Cells Express Estrogen Receptors Research Articles

The Female Preponderance to Cough Hypersensitivity Syndrome: Another Clue Pointing to the Role of TRPV1 in Cough

In a recently published study by Morice et al. [1] cough hypersensitivity syndrome (CHS) is put forward as a distinct disease entity, an exciting concept. Within their definition, the lion’s share of patients referred to a specialized cough clinic would fit the diagnosis of CHS (as assessed by the newly presented Hull Airway Reflux Questionnaire [1]). This single clinical entity includes several phenotypes like rhinitis-dominant CHS or the predominantly Th2 response CHS (also known as cough-variant asthma). However, the hypothesis is that a single precipitant predisposes these patients to cough. The authors speculate that this is a gaseous nonacid reflux that has been undetectable thus far with the currently available techniques. An alternative (or complementary) predisposing factor one could think of is upregulation of the receptor-channel transient receptor potential vanilloid (TRPV) type 1. Morice et al. have done a vast amount of work exploring the role of this ion channel in chronic cough and so have others [2–4]. This channel has gained popularity as a “cough-mediating receptor” since it triggers afferent nerve activity in response to cough-provoking stimuli, including heat, acid, and certain arachidonic acid derivatives. The channel is also indirectly activated by inflammatory mediators like bradykinin, nerve growth factor, and prostaglandin [5]. Furthermore, TRPV1 expression is increased in chronic coughers and the channel is upregulated in reaction to inflammation [3, 4]. From the data presented in Morice et al., I think another clue to the involvement of TRPV1 might be derived. As shown before by others [6, 7], the study by Morice et al. shows a female preponderance for developing CHS. Hitherto this female predilection to CHS has not been elucidated. However, the literature offers the ingredients for a plausible hypothesis which is in line with a role for TRPV1 in CHS. A female predominance for afflictions involving ion channels is not unusual in nature. The influence of sex hormones on ion channels has long been recognized, explaining many pathogenic differences between men and women. For example, women are at greater risk for drug-induced cardiac arrhythmias, and the female gender itself is an independent risk factor for sudden death. This is, at least in part, explained by an effect of estrogen on mRNA levels of several potassium and calcium channels (reviewed in [8]). Estrogen also influences TRPV1 activation/sensitization. Although some controversy exists, most studies describe a gain of function. Not surprisingly, most reports on this subject stem from the field of gynaecology and describe the importance of the channel in activating afferent nerve fibers in gynecologic pain. Yan et al. [9] show that in ovariectomized rats TRPV1 becomes important only in activating afferent nerve fibers in response to uterine cervical distension after estrogen replacement therapy. Peng et al. [10] show that the sensitization of TRPV1 as a nociceptive activator of C fibers in rats is dependent on the estrous cycle. Moreover, they demonstrated that capsaicin-induced activation of C fibers is significantly greater in the proestrus stage (high estradiol, low progesterone) than in the metestrus stage (low estradiol, high progesterone), favoring estradiol over progesterone as a hormonal mediator of TRPV1. Kiasalari et al. [11] identified a population of C-fiber cells that express high levels of TRPV1 and more than 75% of these cells express estrogen receptor α, suggesting a role for estrogen in regulating these cells. From the above, it is hypothesized that the influence of estrogen on TRPV1 predisposes the female sex to CHS, thus explaining the higher prevalence of CHS among women. This association would strengthen the hypothesis that TRPV1 is important in CHS.

Gene Alterations of Ovarian Cancer Cells Expressing Estrogen Receptors by Estrogen and Bisphenol A Using Microarray Analysis

Since endocrine disrupting chemicals (EDCs) may interfere with the endocrine system(s) of our body and have an estrogenicity, we evaluated the effect(s) of bisphenol A (BPA) on the transcriptional levels of altered genes in estrogen receptor (ER)-positive BG-1 ovarian cancer cells by microarray and real-time polymerase-chain reaction. In this study, treatment with 17β-estradiol (E2) or BPA increased mRNA levels of E2-responsive genes related to apoptosis, cancer and cell cycle, signal transduction and nucleic acid binding etc. In parallel with their microarray data, the mRNA levels of some altered genes including RAB31_MEMBER RAS ONCOGENE FAMILY (U59877), CYCLIN D1 (X59798), CYCLIN-DEPENDENT KINASE 4 (U37022), IGF-BINDING PROTEIN 4 (U20982), and ANTI-MULLERIAN HORMONE (NM_000479) were significantly induced by E2 or BPA in this cell model. These results indicate that BPA in parallel with E2 induced the transcriptional levels of E2-responsive genes in an estrogen receptor (ER)-positive BG-1 cells. In conclusion, these microarray and real-time polymerase-chain reaction results indicate that BPA, a potential weak estrogen, may have estrogenic effect by regulating E2-responsive genes in ER-positive BG-1 cells and BG-1 cells would be the best in vitro model to detect these estrogenic EDCs.

