Expression Of Estrogen Target Genes Research Articles

Decoding the Therapeutic Implications of the ERα Stability and Subcellular Distribution in Breast Cancer.

Approximately 70% of all breast cancer cases are estrogen receptor-alpha positive (ERα+) and any ERα signaling pathways deregulation is critical for the progression of malignant mammary neoplasia. ERα acts as a transcription factor that promotes the expression of estrogen target genes associated with pro-tumor activity in breast cancer cells. Furthermore, ERα is also part of extranuclear signaling pathways related to endocrine resistance. The regulation of ERα subcellular distribution and protein stability is critical to regulate its functions and, consequently, influence the response to endocrine therapies and progression of this pathology. This minireview highlights studies that have deciphered the molecular mechanisms implicated in controlling ERα stability and nucleo-cytoplasmic transport. These mechanisms offer information about novel biomarkers, therapeutic targets, and promising strategies for breast cancer treatment.

Osteoporosis Due to Hormone Imbalance: An Overview of the Effects of Estrogen Deficiency and Glucocorticoid Overuse on Bone Turnover.

Osteoporosis is a serious health issue among aging postmenopausal women. The majority of postmenopausal women with osteoporosis have bone loss related to estrogen deficiency. The rapid bone loss results from an increase in bone turnover with an imbalance between bone resorption and bone formation. Osteoporosis can also result from excessive glucocorticoid usage, which induces bone demineralization with significant changes of spatial heterogeneities of bone at microscale, indicating potential risk of fracture. This review is a summary of current literature about the molecular mechanisms of actions, the risk factors, and treatment of estrogen deficiency related osteoporosis (EDOP) and glucocorticoid induced osteoporosis (GIOP). Estrogen binds with estrogen receptor to promote the expression of osteoprotegerin (OPG), and to suppress the action of nuclear factor-κβ ligand (RANKL), thus inhibiting osteoclast formation and bone resorptive activity. It can also activate Wnt/β-catenin signaling to increase osteogenesis, and upregulate BMP signaling to promote mesenchymal stem cell differentiation from pre-osteoblasts to osteoblasts, rather than adipocytes. The lack of estrogen will alter the expression of estrogen target genes, increasing the secretion of IL-1, IL-6, and tumor necrosis factor (TNF). On the other hand, excessive glucocorticoids interfere the canonical BMP pathway and inhibit Wnt protein production, causing mesenchymal progenitor cells to differentiate toward adipocytes rather than osteoblasts. It can also increase RANKL/OPG ratio to promote bone resorption by enhancing the maturation and activation of osteoclast. Moreover, excess glucocorticoids are associated with osteoblast and osteocyte apoptosis, resulting in declined bone formation. The main focuses of treatment for EDOP and GIOP are somewhat different. Avoiding excessive glucocorticoid use is mandatory in patients with GIOP. In contrast, appropriate estrogen supplement is deemed the primary treatment for females with EDOP of various causes. Other pharmacological treatments include bisphosphonate, teriparatide, and RANKL inhibitors. Nevertheless, more detailed actions of EDOP and GIOP along with the safety and effectiveness of medications for treating osteoporosis warrant further investigation.

Analysis of genomic and non-genomic signaling of estrogen receptor in PDX models of breast cancer treated with a combination of the PI3K inhibitor alpelisib (BYL719) and fulvestrant

BackgroundEndocrine therapies targeting estrogen signaling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signaling, estrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumor response in vivo.MethodsThe interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analyzed their anti-proliferative effects on 6 ERα+ and 3 ERα− PDX models. Genomic and non-genomic estrogen signaling were assessed by measuring ERα/PI3K interaction by PLA and the expression of estrogen target genes by RT-QPCR, respectively.ResultsWe confirmed that ERα/Src and ERα/PI3K interactions were associated with a trend to poorer survival, the latter displaying the most significant effects. In ERα+ tumors, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status, or ERα/PI3K targeting. Remarkably, resistance to fulvestrant was associated with non-genomic ERα signaling, since genomic degradation of ERα was unaltered in these tumors, whereas the treatment did not diminish the level of ERα/PI3K interaction. Interestingly, in 2 ERα− models, fulvestrant alone impacted tumor growth, and this was associated with a decrease in ERα/PI3K interaction.ConclusionsOur results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant in ERα+ tumors was associated with a lack of impairment of ERα/PI3K interaction in the cytoplasm. In addition, an efficient targeting of ERα/PI3K in ERα− tumors could constitute a promising therapeutic option.

