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The estrogen receptor (ER) is a ligand-activated transcription factor that regulates gene expression. The classical mechanisms of nuclear ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in promoters of target genes. In addition, non-genomic pathways of ER action have also been identified in breast cancer cells. Cdc25A is a tyrosine phosphatase that catalyzes dephosphorylation of cyclin/cyclin-dependent kinase complexes to regulate G1- to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17β-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the Cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of Cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs, and E2F sites. Studies with inhibitors and dominant negative expression plasmids show that E2 activates Cdc25A expression through activation of genomic ERα/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of Cdc25A in breast cancer cells. The PIAS family was initially identified as cytokine-induced inhibitors of STATs which contain several conserved domains involved in binding to other nuclear coactivators. In this study we have investigated coactivation of ERα by PIAS3 in breast cancer cell lines transiently cotransfected with the pERE3 constructs which contain three tandem EREs linked to a luciferase reporter gene. PIAS3 coactivated ERα-mediated transactivation in cells cotransfected with pERE3 and wild-type ERα. In contrast to many other coactivators, PIAS3 also enhanced transactivation of ERα when cells were cotransfected with the TAF1 ERα mutant. In addition, PIAS3 does not interact with activation function 2 (AF2) domain of ERα in a mammalian two-hybrid assay. These data indicate that coactivation of ERα by PIAS3 was AF2-domain independent. Analysis of several PIAS3 deletion mutants showed that the region containing amino acids 274 to 416 of PIAS3 are required for coactivation suggesting that the RING finger domain and acidic region of PIAS3 are important for interactions with wild-type ERα. These results demonstrate that PIAS3 coactivated ERα and this represents a non-classical LXXLL-independent coactivation pathway.

Vitamin D interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17?-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE3), DRIP205 coactivates ER?-mediated transactivation. DRIP205?587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205?587-636 also coactivates ER?. Moreover, both wild-type and variant DRIP205 also colocalize with ER? in the nuclei of transfected cells. AF1 and AF2 of ER? are both required for DRIP205 coactivation. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ER? and in interactions with ER?. On the other hand, both DRIP205 and DRIP205?587-636 coactivate E2-induced transactivation of ER?/Sp1 in cells transfected with a construct containing three GC-rich sites (pSp13). Coactivation of ER?/Sp1 by DRIP205 is dependent on AF1 of ER?. Enhancement of ER? and ER?/Sp1 by DRIP205 does not require NR boxes of DRIP205, and deletion mutants DRIP205 (1-714) and DRIP205 (516-1566) significantly coactivate ER? and ER?/Sp1. RNA interference study showed that DRIP205 coactivation of ER?/Sp was abolished in cells transfected with iSp3 and iSp4, suggesting that Sp3 and Sp4 are required for coactivation of ER?/Sp by DRIP205 in ZR-75 cells.

Estrogen Receptor ? (ER?)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on the activation function 1 (AF1) of ER?. This study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ER? on activation of ER?/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ER? (hER? or MOR), but not ER? and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hER?/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ER?. Antiestrogen-induced activation of hER?/Sp1 was lost using hER? mutants deleted in zinc finger 1 (amino acids (aa) 185-205), zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hER?/Sp1 required the C-terminal F domain (aa 579-595), which contains a ?-strand structural motif. Moreover, in peptide competition experiments overexpression of NR-box peptides inhibits E2-induced luciferase activity of pERE3, which contains three tandem repeats of consensus ERE sites, whereas E2-induced hER?/Sp1 action was not inhibited by NR-box peptide expression. In contrast, overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hER?/Sp1, but not on activation of pERE3, suggesting that F domain interactions with nuclear cofactors are specifically required for ER?/Sp1 action. Furthermore, direct physical interactions between hER? and Sp1 protein in vivo have been investigated by using Fluorescence Resonance Energy Transfer (FRET) microscopy and image analysis. Consistent with results from transient transfection assay, E2, 4OHT, and ICI enhanced hER?/Sp1 interactions in living cells and these interactions were also confirmed by coimmunoprecipitation. In addition, endogenous hER?/Sp1 action was evaluated by using si RNA for Sp1 and a significant decrease in ligand-induced hER?/Sp1 action was observed after decreased Sp1 expression.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Pharmacology. Includes bibliographical references.

