Efeitos do ICI 182,780 na expressão de receptores de andrógeno e estrógeno em próstata ventral de ratos
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Data
2009-06-24
Autores
Fernandes, Sheilla Alessandra Ferreira [UNIFESP]
Orientadores
Porto, Catarina Segreti [UNIFESP]
Tipo
Dissertação de mestrado
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O papel dos andrógenos na próstata está bem estabelecido na literatura. Recentemente, usando diferentes modelos experimentais, foi possível mostrar que os estrógenos têm um papel no desenvolvimento e na função da próstata. Assim, o presente estudo foi proposto com o objetivo de ampliar os conhecimentos sobre as ações dos estrógenos na próstata ventral de ratos, analisando o efeito do tratamento com o antiestrógeno ICI 182,780 na expressão - RNA mensageiro e proteína - de receptores de andrógenos (AR) e estrógenos (ERα, ERβ e GPR30) e na expressão e na fosforilação das proteínas ERK1 e ERK2. Os perfis hormonais de ratos machos com 30 dias de idade (idade do início do tratamento), controle e tratados com ICI 182,780 foram previamente determinados em nosso laboratório. Com a maturação sexual houve um aumento significativo do peso corporal dos animais e dos níveis de testosterona e 17β-estradiol plasmáticos. No presente estudo foi mostrado um aumento do peso relativo da próstata ventral. O tratamento de ratos machos com o antiestrógeno ICI 182,780 não interferiu no ganho de peso dos animais e não alterou os níveis de testosterona e 17β-estradiol plasmáticos, como previamente demonstrado. No presente estudo, o peso relativo da próstata ventral não foi alterado, mas houve um aumento de 2,4 vezes do conteúdo de testosterona presente neste tecido, sugerindo um aumento na captação do andrógeno e/ou alteração na síntese da testosterona e/ou de seu metabolismo. Os efeitos da maturação sexual e do tratamento com o ICI 182,780 dos ratos machos na expressão de AR, ERs e GPR30 foram avaliados usando PCR em tempo real (RNA mensageiro) e Western blot (proteína) em próstata ventral de ratos. A maturação sexual ou o tratamento com ICI 182,780 não alterou o RNA mensageiro para o AR na próstata ventral de ratos, mas houve uma diminuição da expressão protéica em ambos os grupos experimentais, sugerindo que mecanismos regulatórios estão envolvidos sobre a tradução da proteína, processamento pós- tradução ou estabilidade do AR. O aumento do conteúdo de andrógeno observado na próstata ventral de ambos os modelos experimentais pode não contribuir para a regulação da expressão do AR na próstata ventral. Porém, o aumento da concentração de compostos estrogênicos com a maturação sexual poderia estar envolvido neste processo. Além disso, o tratamento com o ICI 182,780 diretamente regulou a expressão do AR na próstata ventral. Levando em consideração os resultados da literatura e do presente estudo, é possível sugerir que os efeitos do 17β-estradiol e do ICI 182,780 na regulação da expressão do AR poderiam ser mediados pela interação do ERα e/ou ERβ com o fator de transcrição AP-1. Estudos da literatura mostraram que o 17β-estradiol induziu down-regulation da expressão do AR por um mecanismo independente de ERα e ERβ. Além disso, o ICI 182,780 pode interagir com o GPR30 e ativar diferentes cascatas de sinalização intracelular que convergem para ações genômicas. Assim, foi analisado o RNA mensageiro para este receptor em próstata ventral. A maturação sexual induziu uma diminuição do RNA mensageiro para o GPR30 na próstata ventral. Porém, este transcrito não foi alterado pelo tratamento com o ICI 182,780. Se este receptor está envolvido na regulação do AR precisa ser explorado. É importante enfatizar que a diminuição da expressão do AR tanto pela maturação sexual como pelo tratamento com o ICI 182,780 pode ter consequências funcionais importantes nos efeitos mediados por este receptor na próstata ventral. A participação de cada receptor de estrógeno e dos mecanismos envolvidos nesta regulação ainda precisam ser melhores explorados. A maturação sexual induziu uma diminuição do RNA mensageiro para o ERα na próstata ventral. Porém, o tratamento com o ICI 182,780 não alterou a expressão deste transcrito, mas a expressão protéica foi aumentada em ambos os modelos experimentais, sugerindo que a up-regulation da expressão do ERα ocorre ao nível pós-transcricional. Esses resultados, em conjunto com os da literatura, indicam que a regulação da expressão do ERα por 17β-estradiol ou ICI 182,780 é complexa. Além disso, depende do tecido analisado e na próstata poderia envolver o complexo ERα- AP-1. Se o complexo testosterona-AR tem um papel na regulação da expressão da expressão do ERα na próstata ainda precisa ser explorado. A expressão do ERβ (RNA e proteína) não alterou com a maturação sexual ou com o tratamento com o ICI 182,780. Levando em consideração os resultados da literatura e do presente estudo, é possível sugerir um balanço entre o efeito positivo do andrógeno-AR e estrógeno-ERβ-AP1 e negativo do estrógeno-ERα-AP-1 na regulação do ERβ em próstata ventral.
