Richard J. Miksicek

Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.

The estrogen receptor (ER) can be activated as a transcription factor either by binding of cognate estrogenic ligand or, indirectly, by a variety of other extracellular signals. As a first step towards elucidating the mechanism of ‘steroid‐independent activation’ of the ER by the epidermal growth factor (EGF), we have mapped the ER target domain and determined the signaling pathway. We show that the N‐terminal transcriptional activation function AF‐1, but not the C‐terminal AF‐2, is necessary for the EGF response. Both the EGF‐induced hyperphosphorylation and the transcriptional activation of the unliganded ER depend on a phosphorylatable serine residue at position 118. However, its phosphorylation is not sufficient and, hence, there must be other target domains or proteins which fulfill an additional requirement for EGF signaling through the ER. Using dominant‐negative Ras and MAP kinase kinase (MAPK kinase) and constitutively active MAPK kinase mutants, we show that EGF activates the ER by signaling through the MAPK pathway suggesting that MAPK directly phosphorylates the critical serine 118. Our results also imply that the steroid‐independent activation of a variety of ER mutants, which arise during the malignant progression of breast tumors, may contribute to tamoxifen resistance.

Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene

Two glucocorticoid response elements (GREs) located 2.5 kb upstream of the transcription initiation site of the tyrosine aminotransferase gene were identified by gene transfer experiments and shown to bind to purified glucocorticoid receptor. Although the proximal GRE has no inherent capacity by itself to stimulate transcription, when present in conjunction with the distal GRE, this element synergistically enhances glucocorticoid induction of gene expression. Cooperativity of the two GREs is maintained when they are transposed upstream of a heterologous promoter. An oligonucleotide of 22 bp representing the distal GRE is sufficient to confer glucocorticoid inducibility. As evidenced by the mapping of DNAase I hypersensitive sites, local alterations in the structure of chromatin at the GREs take place as a consequence of hormonal treatment.

Interaction of Naturally Occurring Nonsteroidal Estrogens with Expressed Recombinant Human Estrogen Receptor

The interaction between the recombinant human estrogen receptor and a variety of nonsteroidal estrogens was studied using a transient transfection assay in mammalian cells. Eight naturally occurring compounds were confirmed to stimulate the transcriptional activity of the human estrogen receptor and to compete for the binding of radiolabeled 17 beta-estradiol to this protein. In order of biological potency, these were zearalenone, beta-zearalenol, coumestrol, genistein, daidzein, phloretin, formononetin, and biochanin A. As with steroidal estrogens, the hormonal activity of these compounds was specific for the estrogen receptor and sensitive to inhibition by 4-hydroxytamoxifen and ICI-164,384. Evidence is also presented to indicate that the stimulatory activity of genistein is unrelated to the protein tyrosine kinase inhibitory activity of this isoflavone. These results demonstrate that a significant number of structurally diverse plant and fungal secondary metabolites exist in nature that may contribute to the total estrogen exposure of the human population.

ESTROGENIC FLAVONOIDS : STRUCTURAL REQUIREMENTS FOR BIOLOGICAL ACTIVITY

Abstract A systematic survey of polycyclic phenols has been performed to identify members of this chemical group with estrogenic activity. Twelve compounds were found to be able to stimulate the transcriptional activity of the human estrogen receptor expressed in cultured cells by transient transfection. These natural estrogens belong to several distinct, but chemically related classes including chalcones, flavanones, flavones, flavonols, and isoflavones. Selected examples of estrogenic flavonoids were further analyzed to determine their biological potencies and their relative affinities for binding to the estrogen receptor. These data are interpreted with respect to the molecular structure of polycyclic phenols required for hormonal activity as nonsteroidal estrogens.

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk‐promoter or the MMTV‐promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone‐responsive element in mammary cells.

Glucocorticoid responsiveness of the transcriptional enhancer of Moloney Murine Sarcoma Virus

The long terminal repeat of Moloney Murine Sarcoma Virus (MoMSV) contains an imperfect direct repeat that serves as a strong transcriptional enhancer. The strength of the MoMSV enhancer is strongly dependent on the presence of glucocorticoid hormone. Mapping studies in combination with DNAase I footprinting experiments define the presence of glucocorticoid regulatory elements at the promoter-proximal ends of each enhancer repeat. These elements behave like inducible enhancers: their regulatory activity is independent of position and orientation when they are linked in cis to a heterologous promoter. These data demonstrate the modular nature of the MoMSV enhancer and identify the glucocorticoid receptor as one of the trans-acting factors that interact with the MoMSV enhancer and mediate its transcriptional activity.

Interaction of the TGGCA-binding protein with upstream sequences is required for efficient transcription of mouse mammary tumor virus

A high‐affinity binding site for the TGGCA‐binding protein, also known as nuclear factor I, has previously been shown to reside within the mouse mammary tumor virus (MMTV) long terminal repeat. We have introduced mutations into this binding site to test the importance of this ubiquitous nuclear protein in MMTV transcription. Mutations which abolish the binding of the TGGCA protein in vitro are shown to impair strongly glucocorticoid‐induced transcription from this promoter in vivo. These data demonstrate that the TGGCA‐binding protein is a multifunctional DNA‐binding protein, capable of serving a transcriptional role in the case of MMTV, in addition to its known involvement in the replication of adenovirus.

