Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1alpha in transactivating murine neuroblastic glucose transporter

摘要

We have shown in vitro a hypoxia-induced time-dependent increase in facili-tative glucose transporter isoform 3 (GLUT3) expression in N2A murine neuroblasts. This increase in GLUT3 expression is partially reliant on a transcriptional increase noted in actinomycin D and cycloheximide pretreatment experiments. Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to -857- to -573-bp and -203- to - 177-bp regions. Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1alpha and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays. In addition, the interaction of CBP with Creb and HIF-1alpha and CREST with CBP in hypoxia was detected by coimmunoprecipi-tation. Furthermore, small interference (si)RNA targeting Creb in these cells decreased endogenous Creb concentrations that reduced by twofold hypoxia-induced glut3 gene transcription. Thus, in N2A neuroblasts, phosphorylated HIF-1alpha and Creb mediated the hypoxia-induced increase in glut3 transcription. Coactivation by the Ca~(++)-dependent CREST and CBP proteins may enhance cross-talk between p-Creb-AP-1 and HIF-1alpha/HRE of the glut3 gene. Collectively, these processes can facilitate an adaptive response to hypoxic energy depletion targeted at enhancing glucose transport and minimizing injury while fueling the proliferative potential of neuroblasts.