RESUMO
We have previously reported on the relevance of the prevalence of CD44(+)/CD24(-/low) cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative real-time PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ER alpha negative MDA-MB-231 cells suggesting that ER alpha was necessary. This was further confirmed by ER alpha silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ER alpha into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ER alpha corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ER alpha as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ER alpha and histone deacetylases.
Assuntos
Neoplasias da Mama/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Estrogênios/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
The pattern recognition receptors (PRRs) of the innate immune system are the first defence line of the immune system. Toll-like receptors (TLRs) are the most well known and the best examined of the PR receptors. In the last years TLRs had been studied in different ways resulting in a lot of new insights in the function and signalling pathways of these receptors. However, it was not possible to investigate individual combinations of the TLRs and their specific ligands, because of the complex network in immune signalling resulting in interference with each other. This work shows a new cell-based assay, established for the analysis of single PRRs or heterodimers. For this purpose NIH3T3 (mouse fibroblasts) were stably transfected with the NF-kappaB-inducible reporter gene secreted alkaline phosphatase (SEAP) together with the corresponding combinations of human TLRs and their co-receptors (e.g. TLR1/2, TLR2/6 and TLR4/CD14). The specificity of the respective cell lines was shown by induction with variations of specific and unspecific ligands (immune-stimulating components of microorganisms or synthetic ligands). Analysis via the NF-kappaB-dependent reporter gene SEAP allows a direct way to detect the human TLR-activity. Our results showed that this assay is highly sensitive and specific for the respective ligands. For the synthetic ligands Pam(2)CysSK(4) the assay demonstrates a detection limit of 1 pg/ml for TLR2/6. In summary, this test system allows the investigation of individual human PRR-receptors in a highly specific way, without interference with other immune components opening new avenues for novel insights in the innate immune system and its applications.
Assuntos
Imunidade Inata/imunologia , Testes Imunológicos/métodos , Ligantes , Pirogênios/análise , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/imunologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diglicerídeos/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Lipídeo A/farmacologia , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Células NIH 3T3 , Nitrilas/farmacologia , Oligopeptídeos/farmacologia , Pirogênios/farmacologia , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Receptor 1 Toll-Like/agonistas , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor 6 Toll-Like/agonistas , Receptor 6 Toll-Like/genética , Receptor 6 Toll-Like/metabolismo , Transfecção , Zimosan/farmacologiaRESUMO
We have shown previously that high expression levels of microsomal epoxide hydrolase (mEH) correlate with a poor prognosis of breast cancer patients receiving tamoxifen, suggesting that enhanced mEH expression could lead to antiestrogen resistance (Fritz et al. in J Clin Oncol 19:3-9, 2001). Thus, the purpose of this study was to gain insights into the role of mEH in hormone-responsive tissues. We analyzed biopsy samples of the endometrium by immunohistochemical staining, pointing to a regulation of mEH during the menstrual cycle: during the first half mEH expression was low, increased during the second half and reached highest levels during pregnancy. Additionally, the progesterone receptor (PR) positive human endometrial cell lines IKPRAB-36 (estrogene receptor alpha [ERalpha] negative) and ECC1-PRAB72 (ERalpha positive) were chosen to further investigate the hormonal regulation of mEH expression. Western Blot and quantitative RT-PCR analysis revealed an increase of mEH expression after treatment with medroxy-progesterone 17-acetate (MPA) in the ERalpha containing ECC1-PRAB72 cells. In contrast our results suggest that MPA had no influence on the mEH protein level in the ERalpha- IKPRAB-36 cells. In conclusion, mEH expression is regulated by progesterone in the presence of both PRs and ERalpha.