RESUMO
Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H(2)O(2)-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.
Assuntos
Apoptose/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Diferenciação Celular , Clonagem Molecular , Proteínas de Ligação a DNA/química , Humanos , Peróxido de Hidrogênio/farmacologia , Hipóxia/genética , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Hibridização In Situ , Lipossomos/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Dados de Sequência Molecular , Células PC12 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras , Homologia de Sequência de Aminoácidos , Acidente Vascular Cerebral/genética , Fatores de Transcrição/química , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The role of amino acids (AA) on translational regulation in mammary epithelial cells cultured under lactogenic conditions was studied. The rates of total protein synthesis and beta-lactoglobulin (BLG) synthesis in mouse CID-9 cells were 2.1- or 3.1-fold higher, respectively, than in their bovine L-1 counterparts. Total AA deprivation or selective deprivation of Leu had a negative protein-specific effect on BLG synthesis that was more pronounced in bovine cells than in murine cells. Dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase (S6K1) on Thr(389) but not on Ser(411) was also more prominent in bovine cells. Noteably, deprivation of Leu had a less marked effect on BLG synthesis and 4E-BP1 or S6K1 phosphorylation than deprivation of all AA. In AA-deprived CID-9 cells, Leu specifically restored BLG synthesis from pre-existing mRNA whereas AA also restored total protein synthesis. This restoration was associated with a more pronounced effect on 4E-BP1 and S6K1 phosphorylation in bovine versus murine cells. Rapamycin specifically reduced Leu- and AA-stimulated BLG translation initiation in a dose-dependent manner. A further reduction was observed for Leu-treated cells in the presence of LY294002, a PI3K (phosphatidylinositol 3-kinase) inhibitor, which also reduced total protein synthesis. These findings suggest that direct signaling from AA to the translational machinery is involved in determining the rates of milk protein synthesis in mammary epithelial cells.