antibody detects the formation of an activated dimer, which is the only form retained in the nuclei. The H222 antibody, whose epitope is in the hormone-binding domain, reacts with all the hormone-binding subunits present in the non-activated receptor. However, only one H222 epitope is accessible in the activated dimer, suggesting that this epitope and consequently the hormone-binding domain, are located close to the dimerization site. According to these results, the activation process involves a structural transformation of the receptor that modifies the exposure of functional domains.

Studies on the non-activated and activated forms of the estrogen receptor.

The activation of the steroid receptor is a necessary process for the biological role of the receptor. Many factors are involved in this mechanism; in addition to time, temperature, and salt concentration, RNA and RNAase can also affect the transformation of the non-activated to the activated form of the receptor. Using as a model the estrogen receptor of fetal uterus of guinea-pig, the studies of the interaction with three different monoclonal antibodies (D547, H222 and H226) reveal structural transformation during the process of the receptor activation. These conformational transformations suggest that a change in the exposure of the functional domains of the estrogen receptor occurs during activation.

Interaction of three monoclonal antibodies with the nonactivated and activated forms of the estrogen receptor.

The 9S nonactivated oligomeric estrogen receptor from fetal guinea pig uterus interacts with the H222 monoclonal antibody (whose epitope is located in the hormone-binding domain) to yield an 11S complex and with the H226 monoclonal antibody (whose epitope is located in the A/B region, just N-terminal to the DNA-binding domain) to yield a 9.4S complex. No reaction was detected with the D547 monoclonal antibody (whose epitope is located between the hormone-binding and DNA-binding domains). In high salt gradients, the 11S oligomeric receptor-H222 complex dissociates to a 8S monomer-H222 complex and the 9.4S oligomeric receptor-H226 complex to a 7S monomer-H226 complex plus the 4.5S monomer receptor not bound to the antibody. These observations suggest that the nonactivated oligomeric receptor contains more than one estradiol-binding subunit with a structure such that the H222 epitopes are fully accessible, the H226 epitopes are partially accessible, and the D547 epitopes are masked. The temperature-activated receptor reacts with the H226 antibody to yield two complexes that sediment at 7S and 9S in high salt gradients. The 9S complex corresponds to a receptor form complexed with more than one antibody molecule; therefore, this suggests the formation of a receptor homodimer where the two H226 epitopes are exposed. However, a single 8S peak is observed when the H222 antibody reacts with the activated receptor, suggesting that only one H222 epitope is accessible in the dimeric receptor. In addition, binding to the H222 antibody before activation prevents dimerization. Thus, the H222 appears to be close to the dimerization domain. The activated receptor reacts with the D547 antibody to yield a 8S complex that apparently contains only one antibody molecule. On the other hand, the receptor extracted from nuclei was found to be a single 5.5S form with the same immunological characteristics as the receptor activated in cytosol. In conclusion, interaction of these monoclonal antibodies with the different forms of the estrogen receptor reveals a structural transformation during activation, with a concomitant change in the exposure of the receptor functional domains.

Histone acetylation decreased by estradiol in the MCF-7 human mammary cancer cell line.

The effect of estradiol (E2) on the [3H]-acetylation of nuclear histones was studied in the MCF-7 human mammary cancer cell line in culture. Cells (approximately 10(8) were incubated with 8 x 10(-6) M [3H]-acetate in the absence (control) or in the presence of estradiol (10(-5)-10(-8) M). After 20 min incubation, the nuclear histones were extracted and separated by electrophoresis, and each histone band was measured and calculated in DPM/mg protein. It was observed that only the H2 + H3 and H4 histones were associated with the [3H]-acetate. Estradiol (10(-6)-10(-8) M) provoked a significant inhibition in the incorporation of the acetate. The negative effect, in percentage of the non-treated cell value, was as follows: in E2 (10(-6) M): -25 +/- 10 (SE) for H2 + H3 and -26 +/- 5 for H4; in E2 (10(-7) M): -35 +/- 9 and -39 +/- 10; and in E2 (10(-8) M): -56 +/- 22 and -30 +/- 13 respectively. It is concluded that estradiol has a negative effect in the acetylation of H2, H3 and H4 histones of this mammary cancer cell; no acetylation or effect is observed in H1 histones. The relationship of this finding to the biological responses of the hormone is to be explored.