The role of kisspeptin and gonadotropin inhibitory hormone (GnIH) in the seasonality of reproduction in sheep

Sheep are seasonal breeders and reproductive status is controlled by photoperiod. Recent recognition of the significant role for kisspeptin and gonadotropin inhibitory hormone (GnIH) in the regulation of gonadotropin releasing hormone (GnRH) cells has provided a new perspective in the seasonal regulation of reproductive activity. Virtually all kisspeptin cells express estrogen receptors and kisspeptin is a potent stimulator of GnRH secretion. Thus, kisspeptin cells provide a conduit by which changes in estrogen feedback effects may be exerted upon GnRH cells. Changes in the activity of kisspeptin cells with season indicate a major role in the seasonal changes in reproductive activity in the ewe. GnIH is an inhibitor of reproductive function and there is mounting evidence that changing activity of this system is also an important determinant of reproductive status. Reciprocal changes in kisspeptin and GnIH activity explain seasonal changes in the function of GnRH cells.

Inflammatory pseudotumor-like follicular dendritic cell tumor of the liver with expression of estrogen receptor suggests a pathogenic mechanism: a case report and review of the literature

The term inflammatory pseudotumor-like follicular dendritic cell tumor has been suggested for a follicular dendritic cell tumor of the liver associated with a dense inflammatory infiltrate and Epstein–Barr virus infection. This tumor is less aggressive than usual intraabdominal follicular dendritic cell sarcoma and shows a strong female predominance; however, the pathogenesis is uncertain. We report the case of a 59-year-old woman who presented with a 6-cm mass in the liver. Histological examination showed spindle cells with a storiform pattern and prominent inflammatory infiltrate mimicking inflammatory pseudotumor. Spindle cells expressed follicular dendritic cell markers CD21 and CD35, and were positive for Epstein–Barr virus-encoded RNA and LMP-1 protein. In addition, neoplastic cells expressed estrogen receptor (ER). Fifteen cases of inflammatory pseudotumor-like follicular dendritic cell tumor of the liver including the case we describe here have been reported in the literature. A review of the literature disclosed that normal follicular dendritic cells may express ER; however, extensive testing has not been previously reported in follicular dendritic cell sarcoma. The female predominance and the expression of ER in the case we report are suggestive of a hormonal role in the pathogenesis and therapy of inflammatory pseudotumor-like follicular dendritic cell tumor of the liver.

Estradiol-17β Stimulates Specific Receptor and Endogenous Nitric Oxide-Dependent Dynamic Endothelial Protein S-Nitrosylation: Analysis of Endothelial Nitrosyl-Proteome

Covalent adduction of a nitrosyl group to cysteines [S-nitrosylation (S-NO)] is emerging as a key route for nitric oxide (NO) to directly modulate protein functions. Here, we studied the effects of estrogens on endothelial protein S-NO and analyzed the nitrosyl-proteomes by biotin/CyDye switch technique combined with two-dimensional fluorescence difference gel electrophoresis and identified nitrosoproteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Estradiol-17beta (E2) rapidly stimulated protein S-NO in human umbilical vein endothelial cells, maximizing within 10- to 30-min post-E2 (10 nm) exposure. E2-BSA also rapidly stimulated protein S-NO. Both E2 and E2-BSA-induced protein S-NO was blocked by ICI 182,780 and N-nitro-l-arginine-methylester. Human umbilical vein endothelial cells expressed estrogen receptor (ER)alpha and ERbeta; both seemed to be required for E2 stimulation of protein S-NO because: 1) neither ERalpha or ERbeta agonist alone, but their combination, stimulated protein S-NO; and 2) either ERalpha or ERbeta antagonist blocked E2-induced protein S-NO. Numerous nitrosoproteins (spots) were observed on two-dimensional fluorescence difference gel. One hundred spots of interest were picked up; 58 were identified and, of which 15 were novel nitrosoproteins, 28 were up-regulated, 11 were decreased, and the rest were unchanged by E2. Pathway analysis suggested that nitrosoproteins are involved in regulating various endothelial functions, including apoptosis, cell structure and metabolism, redox homeostasis, etc. Thus, estrogens stimulate dynamic endothelial protein S-NO via mechanisms linked to specific ERs possibly on the plasma membrane and endogenous NO. These findings signify a critical next step for the understanding of the biological targets of enhanced NO production by estrogens.

