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2.
Oncogene ; 35(44): 5722-5734, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27065334

RESUMO

Tamoxifen has shown great success in the treatment of breast cancer; however, long-term treatment can lead to acquired tamoxifen (TOT) resistance and relapse. TOT classically antagonizes estradiol (E2) -dependent breast cancer cell growth, but exerts partial agonist/antagonist behavior on gene expression. Although both E2 and TOT treatment of breast cancer cells results in recruitment of the estrogen receptor (ER) to common and distinct genomic sites, the mechanisms and proteins underlying TOT preferential recruitment of the ER remains poorly defined. To this end, we performed in silico motif-enrichment analyses within the ER-binding peaks in response to E2 or TOT, to identify factors that would specifically recruit ER to genomic binding sites in the presence of TOT as compared to E2. Intriguingly, we found Nkx3-1 and Oct-transcription factor homodimer motifs to be enriched in TOT preferential binding sites and confirmed the critical role of Oct-3/4 (aka Oct-4) in directing ER recruitment to TOT preferential genomic binding sites, by chromatin immunoprecipitation (ChIP) analyses. Further investigation revealed Oct-4 expression to be basally repressed by Nkx3-1 in MCF-7 cells and TOT treatment appeared to elevate Nkx3-1 degradation through a p38MAPK-dependent phosphorylation of the E3 ligase, Skp2 at serine-64 residue, as observed by quantitative mass-spectrometry analyses. Consistently, Oct-4 upon induction by phospho-Ser64-Skp2-mediated proteasomal degradation of Nkx3-1, participated in ER transcriptional complexes along with p38MAPK and Skp2 in a tamoxifen-dependent manner leading to TOT-dependent gene activation and cell proliferation of the TOT-resistant MCF-7-tamr breast cancer cells. Notably, Oct-4 levels were highly elevated in MCF-7-tamr cells, and appeared critical for their TOT sensitivity in cell proliferation assays. Furthermore, overexpression of Oct-4 enhanced tumor growth in the presence of tamoxifen in mice in vivo. Collectively, our work presents a novel mechanism for tamoxifen-specific gene activation by ER, secondary to its TOT preferential recruitment to genomic sites by specific activation of Oct-4, a phenomenon that appears to underlie tamoxifen resistance in breast cancer cells and in xenograft tumor models, and could be useful in designing therapeutic interventions to improve treatment outcome.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Tamoxifeno/farmacologia , Animais , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Modelos Biológicos , Motivos de Nucleotídeos , Fator 3 de Transcrição de Octâmero/genética , Fosforilação , Ligação Proteica , Elementos de Resposta , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
3.
Nat Commun ; 6: 8303, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26383514

RESUMO

Cell-cell interactions promote juxtacrine signals in specific subcellular domains, which are difficult to capture in the complexity of the nervous system. For example, contact between axons and Schwann cells triggers signals required for radial sorting and myelination. Failure in this interaction causes dysmyelination and axonal degeneration. Despite its importance, few molecules at the axo-glial surface are known. To identify novel molecules in axo-glial interactions, we modified the 'pseudopodia' sub-fractionation system and isolated the projections that glia extend when they receive juxtacrine signals from axons. By proteomics we identified the signalling networks present at the glial-leading edge, and novel proteins, including members of the Prohibitin family. Glial-specific deletion of Prohibitin-2 in mice impairs axo-glial interactions and myelination. We thus validate a novel method to model morphogenesis and juxtacrine signalling, provide insights into the molecular organization of the axo-glial contact, and identify a novel class of molecules in myelination.


Assuntos
Axônios/metabolismo , Bainha de Mielina/metabolismo , Comunicação Parácrina , Pseudópodes/metabolismo , Proteínas Repressoras/metabolismo , Células de Schwann/metabolismo , Animais , Western Blotting , Imunofluorescência , Gânglios Espinais/citologia , Imuno-Histoquímica , Camundongos , Células NIH 3T3 , Neuroglia/metabolismo , Proibitinas , Proteômica , Ratos
4.
Oncogene ; 31(1): 39-47, 2012 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-21666713