RNA-induced transformation of the estrogen receptor detected by a monoclonal antibody which recognizes the activated receptor.

The effect of RNA and polyribonucleotides on the estrogen receptor from fetal guinea pig uterus was studied through the analysis of the sedimentation properties of this receptor and its interaction with the monoclonal antibody D547. Different exogenous RNAs (calf thymus RNA, yeast RNA and rabbit liver transfer RNA) were able to induce a transformation of the 9S native receptor to 4.5-7S sedimenting forms in low salt sucrose density gradients, as activating factors such as temperature and time do. This transformation was prevented by 20mM sodium molybdate. Moreover, the RNA treated receptor was partially recognized by the monoclonal antibody D547. This antibody, as was demonstrated previously, selectively reacts with the activated form of this receptor. When different homo-polyribonucleotides were tested, the effect depended on their composition. In contrast, DNA did not affect either the sedimentation properties of the receptor or its reaction with the antibody. These observations suggest that RNA induces a dissociation of the 9S receptor and that at least one of the resulting forms is the activated receptor. However, RNA and polyribonucleotides inhibited the receptor binding to DNA-cellulose apparently by competing with DNA. The data suggest a role of RNA in estrogen receptor activation.

Transformation of the estrogen receptor detected by two monoclonal antibodies.

The estrogen receptor from fetal guinea-pig uterus is recognised by two monoclonal antibodies (H222 and H226) developed against the human estrogen receptor but it interacts differently with each of them. The H222 antibody, whose epitope is located in the hormone-binding domain of the receptor, shifts the sedimentation coefficient of the nonactivated oligomeric receptor in low salt sucrose gradients from 9S to 11S. When this oligomeric receptor-H222 complex is centrifuged in high salt gradients, it dissociates to an 8S monomer-H222 complex, indicating that all the estradiol-binding units present in the nonactivated receptor can bind the H222 antibody. In contrast, the H226 antibody, whose epitope is located close to the DNA-binding domain, shifts the sedimentation coefficient of the nonactivated receptor only to 9.4S and when this complex sediments in high salt gradients, it dissociates to a 7S monomer-H226 complex plus a 4.5S monomeric receptor not bound to the antibody. This observation suggests that not all the H226 epitopes are accessible in the nonactivated receptor. On the other hand, the temperature-activated receptor reacts with the H226 antibody to form two complexes sedimenting at 7S and 9S in high salt gradients. This 9S complex indicates the formation of a homodimer that binds two molecules of the H226 antibody. However, only one H222 epitope seems to be accessible in this dimeric form of the receptor, since only one 8S complex is observed when the activated receptor reacts with the H222 antibody. In addition, binding to the H222 antibody before activation prevents the dimerisation. This suggests that the H222 epitope is near or directly involved in the dimerisation domain. Interaction of the H222 and H226 antibodies with the estrogen receptor reveals modifications of its structure during activation, and consequently of the exposure of its functional domains.

Pharmacodynamic and biological effects of anti-estrogens in different models.

The biological response to anti-estrogens is very variable and depends on the animal species considered, the target organ, the parameter studied, and the experimental conditions. Anti-estrogens can bind specifically, (1) to the estrogen receptor, (2) to the typical anti-estrogen specific binding site, and (3) to low density lipoproteins in the plasma. Using a monoclonal antibody against the estrogen receptor, different immunological characteristics of the anti-estrogen-receptor complex can be observed. This difference could explain some of the different biological effects. Studies using different human mammary cancer cell lines (hormone-dependent) show that anti-estrogens are active in decreasing cell proliferation. Also, anti-estrogens can block proteins specifically produced by these cells. Some of these proteins could act as growth or inhibitory factors. Estrogen sulfates are the main precursors of estradiol in breast tissues and this conversion is significantly decreased by anti-estrogens. It is accepted that the main pathway of action of anti-estrogens is through the estrogen receptor, but recent information suggests the possibility that this is not the only step in the mechanism of action of anti-estrogens.