Estrogen Receptor α Controls a Gene Network in Luminal-Like Breast Cancer Cells Comprising Multiple Transcription Factors and MicroRNAs

Luminal-like breast tumor cells express estrogen receptor alpha (ERalpha), a member of the nuclear receptor family of ligand-activated transcription factors that controls their proliferation, survival, and functional status. To identify the molecular determinants of this hormone-responsive tumor phenotype, a comprehensive genome-wide analysis was performed in estrogen stimulated MCF-7 and ZR-75.1 cells by integrating time-course mRNA expression profiling with global mapping of genomic ERalpha binding sites by chromatin immunoprecipitation coupled to massively parallel sequencing, microRNA expression profiling, and in silico analysis of transcription units and receptor binding regions identified. All 1270 genes that were found to respond to 17beta-estradiol in both cell lines cluster in 33 highly concordant groups, each of which showed defined kinetics of RNA changes. This hormone-responsive gene set includes several direct targets of ERalpha and is organized in a gene regulation cascade, stemming from ligand-activated receptor and reaching a large number of downstream targets via AP-2gamma, B-cell activating transcription factor, E2F1 and 2, E74-like factor 3, GTF2IRD1, hairy and enhancer of split homologue-1, MYB, SMAD3, RARalpha, and RXRalpha transcription factors. MicroRNAs are also integral components of this gene regulation network because miR-107, miR-424, miR-570, miR-618, and miR-760 are regulated by 17beta-estradiol along with other microRNAs that can target a significant number of transcripts belonging to one or more estrogen-responsive gene clusters.

Abstract 1569: Antitumor activity of SR16388 against human multiple myeloma in vitro and in vivo

Abstract Multiple myeloma (MM) is the cancer of plasma cells that derive from a clonal disorder of terminally differentiated B cells in the bone marrow. MM is generally thought to be refractory against chemotherapeutics and is incurable. Development of novel agents for the treatment of this disease is urgently needed. Previous studies have indicated that MM cells express estrogen receptors (ERs), and anti-estrogens such as ICI 182,780 and Tamoxifen have demonstrated inhibitory effects on proliferation of MM cell lines as well as on MM cells isolated from patients. MM cells also over-express proangiogenic factors such as VEGF to induce angiogenesis and support their growth and survival. At SRI International, we have developed a steroidal anti-angiogenic molecule (SR16388) that potently inhibits angiogenesis in lung and prostate cancers, and it also binds to ER-α and ER-ß with high affinities in the nanomolar range. In this study, we examined the in vitro and in vivo effects of SR16388 on growth of MM cells. In RPMI-8226 multiple myeloma cells, SR16388 inhibited cell proliferation with an IC50 value of 0.9 µM. Cell cycle analysis revealed that SR16388 arrested the RPMI-8226 cells in G2 phase. In an in vivo murine xenograft model, SR16388 significantly inhibited the growth of RPMI-8226 tumors at the dose levels of 50 and 100 mg/kg with P < 0.05 and P < 0.02 respectively in a dose dependent manner. We are currently examining the molecular effects of SR16388 that contribute to its anti-proliferative and anti-angiogenic effects on MM and other human tumor types. Based on our in vitro and in vivo data, we believe that SR16388 is a promising new therapeutic molecule for the treatment of MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1569.

