Structurally similar estradiol analogs uniquely alter the regulation of intracellular signaling pathways (original) (raw)
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Journal of Biological Chemistry, 2006
Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/ c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ER␣, a mutant ER␣ (E207A/G208A) that selectively lacks ERE binding, or ER␣ null. In the compound heterozygote (AA/؊) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17-estradiol (E 2). The uterine epithelial proliferative response to E 2 and 4-hydroxytamoxifen was retained in the AA/؊ females, and uterine luminal epithelial height increased commensurate with the extent of ER␣ signaling. This proliferative response was confirmed by 5-bromo-2-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathwayresponsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ER␣ (E207A/G208A) mutant. These results indicate that nonclassical ER␣ signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ER␣ plays a critical physiologic role in the uterine response to estrogen.
PLOS One, 2009
Estrogens produce biological effects by interacting with two estrogen receptors, ERa and ERb. Drugs that selectively target ERa or ERb might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERb-selective compounds have been identified. One class of ERb-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERb much greater than ERa. A second class of ERb-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERb. Diarylpropionitrile represents a third class of ERb-selective compounds because its selectivity is due to a combination of greater binding to ERb and transcriptional activity. However, it is unclear if these three classes of ERb-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERb selectivity and pattern of gene expression of these three classes of ERb-selective compounds compared to estradiol (E 2 ), which is a non-selective ER agonist. U2OS cells stably transfected with ERa or ERb were treated with E 2 or the ERb-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERb-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERb, which is consistent with the finding that most genes regulated by the ERb-selective compounds were similar to each other and E 2 . However, there were some classes of genes differentially regulated by the ERb agonists and E 2 . Two ERb-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERb. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERb-selective agonists and E 2 , there were some genes regulated that were distinct from each other and E 2 , suggesting that different ERb-selective agonists might produce distinct biological and clinical effects.
PLOS One, 2009
Estrogens produce biological effects by interacting with two estrogen receptors, ERa and ERb. Drugs that selectively target ERa or ERb might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERb-selective compounds have been identified. One class of ERb-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERb much greater than ERa. A second class of ERb-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERb. Diarylpropionitrile represents a third class of ERb-selective compounds because its selectivity is due to a combination of greater binding to ERb and transcriptional activity. However, it is unclear if these three classes of ERb-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERb selectivity and pattern of gene expression of these three classes of ERb-selective compounds compared to estradiol (E 2 ), which is a non-selective ER agonist. U2OS cells stably transfected with ERa or ERb were treated with E 2 or the ERb-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERb-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERb, which is consistent with the finding that most genes regulated by the ERb-selective compounds were similar to each other and E 2 . However, there were some classes of genes differentially regulated by the ERb agonists and E 2 . Two ERb-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERb. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERb-selective agonists and E 2 , there were some genes regulated that were distinct from each other and E 2 , suggesting that different ERb-selective agonists might produce distinct biological and clinical effects.
The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling
Journal of Biological Chemistry, 2001
The steroid hormone 17-estradiol (E 2 ) is a key regulator of growth, differentiation, and function in a wide array of target tissues, including the male and female reproductive tracts, mammary gland, and skeletal and cardiovascular systems. The predominant biological effects of E 2 are mediated through two distinct intracellular receptors, ER␣ 1 and ER, each encoded by unique genes (1) but possessing the hallmark modular structure of functional domains characteristic of the steroid/thyroid hormone superfamily of nuclear receptors (introduced in the Minireview Prologue (54)). Certain functional domains of the ER␣ and ER exhibit a high degree of homology, namely the DNA-and ligand-binding domains, at 97 and 60%, respectively, whereas considerable divergence is apparent in the N terminus (18% homology). Hence, ER␣ and ER interact with identical DNA response elements and exhibit a similar binding affinity profile for an array of endogenous, synthetic, and naturally occurring estrogens when assayed in vitro (2). In vitro studies also suggest the two receptors may play redundant roles in estrogen signaling; however, tissue localization studies have revealed distinct expression patterns for each receptor that suggest otherwise. Whereas ER␣ is the predominant subtype expressed in the breast, uterus, cervix, vagina, and several additional target organs, ER exhibits a more limited expression pattern and is primarily detected in the ovary, prostate, testis, spleen, lung, hypothalamus, and thymus (3). Regional expression differences of the two receptors have been identified in the brain (4). Further evidence of distinct biological functions for the ERs is revealed by the contrasting phenotypes observed in the individual lines of ER knockout mice, the ␣ERKO and ERKO, which exhibit phenotypes that generally mirror the respective ER expression patterns (5). The most striking phenotypes in the female ␣ERKO mice include estrogen insensitivity (leading to hypoplasia) in the reproductive tract, hypergonadotropic hypergonadism, lack of pubertal mammary gland development, and excess adipose tissue, whereas in the male, testicular degeneration and epididymal dysfunction are major factors (5). These phenotypes combined with severe deficits in sexual behavior result in complete infertility in both sexes of the ␣ERKO. In contrast, ERKO males are fertile and to date show no obvious phenotypes; however, ERKO females exhibit inefficient ovarian function and subfertility. Interestingly, compound knockout mice (␣ERKO) exhibit phenotypes that most heavily resemble those of the ␣ERKO, with the exception of the ovarian phenotype, characterized by progressive germ cell loss accompanied by redifferentiation of the surrounding somatic cells, suggesting a requisite role for both ER forms in this tissue .