Estrogen and glucocorticoid receptors (ER and GR) are both members of the same subfamily of steroid nuclear receptors, and can both signal classically as ligand-activated transcription factors. However, many responses to estrogen and glucocorticoid exposure occur through the non-classical pathways, which include rapid activation of kinase cascades, activation of membrane-associated receptors, gene regulation through transcription by non-classical transcription factors, and protein regulation by translation and post-translational modification. Male territorial aggression is a hypothalamically-mediated steroid hormone-dependent adaptive behavior in mice. The hypothalamus, which expresses multiple ERs and GRs, is also responsive to estrogen and glucocorticoid treatment at a cellular level. Experiments were conducted to test the effects of estrogen and glucocorticoid interactions on spinogenesis in the ventromedial hypothalamus (VMH) and on male territorial aggression through the resident-intruder paradigm. Studies in male postnatal primary hypothalamic cell cultures demonstrate the expression of classic ERα, the variant ERα-36, and GPR30. PSD-95 protein, a marker for dendritic spines, is increased in response to 12 hours of treatment with the GPR30 agonist G-1 in an ERK/MAPK-dependent manner. Further work in immortalized embryonic hypothalamic cell lines (mHypoE-11 and mHypoE-42) demonstrate non-classical effects of a membrane-limited glucocorticoid on rapid nuclear translocation of the intracellular GR. Additionally, pharmacological inhibition of the ERK/MAPK pathway results in similar GR translocation in the absence of a ligand. Male postnatal primary hypothalamic cell cultures also respond to glucocorticoid exposure with increased 17Î_-E synthesis, suggesting crosstalk between GR signaling and estrogen signaling. Spine density in the gonadally intact adult male VMH decreases following suppression of estrogen synthesis with the aromatase inhibitor letrozole, suggesting estrogen is necessary to maintain spine density. In vivo studies in adult male mice demonstrate that estrogen is necessary to maintain basal peripheral CORT synthesis. Behavior testing using the resident-intruder paradigm showed that dexamethasone-suppression of adrenal CORT synthesis increases the amount of time resident mice spent engaged in aggressive bouts, and CORT treatment 20 minutes prior to aggression testing abolished this effect. The findings presented here provide support for the importance of the interactions between classical and non-classical estrogen and glucocorticoid signaling pathways on hypothalamic spinogenesis and male territorial aggression. / 1 / Jennifer Rainville