Abstract 1107: Mutant p53 increases epithelial mesenchymal transition, estrogen receptor alpha and erbB2/Her2/neu

Abstract OBJECTIVES.- Progression of carcinomas involves transition from an epithelial cobblestone-like cytoarchitecture to an elongated, mesenchymal phenotype., leading to an epithelial to mesenchymal transdifferentiation (EMT) (1). Since the p53 tumor suppressor pathway is inactivated in most human cancers due to gene mutations or dysfunction, we now investigated in human wt p53 breast carcinoma cells, whether EMT and cell proliferation are influenced by introducing a dominant-negative (DN) R175H mutant p53 (2). This mutation occurring in the p53 zinc-binding domain, is common to breast cancer patients poorly responsive to therapy (2) METHODS.- .- Fluorescence microscopy was used to determine EMT-linked decreases in E-cadherin and cytokeratin 18. These changes and others associated with EMT, were verified by immune blotting. Laser scanning cytometry was used to measure expression of ErbB2 and estrogen receptor alpha, cell cycle profile and proliferation (2). RESULTS.- Cells transduced with the DN-R175H mutant p53, increased expression of the mesenchymal marker vimentin and proliferation-associated cyclin A, also showing a greater G2 population which correlated with increased c-neu/Her2/ErbB2. Joint cytometric analysis of the Ki67proliferation marker and estrogen receptor alpha revealed a greater number of estrogen receptor-positive proliferating cells (3) among the mesenchymal population harbouring the DN-R175H mutant p53 .CONCLUSIONS.- These results suggest that EMT induced by the DN-R175H mutant p53 promotes a gain of pro-oncogenic function by increasing the % of proliferating cells expressing estrogen receptor alpha (3) and the expression of erbB2 (4) REFERENCES 1.-Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest. 2009 119: 1420-8. 2.- Rieber M, Strasberg Rieber M. DN-R175H p53 mutation is more effective than p53 interference in inducing epithelial disorganization and activation of proliferation signals in human carcinoma cells : role of E-cadherin. Int J Cancer. 2009; 125:1604-12. 3.- Shoker BS, Jarvis C, Clarke RB, Anderson E, Hewlett J, Davies MP, Sibson DR, Sloane JP. Estrogen receptor-positive proliferating cells in the normal and precancerous breast. Am J Pathol 1999, 155:1811-1815. 4.- Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, Brisken C, Minucci S, Di Fiore PP, Pelicci PG. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell. 2009; 138:1083-95. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1107.

Identification and characterization of a spontaneous ovarian carcinoma in Lewis rats

BackgroundOvarian carcinoma is the fourth most common cause of death from cancer in women. Limited progress has been made toward improving the survival rate of patients with this disease in part because of the lack of a good animal model. We present here a model of spontaneous ovarian carcinoma arising in a normal Lewis rat.MethodsA spontaneously occurring tumor of the left ovary was found in a normal Lewis rat during necropsy, which was sectioned for histological examination and placed into single cell suspension. Tumor cells were passaged in vivo by intraperitoneal injection into immunocompetent Lewis rats, and in vitro culture resulted in generation of a cell line. Tumor cells were examined by flow cytometry for expression of estrogen receptor α, progesterone receptor, androgen receptor, her-2/neu, epithelial cell adhesion molecule, and CA125. β-catenin expression and cellular localization was assessed by immunocytochemistry. RNA was harvested for gene expression profiling and studying the expression of cytokines.ResultsThe tumor, designated FNAR, could be serially transplanted into Lewis rats and propagated as a cell line in vitro, maintaining the properties of the original tumor. The FNAR cells displayed striking morphologic similarities to human ovarian carcinoma, resembling the endometrioid carcinoma subtype of surface epithelial neoplasms. The cells expressed estrogen receptor α, progesterone receptor, androgen receptor, her-2/neu, epithelial cell adhesion molecule, CA125, and nuclear β-catenin. A gene expression profile showed upregulation of a number of genes that are also upregulated in human ovarian carcinoma.ConclusionThis reliable model of ovarian carcinoma should be helpful in better understanding the biology of the disease as well as the development of novel treatment strategies.