Traditional Chinese medicine Dingkun Pill facilitates uterine receptivity for implantation in mice†.

Dingkun Pill (DK) is one of the representative traditional Chinese medicines, which has been used in the treatment of gynecological diseases for hundreds of years. Accumulative observations and evidence have shown the beneficial effects of DK, including enhancing the function of hypothalamic-pituitary-ovarian axis. However, the underlying mechanisms remain elusive. In this study, the effects of DK on uterine receptivity and implantation were explored by a series of studies with different mouse models. The results showed that DK can advance the time of implantation by influencing the expression of estrogen target genes to facilitate embryo implantation. DK was efficient to activate embryo implantation at the presence of suboptimal estrogen in delayed implantation mouse model. Our further study revealed that the improvement of DK on receptivity establishment is attributed to the differential regulation of DK on implantation-associated genes. This study provides previously unappreciated molecular mechanism of DK in embryo implantation and benefits the potential clinical application of DK in human reproduction improvement.

Role of SUMOylation in differential ER\u03b1 transcriptional repression by tamoxifen and fulvestrant in breast cancer cells

Antiestrogens (AEs) are widely used for treatment of estrogen receptor alpha (ERα)-positive breast cancer, but display variable degrees of partial agonism in estrogen target tissues and breast cancer (BC) cells. The fact that BC cells resistant to selective ER modulators (SERMs) like tamoxifen (Tam) can still be sensitive to pure AEs, also called selective ER downregulators, suggests different mechanisms of action, some of which may contribute to the more complete suppression of estrogen target genes by pure AEs. We report herein that pure AEs such as fulvestrant induce transient binding of ERα to DNA, followed by rapid release after 30–40 min without loss of nuclear localization. Loss of DNA binding preceded receptor degradation and was not prevented by proteasome inhibition. Chromatin was less accessible in the presence of fulvestrant than with estradiol or Tam as early as 20 min following treatment, suggesting that chromatin remodeling by pure AEs at ERα target regions prevents transcription in spite of receptor binding. SUMO2/3 marks were detected on chromatin at the peak of ERα binding in cells treated with pure AEs, but not SERMs. Furthermore, decreasing SUMOylation by overexpressing the deSUMOylase SENP1 significantly delayed receptor release from DNA and de-repressed expression of estrogen target genes in the presence of fulvestrant, both in ERα-expressing MCF-7 cells and in transiently transfected ER-negative SK-BR-3 cells. Finally, mutation V534E, identified in a breast metastasis resistant to hormonal therapies, prevented ERα modification and resulted in increased transcriptional activity of estrogen target genes in the presence of fulvestrant in SK-BR-3 cells. Together, our results establish a role for SUMOylation in achieving a more complete transcriptional shut-off of estrogen target genes by pure AEs vs. SERMs in BC cells.

Structurally Novel Antiestrogens Elicit Differential Responses from Constitutively Active Mutant Estrogen Receptors in Breast Cancer Cells and Tumors.

Many estrogen receptor α (ERα)-positive breast cancers develop resistance to endocrine therapy via mutation of ERs whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ERs, we describe here our development and characterization of three chemically novel AEs that effectively suppress proliferation of breast cancer cells and tumors. Our AEs are effective against wild-type and Y537S and D538G ERs, the two most commonly occurring constitutively active ERs. The three new AEs suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared with WT ER, mutants exhibited approximately 10- to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER-containing MCF7 and T47D cells revealed that AE responses were compound, cell-type, and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER-expressing tumor xenografts in NOD/SCID-γ mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ERs to different AEs and make clear the value of having a toolkit of AEs for treatment of endocrine therapy-resistant tumors driven by different constitutively active ERs. Cancer Res; 77(20); 5602-13. ©2017 AACR.