Immunological differences between the estradiol-, tamoxifen- and 4-hydroxy-tamoxifen-estrogen receptor complexes detected by two monoclonal antibodies.

Two monoclonal antibodies (D547 and H222), obtained against the estrogen receptor from MCF-7 breast cancer cells, were used to study the estrogen receptor from fetal guinea-pig uterus bound to estradiol or to the antiestrogens tamoxifen and 4-hydroxytamoxifen. The estradiol-receptor complex binds partially to the monoclonal antibody D547, shifting its sedimentation coefficient in high salt sucrose density gradients from 4.5S to 7.5S. Recently, we demonstrated that the form selectively recognized by this monoclonal antibody is the activated form of the receptor. The estrogen receptor complexed with tamoxifen or 4-hydroxytamoxifen is also partially recognized by this monoclonal antibody but the fraction of total receptor bound to the antibody is significantly less than for the receptor complexed with estradiol. Another series of experiments showed that the monoclonal antibody H222, which recognizes a different antigenic site on the receptor molecule, binds all the estradiol-receptor complex (independently of the degree of activation), shifting its sedimentation coefficient to 7.5S. However, even if all the 4-hydroxytamoxifen-receptor complex is bound by this antibody, only a fraction of the receptor is recognized when it is complexed with tamoxifen. These data show different interactions between the estradiol-, tamoxifen- and 4-hydroxytamoxifen-receptor complexes and the two monoclonal antibodies tested and suggest that these compounds induce different conformational modifications of the estrogen receptor molecule.

Immunological difference between ribonuclease and temperature, time and salt-induced forms of the estrogen receptor detected by a monoclonal antibody.

The effect of RNAase A on the activation of the estrogen receptor from fetal guinea pig uterus was studied by DNA-cellulose binding assay and immunorecognition of the estradiol-receptor complex by the monoclonal antibody D547 raised against the human estrogen receptor. After RNAase treatment at 4 degrees C or 25 degrees C the binding of the receptor to DNA-cellulose doubled. This stimulation was partially prevented by sodium molybdate. RNAase treatment did not modify the interaction of the receptor with the monoclonal antibody D547; this antibody, as was demonstrated previously, selectively recognizes the activated form of the receptor when activation has been induced by temperature, time or high salt concentrations. In addition, RNAase had little or no effect on the transformation of the 8-9 S receptor to more slowly sedimenting forms under low salt concentrations. These observations suggest that even if RNAase induces receptor activation, which can be inferred from the increase in its binding to DNA-cellulose, the conformational modifications of the receptor molecule involved in this process are apparently different from those induced by factors such as temperature, time or high-salt concentrations.

The complexity of anti-estrogen responses.

The actions and biological responses of anti-estrogens are a function of: the experimental conditions, the parameters, the organ and the animal species considered. Target tissues for estrogens in the guinea-pig during the perinatal period are interesting models to explore the action of anti-estrogens. The summary of the data indicates: (1) In the fetal uterus of guinea-pig in in vivo experiments (after injection to the maternal compartment) tamoxifen acts as a real agonist concerning growth, as a partial agonist concerning the stimulation of the progesterone receptor. (2) In in vitro experiments (in organ culture of fetal uterus or in isolated cells) anti-estrogens (tamoxifen or 4-hydroxy-tamoxifen) act as antagonists and also inhibit the effects provoked by estrogens. (3) In the uterus and vagina of newborn guinea-pigs, tamoxifen and its derivatives: 4-hydroxytamoxifen and N-desmethyltamoxifen act as real agonists concerning the uterotrophic and vaginotrophic effects, and also stimulate the amount of DNA per organ, but concerning the progesterone receptor in the uterus, in the short treatment anti-estrogens act as partial agonists but they have no effect in the long treatment. In the vagina in the short treatment anti-estrogens provoke no significant effects, but in the long treatment they are full agonists. In neither of the two biological responses studied (growth and progesterone receptor) does tamoxifen and its derivatives block the action of estradiol. (4) The use of a monoclonal antibody to the estrogen receptor revealed quantitative differences in the activation of the estrogen receptor when bound to estradiol or tamoxifen. This observation was in agreement with the lesser extent of binding to DNA-cellulose of the tamoxifen-estrogen receptor complex as compared with the estradiol-estrogen receptor complex. This fact suggests an impaired activation of the estrogen receptor induced by tamoxifen which might be related to the different biological responses provoked by estrogens and anti-estrogens.