RESUMO

Many estrogen receptor (ER)-positive breast cancers respond well initially to endocrine therapies, but often develop resistance during treatment with selective ER modulators (SERMs) such as tamoxifen. We have reported that the 14-3-3 family member and conserved protein, 14-3-3ζ, is upregulated by tamoxifen and that high expression correlated with an early time to disease recurrence. However, the mechanism by which tamoxifen upregulates 14-3-3ζ and may promote the development of endocrine resistance is not known. Our findings herein reveal that the tamoxifen upregulation of 14-3-3ζ results from its ability to rapidly downregulate microRNA (miR)-451 that specifically targets 14-3-3ζ. The levels of 14-3-3ζ and miR-451 were inversely correlated, with 14-3-3ζ being elevated and miR-451 being at a greatly reduced level in tamoxifen-resistant breast cancer cells. Of note, downregulation of miR-451 was selectively elicited by tamoxifen but not by other SERMs, such as raloxifene or ICI182,780 (Fulvestrant). Increasing the level of miR-451 by overexpression, which decreased 14-3-3ζ, suppressed cell proliferation and colony formation, markedly reduced activation of HER2, EGFR and MAPK signaling, increased apoptosis, and, importantly, restored the growth-inhibitory effectiveness of SERMs in endocrine-resistant cells. Opposite effects were elicited by miR-451 knockdown. Thus, we identify tamoxifen downregulation of miR-451, and consequent elevation of the key survival factor 14-3-3ζ, as a mechanistic basis of tamoxifen-associated development of endocrine resistance. These findings suggest that therapeutic approaches to increase expression of this tumor suppressor-like miR should be considered to downregulate 14-3-3ζ and enhance the effectiveness of endocrine therapies. Furthermore, the selective ability of the SERM tamoxifen but not raloxifene to regulate miR-451 and 14-3-3ζ may assist in understanding differences in their activities, as seen in the STAR (Study of Tamoxifen and Raloxifene) breast cancer prevention trial and in other clinical trials.


Assuntos
Proteínas 14-3-3/análise , Neoplasias da Mama/tratamento farmacológico , Regulação para Baixo/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Proteínas 14-3-3/fisiologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspase 7/análise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , MicroRNAs/análise , Cloridrato de Raloxifeno/farmacologia , Receptor ErbB-2/metabolismo
5.
Oncogene ; 31(14): 1825-34, 2012 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-21860415

RESUMO

Estrogen receptor-α (ERα, ESR1) is a pivotal transcriptional regulator of breast cancer physiology and is targeted by endocrine therapies. Loss of ERα activity or expression is an indication of endocrine resistance and is associated with increased risk of tumor recurrence and worse prognosis. In this study, we sought to investigate whether elements of the tumor microenvironment, namely macrophages, would impact on ERα and we found that macrophage-derived factors caused loss of ERα expression in breast cancer cells. Conditioned media from macrophages caused activation of several intracellular pathways in breast cancer cells of which c-Src, protein kinase c and mitogen-activated protein kinase (MAPK) were essential for loss of ERα expression. Moreover, a prolonged hyperactivation of MAPK was observed. The activation of this kinase cascade resulted in recruitment of extracellular signal regulated kinase 2 (ERK2) directly to chromatin at the ESR1 gene locus in a process that was dependent upon activation and recruitment of the c-Jun transcription factor. Thus, we identify a novel mechanism for loss of ERα expression in breast cancer cells via macrophage activation of kinase cascades in the cancer cells causing transcriptional repression of the ESR1 gene by a direct chromatin action of a c-Jun/ERK2 complex. The findings in this study support an alternative mechanism, not intrinsic to the tumor cell but derived from the cross-talk with the tumor microenvironment, that could lead to endocrine resistance and might be targeted therapeutically to prevent loss of ERα expression in breast tumors.


Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/genética , Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo
6.
Oncogene ; 29(44): 5957-68, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-20697349

