RESUMO
OBJECTIVE: The objective of the study was to determine whether cross-talk occurs between estrogen receptors (ERs) and nuclear factor-kappa-B (NF-kappaB), to assess the functional consequences of such an ER/NF-kappaB interaction, and to identify other unknown regulatory proteins that may participate in the NF-kappaB transcriptional complex. STUDY DESIGN: Electromobility gel shifts, reporter gene assays, and mass spectrometry were used to identify proteins interacting with the NF-kappaB deoxyribonucleic acid (DNA) response element. RESULTS: ER and the p65 subunit of NF-kappaB colocalized on DNA. This interaction was inhibitory for ER transcriptional activity. Sequencing of proteins bound to the NF-kappaB/DNA complex identified DNA-modifying enzymes, scaffolding proteins, chaperones, and elements of the nuclear matrix. CONCLUSION: These studies have identified an inhibitory interaction between estrogen receptors and the p65 subunit of NF-kappaB with implications for estrogen action in pregnancy and cancer. New accessory proteins have also been identified that bind to protein complexes on the NF-kappaB DNA response element.
Assuntos
Neoplasias do Endométrio/genética , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Neoplasias do Endométrio/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Genes Reporter , Humanos , NF-kappa B/genética , Reação em Cadeia da Polimerase , Gravidez , Probabilidade , Ligação Proteica , Estudos de Amostragem , Sensibilidade e Especificidade , Transdução de Sinais , Ativação Transcricional , Trofoblastos/citologiaRESUMO
OBJECTIVE: Nuclear factor kappa B (NFkappaB) is a strong anti-apoptotic factor, which is constitutively active in human endometrial cancer cells. Progesterone is the principal growth inhibitory hormone in the endometrial epithelium and promotes apoptosis. To identify the pathways through which progesterone controls NFkappaB function, we explored its genomic and non-genomic effects in endometrial cancer cells. METHODS: PR-negative Hec50co endometrial cancer cells were engineered to express high levels of the A or B isoform of PR (PRA or PRB) by adenoviral infection. Cells were treated with progesterone or vehicle alone, and RNA was isolated. Affymetrix microarrays were performed and transcriptional control of the genes of highest interest was confirmed by semi-quantitative RT-PCR. To assess the non-genomic effects of PR on inflammation associated with NF-kappaB, electromobility shift assays (EMSAs) were performed. RESULTS: Expression analysis demonstrated a significant effect of progesterone after 12- and 24-h treatment on several genes; in particular, A20 and ABIN-2 were induced through PRB. These factors bind in a complex and inhibit NFkappaB transcriptional activity. In addition, EMSAs revealed the complete inhibition of NFkappaB dimer binding to DNA by both PRA and PRB. CONCLUSIONS: Progesterone is the principal differentiating hormone in the endometrium. We have now identified several down-stream pathways of action, one of which is the control of genes involved in NFkappaB activity. The tumorigenic inflammatory and anti-apoptotic effects of NFkappaB are inhibited by progesterone/PRB through the transcriptional control of binding proteins A20 and ABIN-2. This pathway offers interesting targets for future therapeutic development.