A deregulated expression of estrogen-target genes is associated with an altered response to estradiol in aged rats perinatally exposed to bisphenol A

Here we assessed the effects of perinatal exposure to bisphenol A (BPA) on the uterine response to 17β-estradiol (E2) in aged rats. Pregnant rats were orally exposed to 0.5 or 50 μg BPA/kg/day from gestational day 9 until weaning. On postnatal day (PND) 360, the rats were ovariectomized and treated with E2 for three months. The uterine tissue of BPA50 and BPA0.5 rats showed increased density of glands with squamous metaplasia (GSM) and glands with daughter glands respectively. Wnt7a expression was lower in GSM of BPA50 rats than in controls. The expression of estrogen receptor 1 (ESR1) and its 5′- untranslated exons ESR1-O and ESR1-OT was lower in BPA50 rats. Both doses of BPA modified the expression of coactivator proteins and epigenetic regulatory enzymes. Thus, perinatal BPA-exposed rats showed different glandular abnormalities associated with deregulated expression of E2-target genes. Different mechanisms would be involved depending on the BPA dose administered.

Effects of atrazine on estrogen receptor α- and G protein-coupled receptor 30-mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts.

Background: The pesticide atrazine does not bind to or activate the classical estrogen receptor (ER), but it up-regulates the aromatase activity in estrogen-sensitive tumor cells. The G protein estrogen receptor (GPR30/GPER) has been reported to be involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity.Objectives: We aimed to evaluate the potential of atrazine to trigger GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs).Methods and Results: Using gene reporter assays in diverse types of cancer cells, we found that atrazine did not transactivate endogenous ERα or chimeric proteins that encode the ERα and ERβ hormone binding domains. Conversely, atrazine was able to bind to GPER to induce ERK activation and the expression of estrogen target genes, which, interestingly, appeared to rely on both GPER and ERα expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells that depend on GPER and ERα, as evidenced by gene silencing experiments and the use of specific signaling inhibitors. Of note, through GPER, atrazine elicited ERK phosphorylation, gene expression, and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment.Conclusions: Our results suggest a novel mechanism through which atrazine may exert relevant biological effects in cancer cells and CAFs. On the basis of our data, atrazine should be included among the environmental contaminants that may elicit estrogenic activity through GPER-mediated signaling.Citation: Albanito L, Lappano R, Madeo A, Chimento A, Prossnitz ER, Capello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. 2015. Effects of atrazine on estrogen receptor α– and G protein–coupled receptor 30–mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts. Environ Health Perspect 123:493–499; http://dx.doi.org/10.1289/ehp.1408586

Thyroid Hormone Status Interferes with Estrogen Target Gene Expression in Breast Cancer Samples in Menopausal Women

We investigated thyroid hormone levels in menopausal BrC patients and verified the action of triiodothyronine on genes regulated by estrogen and by triiodothyronine itself in BrC tissues. We selected 15 postmenopausal BrC patients and a control group of 18 postmenopausal women without BrC. We measured serum TPO-AB, TSH, FT4, and estradiol, before and after surgery, and used immunohistochemistry to examine estrogen and progesterone receptors. BrC primary tissue cultures received the following treatments: ethanol, triiodothyronine, triiodothyronine plus 4-hydroxytamoxifen, 4-hydroxytamoxifen, estrogen, or estrogen plus 4-hydroxytamoxifen. Genes regulated by estrogen (TGFA, TGFB1, and PGR) and by triiodothyronine (TNFRSF9, BMP-6, and THRA) in vitro were evaluated. TSH levels in BrC patients did not differ from those of the control group (1.34 ± 0.60 versus 2.41 ± 1.10 μU/mL), but FT4 levels of BrC patients were statistically higher than controls (1.78 ± 0.20 versus 0.95 ± 0.16 ng/dL). TGFA was upregulated and downregulated after estrogen and triiodothyronine treatment, respectively. Triiodothyronine increased PGR expression; however 4-hydroxytamoxifen did not block triiodothyronine action on PGR expression. 4-Hydroxytamoxifen, alone or associated with triiodothyronine, modulated gene expression of TNFRSF9, BMP-6, and THRA, similar to triiodothyronine treatment. Thus, our work highlights the importance of thyroid hormone status evaluation and its ability to interfere with estrogen target gene expression in BrC samples in menopausal women.