RESUMO

Myc oncoproteins and histone deacetylases (HDACs) modulate gene transcription and enhance cancer cell proliferation, and HDAC inhibitors are among the most promising new classes of anticancer drugs. Here, we show that N-Myc and c-Myc upregulated HDAC2 gene expression in neuroblastoma and pancreatic cancer cells, respectively, which contributed to N-Myc- and c-Myc-induced cell proliferation. Cyclin G2 (CCNG2) was commonly repressed by N-Myc and HDAC2 in neuroblastoma cells and by c-Myc and HDAC2 in pancreatic cancer cells, and could be reactivated by HDAC inhibitors. 5-bromo-2'-deoxyuridine incorporation assays showed that transcriptional repression of CCNG2 was, in part, responsible for N-Myc-, c-Myc- and HDAC2-induced cell proliferation. Dual crosslinking chromatin immunoprecipitation assay demonstrated that N-Myc acted as a transrepressor by recruiting the HDAC2 protein to Sp1-binding sites at the CCNG2 gene core promoter. Moreover, HDAC2 was upregulated, and CCNG2 downregulated, in pre-cancerous and neuroblastoma tissues from N-Myc transgenic mice, and c-Myc overexpression correlated with upregulation of HDAC2 and repression of CCNG2 in tumour tissues from pancreatic cancer patients. Taken together, our data indicate the critical roles of upregulation of HDAC2 and suppression of CCNG2 in Myc-induced oncogenesis, and have significant implications for the application of HDAC inhibitors in the prevention and treatment of Myc-driven cancers.


Assuntos
Histona Desacetilase 2/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Transcrição Gênica , Regulação para Cima , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Ciclina G2/genética , Primers do DNA , Humanos , Camundongos , Camundongos Transgênicos , Neuroblastoma/patologia , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
J Pediatr Endocrinol Metab ; 22(8): 751-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19845126

RESUMO

BACKGROUND: Premature thelarche is defined as breast development before 8 years of age. This is most often caused by central hormone disregulation and is accompanied by concurrent bone maturation. However, we present a case of premature thelarche with concurrent bone maturation without central hormone disregulation. Genes within the estrogen signaling pathway were examined for genetic changes which might be responsible for the clinical phenotype. PATIENT REPORT: A girl presented with breast development from 18 months of age with undetectable serum estrogens, prepubertal serum gonadotropins, advanced growth and skeletal maturation, but no increase of uterine size, thus presenting a premature thelarche variant. Serum estrogens remained below detectable limits until she entered into an unremarkable puberty at 12.1 years of age. No abnormalities or SNPs were found in the genes tested. CONCLUSION: We describe a case of premature thelarche which cannot be attributed to a central cause of abnormal hormone levels or to alterations in genes suspected for this phenotype. We conclude that other yet to be identified factors are involved in this unique case of premature thelarche.


Assuntos
Mama/crescimento & desenvolvimento , Puberdade Precoce/diagnóstico , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Estrogênios/sangue , Feminino , Genótipo , Humanos , Lactente , Estudos Longitudinais , Polimorfismo de Nucleotídeo Único , Puberdade Precoce/sangue , Puberdade Precoce/etiologia , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais
8.
Oncogene ; 25(12): 1799-806, 2006 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-16158045

RESUMO

The proliferative action of ERalpha largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERbeta displays tumor-suppressor properties, thus supporting the interest to identify compounds that could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) upregulated ERbeta protein levels, whereas it decreased ERalpha expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERbeta more strongly than that of ERalpha. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERbeta expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERbeta and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy.


Assuntos
Inibidores Enzimáticos/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/efeitos dos fármacos , Histona Desacetilases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Células HeLa , Histona Desacetilases/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro
9.
J Neuroendocrinol ; 15(2): 105-8, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12535152

RESUMO

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans, although little is known about the mechanisms and the role of direct action on oestrogen receptors (ER) in this process. We examined the effect of coumestrol, alone and combined with ER antagonists, on gonadotropin-releasing hormone (GnRH) mRNA expression in GT1-7 cells. Coumestrol was found to have an inhibitory effect compared to controls, which was blocked by R,R-THC, a selective ER beta antagonist. These results suggest that ER beta is involved in the suppression of GnRH mRNA expression by coumestrol.