Toxicological Sciences, 2008
We have determined the gene expression profile induced by 17 a-ethynyl estradiol (EE) in Ishikawa cells, a human uterinederived estrogen-sensitive cell line, at various doses (1pM, 100pM, 10nM, and 1mM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose-and timedependent manner (p £ 0.0001). The annotation available for the genes affected indicates that EE exposure results in changes in multiple molecular pathways affecting various biological processes, particularly associated with development, morphogenesis, organogenesis, cell proliferation, cell organization, and biogenesis. All of these processes are also affected by estrogen exposure in the uterus of the rat. Comparison of the response to EE in both the rat uterus and the Ishikawa cells showed that 71 genes are regulated in a similar manner in vivo as well as in vitro. Further, some of the genes that show a robust response to estrogen exposure in Ishikawa cells are well known to be estrogen responsive, in various in vivo studies, such as PGR, MMP7, IGFBP3, IGFBP5, SOX4, MYC, EGR1, FOS, CKB, and CCND2, among others. These results indicate that transcript profiling can serve as a viable tool to select reliable in vitro systems to evaluate potential estrogenic activities of target chemicals and to identify genes that are relevant for the estrogen response.
Steroids, 2000
In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [ 3 H]estradiol (E 2 ). We show here that this property is not restricted to this antiestrogen. [ 3 H]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 [ 3 H]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 [ 3 H]E 2 or [ 3 H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E 2 -induced down regulation of ER, failed to stabilize [ 3 H]E 2 binding (whole cell assay) after an [ 3 H]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.
2003
We have advanced the view that estrogens activate the estrogen receptor (ER) ␣ complex differently. A group of planar (estradiol, genistein, and coumestrol) and nonplanar (methoxychlor and its mono-and didemethylated phenolic metabolites) environmental estrogens, which are all full estrogens in MCF-7 breast cancer cell proliferation assays, was shown to segregate discretely into planar and nonplanar groups. These groups were delineated using a novel assay of mutant ER cDNAs stably transfected into MDA-MB-231 cells and the activation of the transforming growth factor ␣ target gene in situ that putatively describes the external shape of the ER complex. Planar compounds activate estrogen action through the two traditional activation functions (AFs), AF1 and AF2, in the ER. In contrast, nonplanar compounds can activate estrogen action through AF1 and the amino acids Asp-351 and Asp-538, which are exposed when helix 12 silences AF2. The observation that class I (planar) and class II (nonplanar) compounds have different mechanisms of estrogen action may have important implications for tissue selective modulation of the ER.
The Journal of Steroid Biochemistry and Molecular Biology, 1997
Estrogen (E) inhibits the growth of both non-tumorigenic, immortal human mammary epithelial cells (HMEC) and breast cancer cells which stably express exogenous estrogen receptors (ER). The anti-estrogenic compounds 4-hydroxy-tamoxifen (HT) and ICI 164384 (ICI) have different effects on the growth of the ER-~transfectants. HT is a potent growth inhibitor, while ICI has no effect by itself but is able to block the anti-proliferative effects of E and HT. In order to elucidate the mechanism by which E or HT-bound ER inhibit cell growth, we have evaluated the effects of these compounds on the growth of HMEC stably expressing ER with mutations or deletions in the N-terminal A/B domain, the DNA-binding domain (DBD), and the C-terminal ligand-binding domain. These studies revealed that E and HT require different structural domains of the ER for their anti-proliferative activities. The N-teriiiinal A/B domain is required for HT-, but not E-dependent growth inhibition. The DNA-binding domain of the ER is not essential for HT-mediated anti-prollferative effects, but is important for E-dependent activity. The effect of ER mutations on the ligand-inducible expression of the endogenous progesterone receptor (PR) and pS2 genes was also evaluated. Neither gene was induced in the cells containing the ER mutated in the DBD, even though cell growth was inhibited. These results suggest that E and HT use different pathways to elicit their anti-proliferative effects and that this occurs via modulation of genes that are controlled by mechanisms different from those important for activation of the PR and pS2 genes.