RESUMEN

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-beta subunit and an oxygen-regulated HIF-alpha subunit. We have previously defined a hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-beta subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. We show here for the first time that insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, we determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. We demonstrate that stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, we have analyzed in vivo the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. We conclude that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Hipoxia de la Célula/genética , Hipoxia de la Célula/fisiología , Línea Celular , Núcleo Celular/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Drosophila/efectos de los fármacos , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/efectos de los fármacos , Proteínas de Drosophila/genética , Embrión no Mamífero , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Insulina/metabolismo , Oxígeno/farmacología , Proteínas Quinasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN/metabolismo , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR , Transcripción Genética