Androgens have a well-established role in the prostate. Recently, studies using different experimental approaches have revealed that estrogen also play an essential role in the normal development and function of the prostate. Thus, this study was proposed to further investigate the role of estrogens in the prostate, analyzing the effect of ICI 182,780 treatment on the expression of the androgen (AR) and estrogen (ERα, ERβ and GPR30) receptors. Furthermore, the expression and phosphorylation of ERK1 and ERK2 were also investigated in the rat ventral prostate. Previous studies from our laboratory showed that the treatment of rats with ICI 182,780 did not induce any changes in the body weight and in the plasma levels of testosterone and 17β-estradiol. In the present study, it was shown that the treatment with ICI 182,780 did not induce any changes in the ventral prostate weight, but increased 2.4-fold the testosterone levels in this tissue, suggesting an increase in the androgen uptake and/or regulation in the synthesis of testosterone and their metabolites.The effects of sexual maturation of the rats and the treatment of the animals with ICI 182,780 on the expression of AR, ERs and GPR30 in the ventral prostate were evaluated using real time PCR and Western blot. Sexual maturation or the treatment with ICI 182,780 did not change the mRNA levels for AR in the ventral prostate, but the protein level decreased in tissues obtained from both groups, suggesting that regulatory mechanisms such as, protein translation, post-translation processing and AR stability, may be involved. The increase of testosterone in the prostate obtained from both groups may not contribute to AR regulation. However, the increase of estrogen with sexual maturation might be involved in this process. Furthermore, the treatment with ICI 182,780 directly regulated of the AR expression in the ventral prostate. Taken together, data from the literature and the present data, suggest that the effect of 17β-estradiol and ICI 182,780 in the regulation of AR expression might be mediated through the interaction of ERα and/or ERβ with transcriptional complex AP-1. Recent studies from the literature showed that 17β-estradiol induces downregulation of AR expression in the ventral prostate of ERα and ERβ knockout mice, suggesting a mechanism independent of ERs. Furthermore, the binding of ICI 182,780 to GPR30 activates intracellular signaling pathways that converge to genomic actions. Thus, the mRNA for this receptor was evaluated in the ventral prostate. Sexual maturation, but not treatment with ICI 182,780, induced a decrease of mRNA for GPR30 in this tissue. Further experiments are necessary to fully elucidate the role of the GPR30 in the regulation of AR expression. It is important to emphasize that the decrease in the expression of AR by sexual maturation or treatment with ICI 182,780 may play a role in the prostate function. Further experiments are necessary to fully elucidate the contribution of each estrogen receptor and the mechanisms involved in the regulation of AR expression. Sexual maturation induced a decrease of the mRNA for ERα in the ventral prostate. However, the treatment with ICI 182,780 did not change the mRNA levels for ERα, but the protein levels increased in ventral prostate obtained from both groups, suggesting that the up-regulation of ERα occurs at a post-transcription level. These data, together with data from the literature, indicate that the regulation of ERα expression is complex, tissue-dependent and may involve interaction of ERα and/or ERβ with transcriptional complex AP-1. Further experiments are needed to fully elucidate the role of the testosterone-AR in the regulation of ERα expression. ERβ expression (mRNA and protein) did not change with sexual maturation or treatment with ICI 182,780. These data, together with data from the literature, suggest a balance between the positive effects of androgen-AR and estrogen-ERβAP-1 and negative effect of estrogen-ERα-AP-1 in the regulation of the ERβ expression in the ventral prostate. Androgen-AR and estrogen-ERα activate intracellular signaling pathways, such as, mitogen-activated protein kinase (MAPK, ERK1/2). Thus, the expression and activity of ERK1 and ERK2 were evaluated by Western blot in the ventral prostate. Sexual maturation and treatment with ICI 182,780 decreased the phosphorylation of ERK1 and ERK2. Blockade of ERs with ICI 182,780 may block the translocation of these receptors to the plasma membrane and, therefore, decrease the phosphorylation of ERK1 and ERK2. It is important to emphasize, that AR may also regulate these proteins in the ventral prostate from both groups. In conclusion, the present studies suggest that sexual maturation may regulate cell proliferation through a balance between decreased AR expression and the estrogen effects on ERβ in one side and the estrogen effect on increased ERα in the other side. These processes may have beneficial effects to prevent prostatic hyperplasia and hypertrophy. Furthermore, the treatment with ICI 182,780 induced the decrease of AR expression and blocked the ERs and/or their translocation from the nucleus to the plasma membrane and, consequently, decreased ERK1 and ERK2 phosphorylation. Thus, a beneficial anti-proliferative effect in the ventral prostate may be observed with treatment with ICI 182,780. These findings provide a fundamental basis for future studies to determine the physiological roles of estrogen in the ventral prostate and for new approaches in prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostatic cancer.