Tamoxifen and progesterone effects in target tissues during the perinatal period.

The biological effects of tamoxifen (TAM), progesterone (P), or a combination of TAM + P were investigated in the uterus and vagina of newborn guinea pigs after short (2 days) and long (12 days) treatments. In both tissues, tamoxifen provoked a significant trophic effect which is indicated by the increase in weight, protein and DNA content. In the uterus, progesterone also provoked an increase in weight, protein and DNA content, but much less than that provoked by tamoxifen. In contrast, in the vagina progesterone had no effect on the weight, protein and DNA content, but progesterone did not block the agonistic effect provoked by tamoxifen. The situation was different when progesterone receptor was concerned. Tamoxifen in both tissues (particularly in the vagina) stimulated the progesterone receptor very significantly. Progesterone blocked the number of specific binding sites of progesterone and the stimulatory effect provoked by tamoxifen.

[Recognition of the activated form of estrogen receptor bound to estradiol and tamoxifen by a monoclonal antibody]. / Reconnaissance de la forme activée du récepteur oestrogénique lié à l'estradiol et au tamoxifène par un anticorps monoclonal.

Two forms (alpha and beta) of the estrogen receptor were differentiated in the cytosol fraction of fetal guinea pig uterus by using a monoclonal antibody (D 547). Only the alpha form is recognised by the monoclonal antibody, shifting its sedimentation coefficient in high salt sucrose gradients, from 4.5 S to 7.5 S. Dynamic studies of the translocation of the receptor to the nucleus show a correlation between the decrease of the alpha form in the cytosol and the increase of the nuclear receptor concentration, suggesting that the alpha form translocates to the nucleus. Several factors such as time, temperature and high salt concentrations provoke a transformation of the beta form initially found in the cytosol, to the alpha form and induce the activation of the receptor, determined by an increase in its binding to DNA-cellulose. Sodium molybdate inhibits the transformation to the alpha form and the receptor activation. These data suggest that the alpha form, which is recognised by the antibody, is the activated form of the receptor. On the other hand, the estrogen receptor bound to tamoxifen can be activated by time, temperature and high salt concentrations and the activated form of the tamoxifen-receptor complex is also recognised by the monoclonal antibody D 547. However, the interaction with the monoclonal antibody and the binding to DNA-cellulose are less than those of the estradiol-receptor complex. These data support the hypothesis that the action of tamoxifen is related to its interaction with the estrogen receptor.

Activation and immunorecognition by a monoclonal antibody of the tamoxifen-estrogen receptor complex.

The interaction of tamoxifen with the estrogen receptor of fetal guinea pig uterus, the activation of the tamoxifen-estrogen receptor complex and its immunorecognition by a monoclonal antibody raised against the human estrogen receptor is described in the present paper. The results show that: the tamoxifen-receptor complex sediments at 8 S in low-salt and at 4.5 S in high-salt sucrose gradients, this complex is partially recognized by the monoclonal antibody allowing the differentiation of two forms: the alpha form, which binds to the monoclonal antibody, and the beta form, which does not react with it; several factors such as time, temperature and high salt concentrations were capable of activating the tamoxifen-receptor complex, as determined by the increase of its binding to DNA-cellulose; these factors also induced a partial transformation of the beta form to the alpha form; sodium molybdate inhibited both activation and transformation of the beta into the alpha form. The correlation between activation and induction of the alpha form suggests that the monoclonal antibody recognizes selectively the activated form of the tamoxifen-receptor complex. These results indicate similar properties of the estrogen receptor when bound to either tamoxifen or estradiol; however, the differences observed in the behavior of the tamoxifen-receptor complex as compared with the estradiol-receptor complex, though quantitative rather than qualitative, suggest that the estrogen receptor is affected differently by tamoxifen and estradiol.

Estrogen and progesterone receptors in the fetal and newborn vagina of guinea pig: biological, morphological, and ultrastructural responses to tamoxifen and estradiol.