O linfoma de Hodgkin à uma neoplasia com elevados Ãndices de sobrevida global e sobrevida livre de doenÃa e seu comportamento e bases moleculares tÃm sido estudados. Sabe-se que a p53 à uma proteÃna que controla a progressÃo do ciclo celular e mutaÃÃes do gene TP53 prolongam a meia-vida da proteÃna alterando os mecanismos de apoptose e o controle da proliferaÃÃo celular. Cerca de 50% dos tumores humanos tÃm mutaÃÃes no TP53 e acredita-se que nos outros restantes a via de sinalizaÃÃo da p53 esteja comprometida por outros mecanismos. No linfoma de Hodgkin tem sido encontrada expressÃo de p53 e a detecÃÃo de gene TP53 mutado à rara. Ainda nÃo està definido se a p53 serve como marcador prognÃstico no linfoma de Hodgkin. NÃo hà estudos sobre a expressÃo de p53 em linfoma de Hodgkin na populaÃÃo do estado do CearÃ. Os receptores de estrogÃnio(RE) jà foram detectados por imuno-histoquÃmica em cÃlulas de medula Ãssea normal, alÃm de outros tecidos nÃo hematopoiÃticos. Hà evidÃncia de que os estrogÃnios tÃm um papel na diferenciaÃÃo dos linfÃcitos B e regulaÃÃo da apoptose.Sendo o linfoma de Hodgkin uma neoplasia de origem na cÃlula B, onde a resposta imune parece contribuir na sua patogenia, torna-se de particular interesse avaliar o papel do estrogÃnio nesse processo. Ainda nÃo sabemos qual a expressÃo de receptores de estogÃnio nos linfomas e qual sua influÃncia no comportamento dessa neoplasia. Neste trabalho estudamos a expressÃo de p53 e receptor de estrogÃnio em linfoma de Hodgkin no CearÃ. Foram estudados 39 casos de linfoma de Hodgkin confirmado por exame histo-patolÃgico e por imuno-histoquÃmica e um grupo controle de 10 casos de linfonodos com hiperplasia reativa, nÃo relacionados a linfoma. Foi feito estudo imuno-histoquÃmico com marcadores para p53 e REα. A idade dos pacientes variou entre 5 e 63 anos, 21% foram do sexo masculino, a apresentaÃÃo inicial da doenÃa foi em linfonodo cervical em 59% dos casos e 56% dos indivÃduos apresentavam sintomas B. Quanto ao subtipo histolÃgico, 56% dos casos eram de linfoma de Hodgkin(LH) clÃssico esclerose nodular, 27% celularidade mista, 14% rico em linfÃcitos e 3% eram LH predominÃncia linfocitÃria. NÃo houve casos de depleÃÃo linfocitÃria na amostra estudada. A p53 foi positiva em 53% dos casos e no grupo controle em 10%. O RE foi negativo nos casos de LH, com marcaÃÃo persistente dos neutrÃfilos (citoplasma) e marcou em 80% dos casos, em cÃlulas histiocitÃrias, com marcaÃÃo para-nuclear (golgiana) e difusa citoplasmÃtica (fraca). ConcluÃmos que a expressÃo de p53 ocorre em mais de 50% dos casos de LH, conforme jà relatado na literatura e que hà expressÃo de REα citoplasmÃtica em tecido linfÃide, mas nÃo no LH. Hà expressiva marcaÃÃo no citoplasma dos neutrÃfilos. Outros estudos sÃo necessÃrios para avaliar a expressÃo do REβ e seu papel na patogÃnese do LH. / Hodgkinâs lymphoma is a entity where free disease and overal survive is prolonged by treatment and its molecular basis have been studied. In previus literature is described p53 as a protein that controls progress of celular cicle and TP 53 gene mutations prolong half-life of protein, modifing the mechanisms of apoptosis and regulation of celular proliferation. About of 50% of human cancers have TP53 mutations and the investigators believe that in the others the signaling mechanisms of p53 are altered by others ways. Hodgkinâs lymphoma cells stain for p53 frequently, but detection for TP53 gene mutation is rare. Itâs not be well stabilished if p53 is a prognostic factor in Hodgkinâs lymphoma. There is no trial about p53 expression on Hodgkinâs lymphoma in Cearà state. Estrogen receptor were detected by imuno-histochemical analysis in normal bone marrow cells and others non-hematopoietic tissues. There is evidence that estrogen have a role on differentiation of B-cells and on regulation of apoptosis. If is Hodgkinâs lymphoma(HL) a B-cell neoplasm, with immune response seems to contribute in its pathogeny, its interesting to study the role of estrogen on Hodgkinâs lymphoma and its influence on this neoplasm behavior. We studied p53 and estrogen receptor expression in Hodgkinâs lymphoma on Cearà state. Were studied 39 cases of Hodgkinâs lymphoma confirmed by histo-pathological studies and by immuno-histochemical analysis and a control group with 10 cases of benign linfonodal hyperplasia. The simples were stained with p53 and estrogen receptor(ER) α anti-bodies. The age of patitens varied between 5 and 63 years, 21% were males, the initial presentation of disease was cervical adenopathy on 59% of cases and B simptoms were present on 56% of patients. On classification by histological subtipes, 56% were classical HL nodular sclerosis, 27% mixed cellularity, 14% lymphocyte-rich and 3% were HL linfocyte predominance. In this sample, lymphocyte-depleted subtype was not found. Positive staining for p53 occurred on 53% of cases and 10% of control group. Estrogen receptor was negative in all cases of Hodgkinâs lymphoma, but showed positive stainning in neutrophilic cells. In control group was founded ER positivity in 80% of cases, in histiocytic cells, with para-nuclear stainning and cytoplasmic diffuse. In conclusion, p53 expression occurs in more than 50% of Hodgkinâs lymphoma cases, as described previously, and there is expression of ERα in lymphoid tissue, but not on HL. There is persistent positivity in neutrophil cytoplasma. Another investigations are necessary to avaliate ERα and ERβ on RS cells and their role on the pathogenesis of Hodgkinâs lymphoma.