Assuntos
Cumestrol/farmacologia , Estradiol/análogos & derivados , Estrogênios não Esteroides/farmacologia , Hormônio Liberador de Gonadotropina/genética , Isoflavonas , Neurônios/fisiologia , Linhagem Celular , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Fulvestranto , Expressão Gênica/efeitos dos fármacos , Fitoestrógenos , Preparações de Plantas , RNA Mensageiro/análise , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo
10.
Proc Natl Acad Sci U S A ; 98(24): 14132-7, 2001 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-11698654

RESUMO

Estrogen receptor alpha (ER alpha) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na(+)/H(+) exchanger-3 (NHE3) and the rate of (22)Na(+) transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ER alpha knockout (alpha ERKO) efferent ductules. Targeted gene-deficient mice were compared with alpha ERKO, and the NHE3 knockout and CAII-deficient mice showed alpha ERKO-like fluid accumulation, but only the NHE3 knockout and alpha ERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in alpha ERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in alpha ERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in alpha ERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na(+) transport. Finally, these data raise the possibility of targeting ER alpha in developing a contraceptive for the male.


Assuntos
Estradiol/análogos & derivados , Estrogênios/metabolismo , Fertilidade/fisiologia , Trocadores de Sódio-Hidrogênio/fisiologia , Sódio/metabolismo , Ducto Deferente/fisiologia , Absorção , Animais , Aquaporina 1 , Aquaporinas/genética , Aquaporinas/metabolismo , Sequência de Bases , Anidrase Carbônica II/metabolismo , DNA Complementar , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , RNA , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Ducto Deferente/metabolismo , Água/metabolismo
11.
J Med Chem ; 44(24): 4230-51, 2001 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-11708925

RESUMO

Through an effort to develop novel ligands that have subtype selectivity for the estrogen receptors alpha (ERalpha) and beta (ERbeta), we have found that 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) acts as an agonist on both ER subtypes, but has a 70-fold higher relative binding affinity and 170-fold higher relative potency in transcription assays with ERbeta than with ERalpha. To investigate the ERbeta affinity- and potency-selective character of this DPN further, we prepared a series of DPN analogues in which both the ligand core and the aromatic rings were modified by the repositioning of phenolic hydroxy groups and by the addition of alkyl substituents and nitrile groups. We also prepared other series of DPN analogues in which the nitrile functionality was replaced with acetylene groups or polar functions, to mimic the linear geometry or polarity of the nitrile, respectively. To varying degrees, all of the analogues show preferential binding affinity for ERbeta (i.e., they are ERbeta affinity-selective), and many, but not all of them, are also more potent in activating transcription through ERbeta than through ERalpha (i.e., they are ERbeta potency-selective). meso-2,3-Bis(4-hydroxyphenyl)succinonitrile and dl-2,3-bis(4-hydroxyphenyl)succinonitrile are among the highest ERbeta affinity-selective ligands, and they have an ERbeta potency selectivity that is equivalent to that of DPN. The acetylene analogues have higher binding affinities but somewhat lower selectivities than their nitrile counterparts. The polar analogues have lower affinities, and only the fluorinated polar analogues have substantial affinity selectivities. This study suggests that, in this series of ligands, the nitrile functionality is critical to ERbeta selectivity because it provides the optimal combination of linear geometry and polarity. Furthermore, the addition of a second nitrile group beta to the nitrile in DPN or the addition of a methyl substitutent at an ortho position on the beta-aromatic ring increases the affinity and selectivity of these compounds for ERbeta. These ERbeta-selective compounds may prove to be valuable tools in understanding the differences in structure and biological function of ERalpha and ERbeta.


Assuntos
Acetileno/análogos & derivados , Acetileno/síntese química , Derivados de Benzeno/síntese química , Nitrilas/síntese química , Receptores de Estrogênio/metabolismo , Acetileno/química , Acetileno/metabolismo , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Humanos , Ligantes , Nitrilas/química , Nitrilas/metabolismo , Ensaio Radioligante , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica , Células Tumorais Cultivadas
12.
J Med Chem ; 44(23): 3838-48, 2001 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-11689070