Inhibitory effects of melatonin on sulfatase and 17β-hydroxysteroid dehydrogenase activity and expression in glioma cells

Melatonin interacts with estradiol at the estrogen receptor level in different kinds of neoplasias and also regulates the expression and the activity of some enzymes involved in the biosynthesis of estrogens in peripheral tissues. Glioma cells express estrogen receptors and have the ability to synthesize estrogens locally. Since melatonin inhibits the growth of C6 cells, and this indoleamine has been demonstrated to be capable of decreasing aromatase expression and activity in these cells, the aim of the present study was to analyze whether the regulation of the sulfatase, the enzyme that catalyzes the rate-limiting step in the conversion of estrogen sulfates to estrogens, and 17beta-hydroxysteroid dehydrogenase, the enzyme which converts the relatively inactive estrone to the most potent 17beta-estradiol, could be involved in the inhibition of glioma cell growth by melatonin. We found that melatonin decreases the growth of C6 glioma cells and reduces the sulfatase and 17beta-hydroxysteroid dehydrogenase activity. Finally, we demonstrated that melatonin downregulates sulfatase and 17beta-hydroxysteroid dehydrogenase mRNA steady state levels in these glioma cells. By analogy to the implications of these enzymes in other forms of estrogen-sensitive tumors, it is conceivable that their modulation by melatonin may play a role in the growth of glioblastomas.

Estrogenic in vitro assay on mouse embryonic Leydig cells

We and others have reported that mouse embryonic testes contain a subpopulation of somatic cells expressing estrogen receptor alpha (ERalpha). In order to provide evidence for a possible direct estrogen effect on mammalian testes from the early stage of their differentiation, here we devised a method for the in vitro culture of the ERalpha-expressing cells from 12.5 days post coitum mouse testes and their transfection with plasmids containing the classical estrogen responsive element (ERE) or the alternative estrogen AP-1 responsive element upstream of the luciferase reporter gene (ERE-Luc and AP-1-Luc). StAR immunopositivity of the most part of the ERalpha+ cells grown in culture and subjected to the estrogenic assay, allowed their identification as embryonic Leydig cells. Maximum induction of the ERE-Luc activity was achieved with 10 nM 17-beta estradiol (E2), from 1.7 to 3-fold in such cells and from 2.3 to 5.7-fold in MCF-7 cells used for comparison; the anti-estrogen ICI 182.780 abolished such effects. AP-1-Luc was less sensitive to E2 in both cell types (10 nM E2, 1.2 to 2.7-fold increase in embryonic Leydig cells; about 3-fold in MCF-7 cells) and the effect was not ICI-dependent. Eventually, we stimulated the transfected cells with various xenoestrogens such as lindane, bisphenol A or mono-(2-ethylhexyl) pthalate and with the phytoestrogen zeralenone obtaining evidence for ERE-Luc, but not AP-1-Luc stimulation in embryonic Leydig cells. These results represent evidence of functional ERalpha-dependent genomic pathways in embryonic Leydig cells and describe an in vitro assay suitable for evaluating the activity of putative estrogenic compounds on such cells.

Environmental endocrine disruptors promote invasion and metastasis of SK-N-SH human neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial cancer. Metastasis is the main cause of mortality in NB patients. Currently, little is known about the risk factors and their mechanisms that cause metastasis. Environmental endocrine disruptors (EED) are recently identified risk factors associated with various human diseases including malignant tumors. Our previous studies have implicated the role of di(2-ethylhexyl) phthalate (DEHP) and bisphenol A (BPA), two of the most common EED, in neuroblastoma cell proliferation. Here, we further investigated the effects of DEHP, BPA as well as 17beta-estradiol (E2) on the invasion and metastasis of human neuroblastoma SK-N-SH cells in vitro. SK-N-SH cells expressed estrogen receptor (ER)-beta, matrix metalloproteinases-2 (MMP-2), MMP-9 and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) at readily detectable levels. 50 microM DEHP, 0.1 microM BPA and 10 microM E2 exposure all resulted in enhanced motility and invasiveness of SK-N-SH cells (P<0.001), elevated expression of MMP-2 and MMP-9, and decreased expression of TIMP-2 (P<0.01). Furthermore, phosphorylation of Akt (Ser473) was also induced following the exposure (P<0.01). Importantly, both ER antagonist ICI182,780 and phosphoinositide 3-kinase (PI3K) specific inhibitor LY294002 significantly inhibited the DEHP, BPA, or E2-induced cell migration and invasion, as well as the disregulation of MMP-2, MMP-9 and TIMP-2 expression. ICI182,780 may have worked through abolishing Akt (Ser473) phosphorylation. In conclusion, DEHP, BPA, and E2 potently promote invasion and metastasis of neuroblastoma cells through overexpression of MMP-2 and MMP-9 as well as downregulation of TIMP-2. ER-dependent pathway and PI3K/Akt pathway are involved, which may become potential therapeutic targets for neuroblastoma treatment.