Dichloromethane extracts of Sophora japonica L. stimulate osteoblast differentiation in mesenchymal stem cells

Sophora japonica L. fruit prevents bone loss by inhibiting osteoclast activity. We hypothesized that S japonica L. extracts could promote osteoblast differentiation. To test this hypothesis, we investigated the effect of S japonica L. on osteoblast differentiation and identified the bioactive compound(s) from S japonica L. The mature fruit of S japonica L. was partitioned with ethanol, hexane, dichloromethane (DCM), ethyl acetate, and butanol, and their effects were tested on osteoblast differentiation of C3H10T1/2 cells. DCM fractionated extracts were identified as the most osteogenic fractions. DCM fractionated extracts dose-dependently stimulated alkaline phosphatase activity and matrix mineralization. The DCM fractions also induced expression of osteoblast markers such as alkaline phosphatase, osterix, and osteocalcin in C3H10T1/2 and primary bone marrow cells. Genistein was found abundantly in the DCM fractions. Furthermore, the genistein and DCM fractions similarly modulated the expression of estrogen target genes and were both active in transfection assays that measured estrogen agonistic activity. Finally, pharmacological inhibition by treatment with an estrogen receptor antagonist or specific inhibition of gene expression by small interference RNAs targeted to estrogen receptor-β abolished the effects of the DCM extracts, further supporting the idea that the genistein in the DCM extracts mediated the pro-osteogenic effects. Taken together, we identified genistein as the key phytoestrogen responsible for the effects of S japonica L. on osteoblast differentiation.

TOP2β Is Essential for Ovarian Follicles That Are Hypersensitive to Chemotherapeutic Drugs

The mechanisms underlying chemotherapy-induced acceleration of ovarian insufficiency are not fully understood, particularly for ovarian granulosa cells (GCs). We used two widely used cancer chemotherapeutic reagents, bleomycin and VP-16, and an in vivo GC-specific DNA topoisomerase II-β (TOP2β) (Top2b) knockout mouse model to investigate the effects of chemotherapy-induced DNA damage on growing mouse follicles. Bleomycin and VP-16 caused massive double-strand DNA breaks in the GCs of growing follicles in a time-dependent manner as shown by DNA-damage checkpoint activation. This damage was associated with apoptotic GC death and resulted in follicle atresia and ovulation failure. However, FSH-regulated ovarian functions, including estrogen biosynthesis and estrogen target gene expression, were not significantly affected by these genotoxins. TOP2β, a target of several chemotherapeutic drugs including VP-16, was abundantly expressed in the GCs of growing follicles. GC-specific deletion of Top2b using Cyp19-Cre caused DNA damage accumulations in these cells, follicle atresia, and decreased ovulation in response to exogenous gonadotropins. The ovaries of Top2b conditional knockout mice were also more sensitive to low-dose genotoxin treatment than wild-type mice ovaries. Thus, our results indicate that GCs are hypersensitive to genotoxic chemotherapeutic drugs and can activate the canonical DNA-damage checkpoint and the p53-dependent apoptotic pathway in response to insults that damage DNA. We also newly identified TOP2β as a factor involved in regulating GC genomic integrity and follicle atresia. This study has clinical implications for ovarian functional defects both for premenopausal cancer survivors and healthy women.