RESUMO

A variety of nonsteroidal systems can function as ligands for the estrogen receptor (ER), in some cases showing selectivity for one of the two ER subtypes, ER alpha or ER beta. We have prepared a series of heterocycle-based (furans, thiophenes, and pyrroles) ligands for the estrogen receptor and assessed their behavior as ER ligands. An aldehyde enone conjugate addition approach and an enolate alkylation approach were developed to prepare the 1,4-dione systems that were precursors to the trisubstituted and tetrasubstituted systems, respectively. All of the diones were easily converted into the corresponding furans, but formation of the thiophenes and pyrroles from the more highly substituted 1,4-diones was problematical. Of the systems investigated, the tetrasubstituted furans proved to be most interesting. They were ER alpha binding- and potency-selective agents, with the triphenolic 3-alkyl-2,4,5-tris(4-hydroxyphenyl)furans (15a-d) displaying generally higher subtype binding selectivity than the bisphenolic analogues (15f-i). Binding selectivity for ER alpha was as high as 50-70-fold, and transcriptional activation studies showed that several members of this series were ER alpha selective agonists, with the best compound [3-ethyl-2,4,5-tris(4-hydroxyphenyl)furan, 15b] having full transcriptional activity on ER alpha while being inactive on ER beta. Comparative binding affinity analysis and molecular modeling were used to investigate the preferred binding mode adopted by the furan ligands, which appears to have the C(2) phenol mimicking the important role of the A-ring of estradiol. These ligands should be useful in studying the biological roles of both ER alpha and ER beta, and they might form the basis for the development of novel estrogen pharmaceuticals.


Assuntos
Furanos/síntese química , Fenóis/síntese química , Receptores de Estrogênio/metabolismo , Animais , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Furanos/química , Furanos/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fenóis/química , Fenóis/metabolismo , Pirróis/síntese química , Pirróis/química , Ensaio Radioligante , Ovinos , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas , Útero/metabolismo , Útero/ultraestrutura
13.
FEBS Lett ; 502(3): 103-8, 2001 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-11583108

RESUMO

Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERalpha and ERbeta, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERbeta in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ER-specific antagonist R,R-tetrahydrochrysene (R,R-THC). R,R-THC inhibited activation of iNOS/eNOS promoter-luciferase reporter constructs (iNOS/eNOS-Luc) in a dose-dependent fashion in COS7 cells selectively transfected with ERbeta, but failed to influence ERalpha-mediated increase of iNOS/ eNOS-Luc. In neonatal rat cardiomyocytes transfected with eNOS-Luc or iNOS-Luc, incubation with 17betaestradiol (E2, 10(-8) M) for 24 h stimulated expression of eNOS and iNOS. R,R-THC (10(-5) M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R,R-THC as shown by immunoblot analysis. Taken together, these results show that ERbeta mediates transcriptional activation of eNOS and iNOS by E2.


Assuntos
Estradiol/farmacologia , Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Western Blotting , Células COS , Células Cultivadas , Crisenos/farmacologia , Receptor beta de Estrogênio , Coração/efeitos dos fármacos , Immunoblotting , Luciferases/metabolismo , Masculino , Miocárdio/química , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Transfecção , Regulação para Cima
14.
J Cell Biochem ; 83(3): 448-62, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11596113

RESUMO

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.


Assuntos
Estradiol/análogos & derivados , Estrogênios/metabolismo , Estrogênios/fisiologia , Osteoblastos/metabolismo , Isoformas de Proteínas , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Fosfatase Alcalina/metabolismo , Northern Blotting , Western Blotting , Diferenciação Celular , Linhagem Celular , Citocinas/biossíntese , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Estrogênios/farmacologia , Matriz Extracelular/metabolismo , Fulvestranto , Genes Reporter , Humanos , Ligantes , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Tamoxifeno/farmacologia , Fatores de Tempo
16.
Mol Cell Endocrinol ; 181(1-2): 139-50, 2001 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-11476948

RESUMO

Investigation of the role of the second, more recently described estrogen receptor, denoted ERbeta, will be critical in understanding the molecular mechanisms underlying tissue-specific gene regulation by estrogens. Expression of ERbeta in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization and size of the endogenous ERbeta protein, due, in part, to the limited availability of human ERbeta-specific antibodies. Thus, our aim was to generate specific antibodies to human ERbeta and use them to determine the tissue-specific distribution and size(s) of the ERbeta protein. To this end, we have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ERbeta. The antibodies, made in mice against human ERbeta amino acids 256-505 (hormone binding domain lacking the F domain), are designated CFK-E12 (E12), CMK-A9 (A9) and CWK-F12 (F12) and were determined to be the IgG gamma1 isotype for E12, and IgG gamma2b for A9 and F12. All three monoclonal antibodies could be used to detect in vitro translated, baculovirus expressed, and cell transfected and expressed ERbeta protein by Western blot analyses, and all failed to detect ERalpha. A9 and F12 were able to immunoprecipitate efficiently the native form of ERbeta protein in the presence and absence of estradiol. Epitope mapping studies indicate that the E12 and F12 antibodies recognize overlapping peptide sequences in the N-terminal region of the hormone-binding domain, a region that is highly conserved among species. Immunocytochemical studies with these antibodies reveal nuclear-specific localization of the ERbeta protein in granulosa cells of the rat ovary. Nuclear ERbeta is also specifically localized in epithelial and some stromal cells of mouse and rat epididymis. Western blot analysis with protein extracts from ovarian granulosa cells of human, rat, mouse, and pig showed a ca. 52 kDa and an additional ca. 62-64 kDa band in these species. These results indicate the presence of two predominant molecular size forms of the ERbeta protein in ovarian granulosa cells and demonstrate the utility of these antibodies for detection of ERbeta in the human and in several other mammalian species.