Identification of perineal sensory neurons activated by innocuous heat

C-fiber sensory neurons comprise nociceptors and smaller populations of cells detecting innocuous thermal and light tactile stimuli. Markers identify subpopulations of these cells, aiding our understanding of their physiological roles. The transient receptor potential vanilloid 1 (TRPV1) cation channel is characteristic of polymodal C-fiber nociceptors and is sensitive to noxious heat, irritant vanilloids, and protons. By using immunohistochemistry, in situ hybridization, and retrograde tracing, we anatomically characterize a small subpopulation of C-fiber cells that express high levels of TRPV1 (HE TRPV1 cells). These cells do not express molecular markers normally associated with C-fiber nociceptors. Furthermore, they express a unique complement of neurotrophic factor receptors, namely, the trkC receptor for neurotrophin 3, as well as receptors for neurturin and glial cell line-derived neurotrophic factor. HE TRPV1 cells are distributed in sensory ganglia throughout the neuraxis, with higher numbers noted in the sixth lumbar ganglion. In this ganglion and others of the lumbar and sacral regions, 75% or more of such HE TRPV1 cells express estrogen receptor alpha, suggestive of their regulation by estrogen and a role in afferent sensation related to reproduction. Afferents from these cells provide innervation to the hairy skin of the perineal region and can be activated by thermal stimuli from 38 degrees C, with a maximal response at 42 degrees C, as indicated by induction of extracellular signal-regulated kinase phosphorylation. We hypothesize that apart from participating in normal thermal sensation relevant to thermoregulation and reproductive functions, HE TRPV1 cells may mediate burning pain in chronic pain syndromes with perineal localization.

Spiradenocylindrocarcinoma. Report of a Case With a Low-Grade Component of Spiradenocarcinoma and an Immunohistochemical Study

Spiradenocylindrocarcinoma is a very rare malignant cutaneous neoplasm of the folliculosebaceous-apocrine unit. We report a case of this hybrid tumor in a 42-year-old woman. The tumor consisted of 2 circumscribed nodules with areas of cylindrocarcinoma and low-grade spiradenocarcinoma. In the overlapping areas, both spiradenomatous and cylindromatous features were observed. Expansion of the tumor beyond the fibrous pseudocapsule into the adjacent tissue was present. Furthermore, tumor cells were demonstrating mild to moderate pleomorphism and an increased mitotic index. p53 and ki-67 were among the positive immunohistochemical markers. A relatively small number of tumor cells expressed estrogen receptors. The aim of this study was to investigate the nature of this rare tumor of the skin appendages.

Regulation of GNRH production by estrogen and bone morphogenetic proteins in GT1-7 hypothalamic cells

Recent studies have shown that bone morphogenetic proteins (BMPs) are important regulators in the pituitary-gonadal endocrine axis. We here investigated the effects of BMPs on GNRH production controlled by estrogen using murine GT1-7 hypothalamic neuron cells. GT1-7 cells expressed estrogen receptor alpha (ERalpha; ESR1 as listed in MGI Database), ERbeta (ESR2 as listed in MGI Database), BMP receptors, SMADs, and a binding protein follistatin. Treatment with BMP2 and BMP4 had no effect on Gnrh mRNA expression; however, BMP6 and BMP7 significantly increased Gnrh mRNA expression as well as GnRH production by GT1-7 cells. Notably, the reduction of Gnrh expression caused by estradiol (E(2)) was restored by cotreatment with BMP2 and BMP4, whereas it was not affected by BMP6 or BMP7. E(2) activated extracellular signal-regulated kinase (ERK) 1/2 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) signaling but did not activate p38-mitogen-activated protein kinase (MAPK) signaling in GT1-7 cells. Inhibition of ERK1/ERK2 reversed the inhibitory effect of estrogen on Gnrh expression, whereas SAPK/JNK inhibition did not affect the E(2) actions. Expression levels of Eralpha and Erbeta were reduced by BMP2 and BMP4, but were increased by BMP6 and BMP7. Treatment with an ER antagonist inhibited the E(2) effects on Gnrh suppression including reduction of E(2)-induced ERK phosphorylation, suggesting the involvement of genomic ER actions in Gnrh suppression. BMP2 and BMP4 also suppressed estrogen-induced phosphorylation of ERK1/ERK2 and SAPK/JNK signaling, suggesting that BMP2 and BMP4 downregulate estrogen effects by attenuating ER-MAPK signaling. Considering that BMP6 and BMP7 increased the expression of alpha1E-subunit of R-type calcium channel (Cacna1e), which is critical for GNRH secretion, it is possible that BMP6 and BMP7 directly stimulate GNRH release by GT1-7 cells. Collectively, a newly uncovered interaction of BMPs and ER may be involved in controlling hypothalamic GNRH production and secretion via an autocrine/paracrine mechanism.