Androgens have a well-established role in the prostate. Recently, studies using different experimental approaches have revealed that estrogen also play an essential role in the normal development and function of the prostate. Thus, this study was proposed to further investigate the role of estrogens in the prostate, analyzing the effect of ICI 182,780 treatment on the expression of the androgen (AR) and estrogen (ERα, ERβ and GPR30) receptors. Furthermore, the expression and phosphorylation of ERK1 and ERK2 were also investigated in the rat ventral prostate. Previous studies from our laboratory showed that the treatment of rats with ICI 182,780 did not induce any changes in the body weight and in the plasma levels of testosterone and 17β-estradiol. In the present study, it was shown that the treatment with ICI 182,780 did not induce any changes in the ventral prostate weight, but increased 2.4-fold the testosterone levels in this tissue, suggesting an increase in the androgen uptake and/or regulation in the synthesis of testosterone and their metabolites.The effects of sexual maturation of the rats and the treatment of the animals with ICI 182,780 on the expression of AR, ERs and GPR30 in the ventral prostate were evaluated using real time PCR and Western blot. Sexual maturation or the treatment with ICI 182,780 did not change the mRNA levels for AR in the ventral prostate, but the protein level decreased in tissues obtained from both groups, suggesting that regulatory mechanisms such as, protein translation, post-translation processing and AR stability, may be involved. The increase of testosterone in the prostate obtained from both groups may not contribute to AR regulation. However, the increase of estrogen with sexual maturation might be involved in this process. Furthermore, the treatment with ICI 182,780 directly regulated of the AR expression in the ventral prostate. Taken together, data from the literature and the present data, suggest that the effect of 17β-estradiol and ICI 182,780 in the regulation of AR expression might be mediated through the interaction of ERα and/or ERβ with transcriptional complex AP-1. Recent studies from the literature showed that 17β-estradiol induces downregulation of AR expression in the ventral prostate of ERα and ERβ knockout mice, suggesting a mechanism independent of ERs. Furthermore, the binding of ICI 182,780 to GPR30 activates intracellular signaling pathways that converge to genomic actions. Thus, the mRNA for this receptor was evaluated in the ventral prostate. Sexual maturation, but not treatment with ICI 182,780, induced a decrease of mRNA for GPR30 in this tissue. Further experiments are necessary to fully elucidate the role of the GPR30 in the regulation of AR expression. It is important to emphasize that the decrease in the expression of AR by sexual maturation or treatment with ICI 182,780 may play a role in the prostate function. Further experiments are necessary to fully elucidate the contribution of each estrogen receptor and the mechanisms involved in the regulation of AR expression. Sexual maturation induced a decrease of the mRNA for ERα in the ventral prostate. However, the treatment with ICI 182,780 did not change the mRNA levels for ERα, but the protein levels increased in ventral prostate obtained from both groups, suggesting that the up-regulation of ERα occurs at a post-transcription level. These data, together with data from the literature, indicate that the regulation of ERα expression is complex, tissue-dependent and may involve interaction of ERα and/or ERβ with transcriptional complex AP-1. Further experiments are needed to fully elucidate the role of the testosterone-AR in the regulation of ERα expression. ERβ expression (mRNA and protein) did not change with sexual maturation or treatment with ICI 182,780. These data, together with data from the literature, suggest a balance between the positive effects of androgen-AR and estrogen-ERβAP-1 and negative effect of estrogen-ERα-AP-1 in the regulation of the ERβ expression in the ventral prostate. Androgen-AR and estrogen-ERα activate intracellular signaling pathways, such as, mitogen-activated protein kinase (MAPK, ERK1/2). Thus, the expression and activity of ERK1 and ERK2 were evaluated by Western blot in the ventral prostate. Sexual maturation and treatment with ICI 182,780 decreased the phosphorylation of ERK1 and ERK2. Blockade of ERs with ICI 182,780 may block the translocation of these receptors to the plasma membrane and, therefore, decrease the phosphorylation of ERK1 and ERK2. It is important to emphasize, that AR may also regulate these proteins in the ventral prostate from both groups. In conclusion, the present studies suggest that sexual maturation may regulate cell proliferation through a balance between decreased AR expression and the estrogen effects on ERβ in one side and the estrogen effect on increased ERα in the other side. These processes may have beneficial effects to prevent prostatic hyperplasia and hypertrophy. Furthermore, the treatment with ICI 182,780 induced the decrease of AR expression and blocked the ERs and/or their translocation from the nucleus to the plasma membrane and, consequently, decreased ERK1 and ERK2 phosphorylation. Thus, a beneficial anti-proliferative effect in the ventral prostate may be observed with treatment with ICI 182,780. These findings provide a fundamental basis for future studies to determine the physiological roles of estrogen in the ventral prostate and for new approaches in prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostatic cancer.
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FERNANDES, Sheilla Alessandra Ferreira. Efeitos do ICI 182,780 na expressão de receptores de andrógeno e estrógeno em próstata ventral de ratos. 2009. Dissertação (Mestrado) - Universidade Federal de São Paulo (UNIFESP), São Paulo, 2009.