DNA Binding and Transactivation Characteristics of the Mosquito Ecdysone Receptor-Ultraspiracle Complex

The steroid hormone 20-hydroxyecdysone is a key regulatory factor, controlling blood-meal triggered egg maturation in mosquitoes. To elucidate the ecdysone hierarchy governing this event, we cloned and characterized the ecdysone receptor (AaEcR) and the nuclear receptorUltraspiracle (AaUSP), a retinoid X receptor homologue, from the mosquito, Aedes aegypti, which form a functional complex capable of ligand and DNA binding. Here we analyzed the DNA-binding properties of the AaEcR·AaUSP heterodimer with respect to the effects of nucleotide sequence, orientation, and spacing between half-sites in natural Drosophila and synthetic ecdysone response element (EcREs). By using an electrophoretic gel mobility shift assay, we showed that AaEcR·AaUSP exhibits a broad binding specificity, forming complexes with inverted (IR) and direct (DR) repeats of the nuclear receptor response element half-site consensus sequence AGGTCA separated by spacers of variable length. A single nucleotide spacer was optimal for both imperfect (IRhsp-1) and perfect (IRper-1) inverted repeats; adding or removing 1 base pair in an IRhsp-1 spacer practically abolished binding. However, changing the half-site to the consensus sequence AGGTCA (IRper-1) increased binding of AaEcR·AaUSP 10-fold over IRhsp-1 and, at the same time, reduced the stringency of the spacer length requirement, with IRper-0 to IRper-5 showing detectable binding. Spacer length was less important in DRs of AGGTCA (DR-0 to DR-5); although 4 bp was optimal, DR-3 and DR-5 bound AaEcR·AaUSP almost as efficiently as DR-4. Furthermore, AaEcR·AaUSP also bound DRs separated by 11–13 nucleotide spacers. Competition experiments and direct estimation of binding affinity (K d ) indicated that, given identical consensus half-sites and an optimal spacer, the AaEcR·AaUSP heterodimer bound an IR with higher affinity than a DR. Co-transfection assays utilizing CV-1 cells demonstrated that the mosquito EcR·USP heterodimer is capable of transactivating reporter constructs containing either IR-1 or DR-4. The levels of transactivation are correlated with the respective binding affinities of the response elements (IRper-1 > DR-4 > IRhsp-1). Taken together, these analyses predict broad variability in the EcREs of mosquito ecdysone-responsive genes.

Leiomyoma primary cultures have elevated transcriptional response to estrogen compared with autologous myometrial cultures.

Objective: We tested the hypothesis that uterine leiomyomas are hypersensitive to estrogen as compared with autologous human myometrium. Methods: The estrogen-induced transcriptional responses of uterine leiomyoma and myometrial primary cultures were determined by transient expression assays. The relative levels of estrogen receptor (ER) in myometrial and leiomyoma tissues were determined by immunoblot. Results: Myometrial and leiomyoma primary cultures were transcriptionally responsive to the estrogen ethinly estradiol (eE2). The partial agonist tamoxifen did not elicit a positive transcriptional response and antagonized estrogen-induced transciption in the cultured cells. The responses of hormone-treated leiomyoma cells averaged 4.5-fold higher than those in controls with no hormone (P = .0001). The myometrical cells from women in the follicular phase exhibited little if any transcriptional response to eE2, whereas myometrial cells from women in the luteal phase had a transcriptional response to eE2 averaging threefold higher than that in no-hormone controls (P = .0083). Differences in response between autologous myometrial and leiomyoma cultures were statistically significant by the two-tailed Wilcoxon paired nonparametric signed-rank test (n = 11; P = .0137). These differences were more pronounced in cultures from women in the follicular or early luteal phase. In addition, the levels of ER increased in follicular and early luteal phase myometrial tissues, which correlated well with the number of days from the last menstrual period (n = 8; r = 0.9046; P = .002). Estrogen-receptor levels in myometrial tissues decreased during the late leuteal phase. Levels in leiomyoma tissues did not follow the same pattern as in the myometrium and were elevated in tissues taken from women in the follicular phase. Conclusions: Autologous leiomyoma cultures have a significantly higher response to estrogen than do matched myometrial cultures, especially if the cultures are derived from the follicular phase. The levels of ER in leiomyoma tissue from women in the follicular phase are significantly elevated.

Exon skipping gives rise to alternatively spliced forms of the estrogen receptor in breast tumor cells.

SummaryWe have previously described three messenger RNA variants coding for the human estrogen receptor (ER) [1]. These variants were identified using the polymerase chain reaction to perform directed cloning of ER cDNAs synthesized from polyadenylated RNA extracted from the human breast cancer cell line T47D. Each of the variants is characterized by the precise deletion of a single exon within the protein coding region of this message and was presumably derived by inaccurate or promiscuous splicing of primary estrogen receptor transcripts. We report here the results of RNAse protection experiments which independently confirm the existence of these splicing variants in T47D cells. Similar analysis of RNA from MCF-7 cells also revealed the presence of variant ER transcripts, suggesting that they may be a common finding in tumor cell lines which express the estrogen receptor. However, attempts to identify splicing variants in a number of nominally ER-negative cell lines using either RNAse protection or PCR amplification were without success.