RESUMO
At eukaryotic promoters, multi-faceted protein-protein and protein-DNA interactions can result in synergistic transcriptional activation. NFAT and AP-1 proteins induce interleukin-2 (IL-2) transcription in stimulated T cells, but the contributions of individual members of these activator families to synergistically activating IL-2 transcription is not known. To investigate the combinatorial regulation of IL-2 transcription we tested the ability of different combinations of NFATc2, NFATc1, cJun, and cFos to synergistically activate transcription from the IL-2 promoter. We found that NFATc2 and cJun are exclusive in their ability to synergistically activate human IL-2 transcription. Protein-protein interaction assays revealed that in the absence of DNA, NFATc2, but not NFATc1, bound directly to cJun/cJun dimers, but not to cFos/cJun heterodimers. A region of NFATc2 C-terminal of the DNA binding domain was necessary and sufficient for interaction with cJun in the absence of DNA, and this same region of NFATc2 was required for the synergistic activation of IL-2 transcription in T cells. Moreover, expression of this C-terminal region of NFATc2 specifically repressed the synergistic activation of IL-2 transcription. These studies show that a previously unidentified interaction between human NFATc2 and cJun is necessary for synergistic activation of IL-2 transcription in T cells.
Assuntos
Interleucina-2/genética , Fatores de Transcrição NFATC/química , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sequência de Bases , Sítios de Ligação , Primers do DNA/genética , Humanos , Células Jurkat , Modelos Biológicos , Fatores de Transcrição NFATC/genética , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativação TranscricionalRESUMO
BACKGROUND: RNA interference is an endogenous cellular mechanism in which short interfering RNAs (siRNAs) direct the sequence specific degradation of a target mRNA. siRNAs can be synthesized with chemical modifications to increase stability and reduce double-stranded RNA-induced immune responses without affecting their ability to elicit degradation of target mRNA. OBJECTIVES: This study examined the use of chemically modified siRNAs in a mouse model of allergen-induced airway hyperresponsiveness. METHODS: Chemically modified siRNAs were designed and screened in a cell-based reporter assay. The most potent siRNAs were then screened in bone marrow-derived mast cells to demonstrate efficacy in primary cells. RESULTS: A candidate siRNA was formulated and administered to sensitized mice just before airway challenge with allergen. Administration of the siRNA was shown to reduce airway resistance significantly in sensitized and challenged mice by 60%, whereas a control siRNA had no effect. CONCLUSION: These data demonstrate the effectiveness of introducing targeted siRNAs to prevent induction of allergen-induced airway dysfunction and suggest potential therapeutic applications.
Assuntos
Hiper-Reatividade Brônquica/terapia , Interleucina-13/metabolismo , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Animais , Células da Medula Óssea , Hiper-Reatividade Brônquica/etiologia , Modelos Animais de Doenças , Feminino , Genes Reporter , Humanos , Interleucina-13/genética , Mastócitos , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Organismos Livres de Patógenos Específicos , Resultado do TratamentoRESUMO
We previously reported that c-Jun binds directly to the N-terminal 163 amino acids of Homo sapiens TATA-binding protein-associated factor-1 (hsTAF1), causing a derepression of transcription factor IID (TFIID)-driven transcription (Lively, T. N., Ferguson, H. A., Galasinski, S. K., Seto, A. G., and Goodrich, J. A. (2001) J. Biol. Chem. 276, 25582-25588). This region of hsTAF1 binds TATA-binding protein to repress TFIID DNA binding and transcription. Here we show that the basic leucine zipper domain of c-Jun, which allows for DNA binding and homodimerization, is necessary and sufficient for interaction with hsTAF1. Interestingly, the isolated basic leucine zipper domain of c-Jun was able to derepress TFIID-directed basal transcription in vitro. Moreover, when the N-terminal region of hsTAF1 was added to in vitro transcription reactions and overexpressed in cells, it blocked c-Jun activation. c-Fos, another basic leucine zipper protein, did not interact with hsTAF1, but c-Fos/c-Jun heterodimers did bind the N terminus of hsTAF1. Our studies show that, in addition to dimerization and DNA binding, the well characterized basic leucine zipper domain of c-Jun functions in transcriptional activation by binding to the N terminus of hsTAF1 to derepress transcription.