Thyroid hormone status interferes with estrogen target gene expression in breast cancer samples of menopausal women

We investigated thyroid hormone levels in menopausal BrC patients, and verified the action of triiodothyronine (T3) on genes regulated by estrogen (E2) and by T3 itself in BrC tissues. We selected 15 postmenopausal BrC patients and a control group of 18 postmenopausal women without BrC. We measured serum thyroid peroxidase antibody, TSH, free thyroxine, and estradiol, before and after surgery, and used immunohistochemistry to examine E2 and progesterone receptors. BrC primary tissue cultures received the following treatments: ethanol, T3, T3+tamoxifen (TAM), TAM, E2, or E2+TAM. Genes regulated by E2 (TGFA, TGFB1, PGR) and by T3 (TNFRSF9, BMP‐6, THRA) in vitro were evaluated in each patient. Three BrC patients presented with clinical hyperthyroidism. Thyroxine levels of BrC patients were statistically higher than controls (1.78±0.20 vs. 0.95±0.16 ng/dl). TGFA and TGFB1 were upregulated and downregulated, respectively, after E2 and T3 treatment. T3 treatment increased PGR expression, however TAM did not block T3 action on PGR expression. TAM, alone or associated with T3, modulated gene expression of TNFRSF9, BMP‐6 and THRA, similar to T3 treatment, showing that TAM can interfere with gene expression modulated by T3. Our results reinforce that the thyroid hormone status of BrC patients can influence E2‐controlled processes, even after TAM intervention and/or in the absence of circulating E2 postmenopause.

Structure–activity relationships of resveratrol and derivatives in breast cancer cells

Resveratrol (RSV) is classified as a phytoestrogen due to its ability to interact with estrogen receptors (ERs). We assessed structure-activity relationships of RSV and the analogs 4,4'-dihydroxystilbene (4,4'-DHS), 3,5-dihydroxystilbene (3,5-DHS), 3,4'-dihydroxystilbene (3,4'-DHS), 4-hydroxystilbene (4-HS) using as model systems the ERalpha-positive and negative MCF7 and SkBr3 breast cancer cells, respectively. In binding assays and transfection experiments RSV and the analogs showed the following order of agonism for ERalpha: 3,4'-DHS > 4,4'-DHS > 4-HS > RSV, while 3,5-DHS did not elicit any ligand properties. Computational docking analysis and real-time PCR revealed for each analog a distinct ERalpha binding orientation and estrogen target gene expression profile. Interestingly, the aforementioned order of ligand activity was confirmed in proliferation assays which also showed the lack of growth stimulation by 3,5-DHS. Our data suggest that subtle changes in the structure of the RSV derivatives examined may be responsible for the different ERalpha-mediated biological responses observed in estrogen-sensitive cancer cells.

G-protein-coupled receptor 30 and estrogen receptor-alpha are involved in the proliferative effects induced by atrazine in ovarian cancer cells.

BackgroundAtrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17β-estradiol and environmental estrogens.ObjectivesGiven the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells.Methods and resultsAtrazine did not transactivate the endogenous ERα in different cancer cell contexts or chimeric proteins encoding the ERα and ERβ hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERα nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30–epidermal growth factor receptor transduction pathway and the involvement of ERα.ConclusionsOur results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action.

Gene expression and estrogen sensitivity in rat uterus after developmental exposure to the polybrominated diphenylether PBDE 99 and PCB

Considering the presence of polybrominated diphenylethers (PBDEs) in human milk and cord blood, and the estrogenic activity of some congeners, it is conceivable that PBDEs may interact with developing neuroendocrine systems. We investigated effects of 2,2′,4,4′,5-pentabromo-DE (PBDE 99), a major congener in human milk, on development of brain and reproductive organs, with focus on estrogen target gene expression. Time-pregnant Long Evans rats were subcutaneously injected with PBDE 99 (1 or 10 mg/kg/day), the PCB mixture Aroclor 1254 (10 mg/kg/day), known to interfere with sexual development, or vehicle, from gestational day (GD) 10 to GD 18. In female offspring, anogenital distance was unaffected by PBDE 99 but increased by Aroclor; puberty (vaginal opening) was not significantly changed. Adult PBDE 99-exposed offspring exhibited unchanged uterine weight but increased ovarian weight. Uterine mRNA levels of estrogen target genes were determined by real-time PCR. Progesterone receptor (PR) mRNA was down-regulated at both PBDE 99 doses, estrogen receptor alpha (ER alpha), ER beta and insulin-like growth factor-I (IGF-I) were up-regulated at the lower dose. Aroclor induced different effect patterns. In order to investigate possible changes in sensitivity of target genes to estrogen, some offspring were ovariectomized at 10 weeks of age, s.c. injected with estradiol-17β (E2, 10 μg/kg) or vehicle at 12 weeks, and sacrificed 6 h later. PBDE 99 dose-dependently reduced the magnitude of IGF-I mRNA induction by E2, and increased the magnitude of ER beta repression. PBDE 99 also influenced baseline levels of PR, IGF-I and ER beta mRNAs in ovariectomized, vehicle-injected controls. These data indicate that developmental exposure to PBDE 99 at doses devoid of general toxicity, affects the regulation of estrogen target genes in uterus. Since PBDE 99 was detected in blood and adipose tissue of adult offspring, these effects may result from interactions with developmental processes, adult functions, or a combination of both.