Assuntos
Anticorpos Monoclonais/imunologia , Epididimo/química , Ovário/química , Receptores de Estrogênio/análise , Receptores de Estrogênio/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Western Blotting , Células CHO , Cricetinae , Epididimo/citologia , Epididimo/imunologia , Mapeamento de Epitopos , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Células da Granulosa/química , Células da Granulosa/citologia , Células da Granulosa/imunologia , Humanos , Hibridomas/citologia , Hibridomas/imunologia , Imuno-Histoquímica , Masculino , Camundongos , Peso Molecular , Ovário/citologia , Ovário/imunologia , Testes de Precipitina , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/química , Especificidade da Espécie , Suínos
17.
Endocrinology ; 142(8): 3493-501, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11459795

RESUMO

Prothymosin alpha (PTalpha), a protein associated with cell proliferation and chromatin remodeling, and found to selectively enhance ER transcriptional activity by interacting with a repressor of ER activity, is shown to be a primary response gene to estrogen. Prothymosin alpha mRNA was rapidly increased by estrogen, followed by a 6-fold increase in prothymosin alpha protein content in ER-containing breast cancer cells. Analysis of the prothymosin alpha promoter and 5'-flanking region, and electrophoretic gel mobility shift studies showed the strong inducibility by the estradiol-ER complex to be mediated by two consensus half-palindromic estrogen response elements at -750 and -1051, which directly bind the ER. Estrogenic stimulation of prothymosin alpha required a DNA binding form of ER with a functional activation function-2 domain. The prothymosin alpha 5'-regulatory region also contains multiple Sp1 sites. Although addition of Sp1 did not further enhance estradiol-ER stimulated prothymosin alpha transcriptional activity in breast cancer cells, transfection and response element mutagenesis studies using Drosophila cells, which are deficient in Sp1, revealed that Sp1 and the estradiol occupied-ER can each activate the prothymosin alpha gene independently of the other and act in an additive manner. These observations, documenting robust prothymosin alpha up-regulation by the estradiol-ER complex via widely spaced half-palindromic estrogen response element motifs, are reminiscent of those shown previously for the ovalbumin gene and suggest that the use of multiple half response elements may be a more common mode for regulation of gene expression by the ER than previously appreciated. In addition, these observations suggest interrelationships between cell proliferation and gene transcriptional activities and indicate a positive mechanism by which PTalpha, which increases ER transcriptional effectiveness, is itself up-regulated by the estrogen-ER complex.


Assuntos
Motivos de Aminoácidos/fisiologia , Neoplasias da Mama/genética , Estrogênios/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Precursores de Proteínas/genética , Receptores de Estrogênio/metabolismo , Timosina/genética , Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Precursores de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Elementos de Resposta/fisiologia , Fator de Transcrição Sp1/fisiologia , Timosina/análogos & derivados , Timosina/metabolismo , Células Tumorais Cultivadas , Regulação para Cima
18.
J Steroid Biochem Mol Biol ; 77(4-5): 193-203, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11457657