Estrous Cycle Modulates Ovarian Carcinoma Growth

The effects of reproductive hormones on ovarian cancer growth are not well understood. Here, we examined the effects of estrous cycle variation and specific reproductive hormones on ovarian cancer growth. We investigated the role of reproductive hormones in ovarian cancer growth using both in vivo and in vitro models of tumor growth. In vivo experiments using the HeyA8 and SKOV3ip1 ovarian cancer models showed that tumor cell inoculation during proestrus significantly increased tumor burden (251-273%) compared with injection during the estrus phase. Treatment of ovariectomized mice with 17beta-estradiol resulted in a 404% to 483% increase in tumor growth compared with controls. Progestins had no significant effect, but did block estrogen-stimulated tumor growth. Tumors collected from mice sacrificed during proestrus showed increased levels of vascular endothelial growth factor (VEGF) and microvessel density compared with mice injected during estrus. HeyA8, SKOV3ip1, and mouse endothelial (MOEC) cells expressed estrogen receptor alpha and beta and progesterone receptor at the protein and mRNA levels, whereas 2774 ovarian cancer cells were estrogen receptor-negative. In vitro assays showed that 17beta-estradiol significantly increased ovarian cancer cell adhesion to collagen in estrogen receptor-positive, but not in estrogen receptor-negative cells. Additionally, 17beta-estradiol increased the migratory potential of MOEC cells, which was abrogated by the mitogen-activated protein kinase (MAPK) inhibitor, PD 09859. Treatment with 17beta-estradiol activated MAPK in MOEC cells, but not in HeyA8 or SKOV3ip1 cells. Our data suggest that estrogen may promote in vivo ovarian cancer growth, both directly and indirectly, by making the tumor microenvironment more conducive for cancer growth.

K005 Estradiol accelerates reendothelialization through a cooperative effect between bone marrow and endothelial cells expressing estrogen receptor alpha

Drug-eluting stents releasing antimitotic drugs are frequently used in patients undergoing percutaneous coronary interventions. Although these stents inhibit proliferation of vascular smooth muscle cells and thus reduce rates of early stenosis, they also impair proliferation of endothelial cells. The resulting delayed arterial healing increases the risk of thrombosis and leads to higher late stent restenosis. Optimization of endothelium repair is then necessary to prevent intimal thickening and in-stent restenosis. Using a mouse model of carotid injury, we previously demonstrated that 17βestradiol (E2) accelerates endothelium regeneration through estrogen receptor ERα but not ERβ (1) and involvement of FGF-2 (2). However, the molecular and cellular mechanisms underlying this beneficial effect still remain poorly understood, mainly due to the lack of appropriate models and tools to visualize endothelium. Here, we employed en face confocal microscopy to follow endothelium repair in longitudinally opened and flattened mouse carotid arteries. We showed that E2 accelerates both migration and proliferation of endothelial cells and increased the recruitment of cells in an uninjured adjacent retrograde zone, leading to enlarged reendothelialized area (3). Many studies suggest that circulating bone marrow (BM) derived cells are involved in arterial healing. Thus, we generated chimeric mice by grafting wild type mice with ERα -/- or ERα+/+ BM and vice versa. The beneficial effect of E2 was abolished when the ERα was absent either in donor or in receiver mice, demonstrating that both BM and non-BM ERα-expressing cells cooperate to mediate E2 regenerative effects. Using a cell-specific inactivation mouse model of ERα, by the loxP/Cre recombination system, we showed that endothelial ERα is absolutely required in the accelerative effect of E2 on reendothelialization. To precise which BM derived cells are involved in the E2 effect, the implication of ERα-expressing macrophages is now under investigation. These results might help us to propose new strategy to optimize post-angioplasty and in-stent reendothelialization. 1. Brouchet L., Circulation, 2001 . 2. Fontaine V. Am J Pathol, 2006 . 3. Filipe C. Am J Physiol Heart Circ Physiol, 2008 .