Transcription of the fish Latent TGFβ-binding protein gene is controlled by estrogen receptor α

In endocrine disruption a key role has been suggested for endocrine receptors, in particular the estrogen receptors (ERs), in the regulation by compounds mimicking natural hormones. The two ERs, ERα and ERβ are transcription factors involved in the regulated expression of estrogen target genes and have been shown to play an essential role in mammalian ovary development. A similar role is to be expected for ERs in fish; little is, however, known in fish about genes regulated by ERs. To begin to address this, we here report the identification and characterization of a novel gene regulated by the fish ERα in response to 17β-estradiol. This gene encodes a fish orthologue of the latent transforming growth factor beta binding protein 3 (LTBP-3) and was identified through a differential display approach from a rainbow trout gonad cell line (RTG-2-ERα). We show that the rainbow trout LTBP (rtLTBP-3) is ERα dependent and is upregulated 5-fold in response to 17β-estradiol addition. The rtLTBP shows 61% amino acid similarity to human LTBP-3 and 48%, 44% and 41% to LTBP-1, LTBP-2 and LTBP-4, respectively. The highly conserved TB2 domain of rtLTBP shows 87% and 66% identity to the TB domains of human LTBP-3 and LTBP-1, respectively. LTBP plays a pivotal role in TGFβ activation in mammals and the high degree of sequence similarity suggests a similar role in fish. This would represent a novel link between nuclear hormone receptors and growth factor (TGFβ) mediated developmental processes, and show new aspects of the role of hormones in developmental biology and endocrine disruption.

Estrogen Target Gene Regulation and Coactivator Expression in Rat Uterus after Developmental Exposure to the Ultraviolet Filter 4-Methylbenzylidene Camphor

Because the estrogen receptor (ER) ligand type influences transactivation, it is important to obtain information on molecular actions of nonclassical ER agonists. UV filters from cosmetics represent new classes of endocrine active chemicals, including the preferential ER beta ligands 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor. We studied estrogen target gene expression in uterus of Long Evans rats after developmental exposure to 4-MBC (0.7, 7, 24, and 47 mg/kg x d) administered in feed to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. 4-MBC altered steady-state levels of mRNAs encoding for ER alpha, ER beta, progesterone receptor (PR), IGF-I, androgen receptor, determined by real-time RT-PCR in uterus of 12-wk-old offspring. Western-blot analyses of the same tissue homogenates indicated changes in ER alpha and PR but not ER beta proteins. To assess sensitivity to estradiol (E2), offspring were ovariectomized on d 70, injected with E2 (10 or 50 microg/kg sc) on d 84, and killed 6 h later. Acute up-regulation of PR and IGF-I and down-regulation of ER alpha and androgen receptor by E2 were dose-dependently reduced in 4-MBC-exposed rats. The reduced response to E2 was accompanied by reduced coactivator SRC-1 mRNA and protein levels. Our data indicate that developmental exposure to 4-MBC affects the regulation of estrogen target genes and the expression of nuclear receptor coregulators in uterus at mRNA and protein levels.