RESUMO

Estrogen receptor (ER) and cAMP signaling pathways interact in a number of estrogen target tissues including mammary and uterine tissues. One aspect of this interaction is that estradiol and protein kinase A (PKA) activators can cooperate synergistically to activate ER-mediated transcription of both endogenous genes and reporter genes containing only estrogen response elements. The purpose of this study was to investigate the molecular mechanism of this interaction between signaling pathways. Site-directed mutagenesis of the potential PKA phosphorylation sites in the ER indicated that phosphorylation of these sites was not necessary for the observed transcriptional synergy. In transient transfection assays in two different cell lines using reporter constructs containing either cAMP response elements, estrogen response elements or both types of elements, with the addition or absence of cAMP response element binding protein (CREB) expression plasmid, we observed that only one of these cell lines exhibited estrogen/PKA transcriptional synergy. Experiments demonstrated that CREB itself was involved in the transcriptional synergy, and that transfection of CREB restored transcriptional synergy in the cell line in which it was lacking. A functional interaction between ER and CREB was also demonstrated using a mammalian cell protein interaction assay; a dominant negative mutant of CREB did not exhibit this interaction. Therefore, these data indicate that CREB protein is required for the transcriptional synergy between cAMP and estrogen signaling pathways. Furthermore, CREB cooperated with the ER on genes that did not contain cAMP response elements, but contained only estrogen response elements. We propose that activated CREB is recruited to estrogen responsive genes by an ER--coactivator complex containing proteins such as CREB binding protein (CBP) and that the interaction of CREB with ER may assist in stabilizing its interaction with CBP and in promoting estrogen-ER and PKA transcriptional synergy.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA/metabolismo , Estradiol/farmacologia , Receptores de Estrogênio/metabolismo , Animais , Células CHO , Cricetinae , DNA/efeitos dos fármacos , Sinergismo Farmacológico , Ativação Enzimática , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Isoformas de Proteínas/metabolismo , Receptores de Estrogênio/genética , Transcrição Gênica , Células Tumorais Cultivadas
19.
Chem Biol ; 8(3): 277-87, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11306352

RESUMO

BACKGROUND: The specificity of hormone action arises from complementary steric and electronic interactions between a hormonal ligand and its cognate receptor. An analysis of such key ligand-receptor contact sites, often delineated by mutational mapping and X-ray crystallographic studies, can suggest ways in which hormone-receptor specificity might be altered. RESULTS: We have altered the hormonal specificity of the estrogen receptor alpha (ER) by making 'coordinated' changes in the A-ring of the ligand estradiol and in the A-ring binding subpocket of ER. These changes were designed to maintain a favorable interaction when both E and ER are changed, but to disfavor interaction when only E or ER is changed. We have evaluated several of these altered ligand and receptor pairs in quantitative ligand binding and reporter gene assays. CONCLUSIONS: In best cases, the new interaction is sufficiently favorable and orthogonal so as to represent the creation of a new hormone specificity, which might be useful in the regulation of transgene activity.


Assuntos
Desenho de Fármacos , Receptores de Estrogênio/química , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Estrogênios/síntese química , Estrogênios/química , Estrogênios/metabolismo , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
20.
Mol Endocrinol ; 15(4): 543-52, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11266506

RESUMO

Estrogen exerts profound effects on mood and mental state. The ability of estrogen to modulate serotonergic function raises the possibility that it may play a role in the mechanism associated with depression and its treatment. A cellular mechanism for estrogen to influence mood might be through the regulation of genes involved at various levels of the serotonin system. Here we report that estrogen can up-regulate the expression of the serotonin-1A receptor via a new mechanism involving synergistic activation by nuclear factor-kappa B (NF-kappa B) with estrogen receptor alpha. Interestingly, we observed that only estrogen receptor-alpha, and not -beta, was able to mediate this effect of estrogens. The partial antiestrogen, 4-hydroxytamoxifen, had the same effect as estrogen. In addition, mutation analysis showed that both the transactivation function of p65 and activation function 1 of estrogen receptor-alpha were essential for this synergistic regulation. Therefore, we propose that NF-kappa B complexes cooperate with estrogen receptor-alpha to recruit cofactors into the complex and thereby synergistically activate the serotonin-1A receptor promoter through nonclassical estrogen response elements by a mechanism that does not involve direct receptor binding to DNA.


Assuntos
Estrogênios/metabolismo , NF-kappa B/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Tamoxifeno/análogos & derivados , Animais , Células Cultivadas , Estradiol/análogos & derivados , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Humanos , Alcamidas Poli-Insaturadas , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Receptores 5-HT1 de Serotonina , Tamoxifeno/farmacologia , Ativação Transcricional
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