Estrogen and progesterone receptors were characterized in the fetal and newborn vagina of guinea pig. The concentrations of estrogen receptor (total sites, cytosol plus nuclei) which were very high [7090 +/- 1700 (+/- SD) fmol/mg DNA] in the fetal vagina (62-64 days gestation) decreased significantly after birth and increased slightly at 2 weeks of age. Tamoxifen (TAM) and estradiol (E2) or the combined treatment (TAM + E2) induced a great increase in weight and DNA content in both the fetal and newborn vagina. After a 12-day treatment to pregnant or newborn guinea pigs, the fetal vagina wet weights (milligrams +/- SD) were as follows: control animals, 87 +/- 14; +TAM, 254 +/- 38; +E2, 177 +/- 19; +(TAM+E2), 218 +/- 41. The values in newborn vaginas were: 155 +/- 40, 434 +/- 75, 477 +/- 49, and 512 +/- 76, respectively. The DNA contents per vagina (in micrograms per +/- SD) in the fetal tissues were as follows: control, 187 +/- 64; +TAM, 563 +/- 74; +E2, 650 +/- 100; and +(TAM+E2), 776 +/- 113; in the newborn vagina these values were: 592 +/- 75, 880 +/- 113; 781 +/- 75, and 901 +/- 15, respectively. Histological studies showed drastic morphological alterations after each of the treatments, particularly in the epithelial cells. Similarly, the ultrastructural examination with transmission electron microscopy showed the alteration of mitochondria, the development of the rough endoplasmic reticulum, and the formation of numerous vacuoles and secretory granules. TAM stimulated the number of specific sites for progesterone, but less intensely than did E2. However, in the combined experiment (TAM+E2), TAM did not block the action of E2. It is concluded that TAM acts in the fetal and newborn vagina of guinea pigs as a real estrogen agonist.

Biological responses of tamoxifen in the fetal and newborn vagina and uterus of the guinea-pig and in the R-27 mammary cancer cell line.

The biological and morphological responses of tamoxifen were studied in two models: the uterus and vagina of fetal and newborn guinea-pigs: R-27 cells--a mammary cancer cell line (tamoxifen resistant) derived from the MCF-7 cancer cell line. Tamoxifen (TAM) alone or in combination with estradiol (E2) was administered to pregnant (50-52 days of gestation) or to newborn (2-day-old) guinea-pigs for a long period (12 days). TAM alone produced a great trophic effect on the uterus and vagina which was markedly enhanced when TAM was administered together with E2. Histological studies showed that TAM provokes morphological changes in both the endometria and the myometria and this effect was also greater when TAM was administered together with E2. In the fetal uterus and vagina, the ultrastructural studies showed that TAM induces morphological alterations in different cytoplasmic organelles. This effect was much more intense in newborns where TAM provoked a significant vacuolization of the epithelial cells. Concerning progesterone receptor (PR) in the fetal or newborn tissues (uterus or vagina) TAM provoked a less intense effect than those provoked by E2, but TAM did not block the effect provoked by E2. It was observed that [3H]TAM binds specifically to the estrogen receptor (ER) of fetal guinea pig uterus and this complex is partially recognized by a monoclonal antibody which recognizes the activated form of this receptor, supporting the suggestion that the biological action of TAM is mediated by the ER. The biological and ultrastructural effects provoked by TAM (1 X 10(-6) M), estriol (E3)(5 X 10(-8) M) and the combination of TAM + E3 were studied in the R-27 mammary cancer cell line in culture. E3 stimulated the PR content by 7-10 times. However, TAM did not provoke a significant decrease in the concentration of PR, and in the mixture of TAM + E3 the concentration of PR was of the same order as that in E3 treatment. Ultrastructural observations indicate an intense concentration of ribosomes in the pericytoplasmic area after exposure to E3 and with exposure to TAM an increase in vacuoles and a significant enlargement of the size of the mitochondria were observed. It is concluded that TAM in the target tissues of fetal and newborn guinea pigs acts as a real estrogen and in the R-27 mammary cancer cell line TAM does not block the effect provoked by E3, however it does provoke intense ultrastructural modifications.