Assessing the estrogenicity of environmental chemicals with a stably transfected lactoferrin gene promoter reporter in HeLa cells

Lactoferrin is an important marker protein of the estrogens. In mice, lactoferrin expression is stimulated in the uterus by ligand-bound estrogen receptors (ERs). With this study we aimed to evaluate the effect of different environmental estrogenic chemicals on the mouse lactoferrin gene expression in a cell-based assay. We constructed a reporter that contains the firefly luciferase gene under hormone-inducible control of a 1.1 kbp fragment of the mouse lactoferrin gene promoter. In an attempt to study the promoter regulation in a chromatin context, we stably transfected the construct (pGL3-mLF-Luc) into HeLa cells, and a stable clone (HeLa-mLF-Luc) incorporating the construct was subsequently generated. Transient transfection of HeLa-mLF-Luc cells with ERα and ERβ expression plasmids showed that both 17β-estradiol (E2) and diethylstilbestrol (DES) at 10 −7 M significantly increased luciferase expression via ERα and ERβ. Xenoestrogens such as bisphenol A, 4-octylphenol, 4-nonylphenol and the phytoestrogen genistein when used at increasing concentrations (10 −8 to 10 −5 M) revealed varying magnitudes of activation (1.96–8-fold). The environmental estrogens showed similar magnitudes of luciferase induction when acting through ERα and ERβ-mediated pathways. Also, in the absence of ERs, the xenoestrogens could not induce luciferase expression thereby reflecting receptor dependency. Taken together, the results indicate a significant responsiveness of the stably transfected mouse lactoferrin promoter to endogenous estrogen and environmental estrogenic compounds through ERs. This cell-based transcription assay system may be useful in understanding the susceptibility of estrogen target gene expression by these chemicals at the chromatin level.

Transcription profiling of estrogen target genes in young and old mouse uterus

The goal of this study was to identify age-related changes in the expression of estrogen target genes in mouse uterus. We developed a novel ‘estrogen response element (ERE) Chip’ microarray bearing 297 genes including both known estrogen target genes and genes identified by searching the mouse genome database to have EREs, AP-1 sites, and Sp1 sites, all targets of estrogen receptor (ER) regulation. 400–500 bp PCR products of these 297 genes were printed onto nylon membranes creating the ‘ERE Chip’ microarray. This microarray is unique because it is the first estrogen-responsive gene-specific microarray to identify changes in uterine gene expression in young versus old mice. Using this ERE microarray we identified 10 uterine genes whose expression was up-regulated in old mice, e.g. β-actin, calcium binding protein 45a, Sp1, and COUP-TFII. In contrast, the expression of only 4 uterine genes, i.e., complement C3, lactoferrin, Muc-1, and 17-beta-hydroxysteroid dehydrogenase 8 (H2-Ke6) was down-regulated in old mice. These changes may reflect an increase in stromal and a decrease in glandular epithelial gene expression, and may be associated with age-related changes in these tissue compartments within the uterus, possibly leading to the decline in reproductive function in C57Bl/6 mice.

Signal cross-talk between estrogen receptor alpha and beta and the peroxisome proliferator-activated receptor gamma1 in MDA-MB-231 and MCF-7 breast cancer cells

We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) is expressed and transcriptionally responsive to both synthetic and natural ligands in a variety of human breast cancer cells. We also observed significant differences in basal and ligand-mediated transactivation of PPARγ in cells with variable expression of the estrogen receptor. While previous reports indicate that PPARγ can mediate the expression of estrogen target genes, no data have suggested that estrogen receptor (ER) expression can alter the transcriptional regulation of PPARγ target gene expression. Here we have demonstrated that the expression of either ERα or β, but not the androgen or aryl hydrocarbon receptors, lowers both basal and stimulated PPARγ-mediated reporter activity. Interestingly, the presence of an ER antagonist does not inhibit this response while estradiol treatment further inhibits the ligand-stimulated transactivation of PPARγ in cells expressing ERα but not ERβ. Cells transfected with ERα deletion mutants demonstrate that the DNA binding domain of the ER is required to repress PPAR transactivation in these cells. Finally, using RNase protection assays we show that the inhibition of PPAR function is not due to a decrease in the expression of PPARγ. These data suggest that signal cross talk exists bidirectionally between PPARγ and ER in breast cancer cells.