The increased risk of age-related cataracts in postmenopausal women and studies in animal models suggest that estrogen may have a protective role in the lens. However, very little is known regarding the role of estrogen and its receptors in the lens. To begin unraveling the estrogen signaling mechanism in the lens, the following aims were investigated: 1) to determine if estrogen receptors are expressed in the lens, [125I]-17beta-estradiol binding and mRNA expression of ERalpha, ERbeta, and GPR30 were examined in the mouse and human lens; 2) to determine if the loss of ERalpha and/or ERbeta receptors will induce spontaneous development of cataracts and to examine their role in an inducible cataract model; and 3) to identify estrogen-regulated genes in the lens that influence cataract development in the ERdelta3-induced cataract model. High-affinity, saturable binding sites for 17beta-estradiol were identified in the nuclear, cytosolic, and membrane fractions of the mouse and human lens. Additionally, detectable binding in the membrane fraction and expression of GPR30 mRNA in the mouse and human lens are the first evidence of this novel transmembrane estrogen receptor in the lens of any species. Transcripts for ERalpha, ERbeta, and GPR30 were expressed in the mouse lens which suggests that one or more of these estrogen receptor subtypes are responsible for the binding detected. With the loss of nuclear ER in the lens, spontaneous cataracts did not occur; however, diminished levels or loss of ERalpha in ERdelta3 female mice increased the severity of cortical cataracts with age. These results suggest that ERalpha in the lens may provide protection against the progression of cataracts in the ERdelta3 model. Together with the cataract induction and gene expression studies, six genes were identified to be differentially expressed with estrogen versus vehicle treatment in the lenses of ERdelta3 mice. The pax6, tcfap2a, tgfbeta2, six3, sox2, and pdgfalpha genes are known to have a critical role in lens development, proliferation, differentiation and maintenance of lens homeostasis. Therefore, future examination of these genes and their pathways in the lens may contribute to the understanding of the mechanisms of estrogen-mediated protection of lens transparency. Knowledge of pathways that function to maintain lens transparency and how estrogen regulated these pathways will assist in the development of estrogen therapies that can be clinically used to delay the onset and/or progression of cataracts. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmacology-Toxicology / PhD; / Dissertation;

Male rat sexual behavior requires aromatization of testosterone (T) to estradiol (E2) in the medial preoptic area (MPO) where estrogen receptors (ER) exist in two isoforms, ERα and ERβ. We hypothesized that E2 acts through estrogen receptor α (ERα) in the MPO to promote male mating behavior. Four groups of male rats were castrated, administered DHT s.c. and bilateral MPO implants delivering either: cholesterol, E2, propyl pyrazole triol (PPT, ERα agonist), diarylpropionitrile (DPN, ER β agonist), or 1-methyl-4-phenyl pyridinium (MPP, ERα antagonist). Additional gonadally intact males received bilateral MPO DPN implants. PPT maintained sexual behavior equally as well as E2, whereas mating was not maintained by cholesterol or DPN MPO implants. Exogenous T did not reinstate mating in animals that received MPP MPO implants. These findings indicate that, in the MPO, ERα is necessary and sufficient to promote copulatory behavior in male rats and ERβ is not sufficient for mating.

Estrogen receptor α (ERα) is a ligand activated transcription factor. Many widely used synthetic compounds and natural chemicals can activate ERα. The compounds investigated in this study include 17β-estradiol (E2), diethylstilbestrol (DES), antiestrogens ICI 182,780, 4-hydroxytamoxifen, the phytoestrogen resveratrol, and the xenoestrogens bisphenol A (BPA), nonylphenol (NP), octylphenol (OP), endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1- trichloroethane (HPTE) and 2,3,4,5-tetrachlorobiphenylol-4-ol (HO-PCB-Cl4). With the exception of the antiestrogens, all the compounds induced transactivation in MCF-7 or MDA-MB-231 cells transfected with wild-type ERα and a construct (pERE3) containing three tandem estrogen responsive elements (EREs) linked to a luciferase gene. However, these compounds differentially activated C-terminal deletion mutants of ERα. For example, neither E2 nor DES induced transactivation in MCF-7 transfected with ERα(1-537) due to partial deletion of helix 12 of ERα; however, OP, NP, resveratrol, kepone and HPTE induced this ERα mutant, demonstrating that the estrogenic activity of these synthetic compounds do not require activation function 2 (AF-2) of ERα. This study also investigated the effects of xenoestrogens on activation of reporter gene activity in MCF-7 and MDA-MB-231 cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to the luciferase gene. In MCF-7 cells, antiestrogen-induced activation of ERα/Sp1 required the zinc finger motifs of ERα, whereas activation by estrogen and some xenoestrogens activation, such as endosulfan, NP and OP required the H12 of ERα. In contrast, xenoestrogens, such as HPTE, BPA, kepone and HO-PCBCl4, significantly induced transactivation of all four ERα deletion mutants tested in this study. Moreover, RNA interference assays demonstrated structuredependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. The in vivo activities of E2, ICI 182,780, BPA and NP were further investigated in a transgenic mouse model containing pSp13 transgene. All the compounds induced luciferase activity in the mouse uterus; however activities observed in the penis and testis of male and stomach of both male and female mice were structure-dependent,. These results demonstrate that various ER ligands differentially activate ERα in breast cancer cells and transgenic mice, and their activities are dependent on ERα variants, promoter-, cell-context and selective use of different Sp proteins, suggesting these structurally diverse compounds are selective ER modulators (SERMs).