Genistein decreases androgen biosynthesis in rat Leydig cells by interference with luteinizing hormone-dependent signaling

Testicular Leydig cells express estrogen receptors and are the predominant source of the male sex steroid hormone testosterone (T). Previous studies demonstrated that genistein acts through estrogen receptors in Leydig cells. In the present study, pre-treatment of Leydig cells isolated from 35 day-old male Long Evans rats with the epidermal growth factor receptor (EGFR) kinase inhibitor AG 1478 abrogated genistein inhibition of T biosynthesis. Also, incubation of Leydig cells in culture medium containing epidermal growth factor (EGF) decreased T secretion (control: 255 ± 16; EGF: 190 ± 17 ng/10 6 cells, 24 h) ( P < 0.05). However, T secretion by genistein-treated Leydig cells (0.1 nM, 10 μM; 24 h) was rescued by post-treatment incubation with forskolin (control: 275 ± 28 versus 325 ± 35; 780 ± 85; ng/10 6 cells, 3 h) and dibutyryl cyclic adenosine 3′-5′-monophosphate (dbcAMP) (control: 370 ± 65 versus 580 ± 75; 2500 ± 200; ng/10 6 cells, 3 h) ( P > 0.05). Furthermore, post-treatment incubation with cholera toxin, an activator of G proteins, caused genistein-treated Leydig cells to produce similar T amounts as untreated control (control: 55 ± 5 versus 52 ± 2 and 47 ± 4; ng/10 6 cells, 3 h) ( P > 0.05). These observations imply that genistein action interferes with coupling of transmembrane luteinizing hormone receptors (LHR) with G proteins. Uncoupling of LHR from G proteins adversely affects adenylate cyclase function and impacts LH-dependent stimulation of Leydig cells. These findings have implications for testicular steroidogenesis in individuals exposed to genistein and soy-based products.

Effects of Estrogen, Raloxifene and Levormeloxifene on α1A-Adrenergic Receptor Expression

We investigated the effect of estrogen, raloxifene and levormeloxifene on alpha1A-adrenergic receptor expression. Postpartum rats underwent intravaginal balloon injury and ovariectomy, and were then treated with estrogen or placebo for 8 weeks. The urethras were examined for alpha1A-adrenergic receptor expression by Western blot analysis and immunohistochemistry. Urethral smooth muscle cells were isolated from untreated female rats and examined for the expression of estrogen receptors alpha and beta by immunofluorescence microscopy. Urethral smooth muscle cells were treated with estrogen, raloxifene or levormeloxifene for 24 hours and examined for alpha1A-adrenergic receptor expression by real-time polymerase chain reaction. The effects of these drugs on alpha1A-adrenergic receptor expression were further examined by promoter assays. Estrogen treatment resulted in decreased alpha1A-adrenergic receptor expression in the urethras. Urethral smooth muscle cells expressed estrogen receptors alpha and beta, the former predominantly in the cytoplasm and the latter in the nucleus. Estrogen significantly down-regulated alpha1A-adrenergic receptor mRNA expression, while raloxifene and levormeloxifene had no significant effect. Estrogen also significantly down-regulated alpha1A-adrenergic receptor promoter in the presence of estrogen receptor alpha or beta. Raloxifene and levormeloxifene up-regulated alpha1A-adrenergic receptor promoter in the presence of estrogen receptor alpha but not beta. Estrogen down-regulated alpha1A-adrenergic receptor expression in the urethral smooth muscle of female rats, while raloxifene and levormeloxifene had no significant effect. These findings represent a possible molecular mechanism through which estrogen, raloxifene and levormeloxifene differentially affect urinary continence.

Biophysical and biochemical characterization of a liposarcoma‐derived recombinant MnSOD protein acting as an anticancer agent

A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild-type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N-terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or (68)Gallium-labelled synthetic peptide. The labelled peptide permeated MCF-7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with (125)I- and (131)I-labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro-apoptotic mechanism.