Trans-activators regulating neuronal glucose transporter isoform-3 gene expression in mammalian neurons

The murine facilitative glucose transporter isoform 3 is developmentally regulated and is predominantly expressed in neurons. By employing the primer extension assay, the transcription start site of the murine Glut 3 gene in the brain was localized to - 305 bp 5' to the ATG translation start codon. Transient transfection assays in N2A neuroblasts using murine GLUT3-luciferase reporter constructs mapped enhancer activities to two regions located at - 203 to - 177 and - 104 to - 29 bp flanking a previously described repressor element (-137 to -130 bp). Dephosphorylated Sp1 and Sp3 proteins from the 1- and 21-day-old mouse brain nuclear extracts bound the repressor elements, whereas both dephosphorylated and phosphorylated cAMP-response element-binding protein ( CREB) in N2A, 1- and 21-day-old mouse brain nuclear extracts bound the 5'-enhancer cis-elements ( - 187 to - 180 bp) of the Glut 3 gene, and the Y box protein MSY-1 bound the sense strand of the - 83- to - 69-bp region. Sp3, CREB, and MSY-1 binding to the GLUT 3 DNA was confirmed by the chromatin immunoprecipitation assay, whereas CREB and MSY-1 interaction was detected by the co-immunoprecipitation assay. Furthermore, small interference RNA targeted at CREB in N2A cells decreased endogenous CREB concentrations, and CREB mediated GLUT 3 transcription. Thus, in the murine brain similar to the N2A cells, phosphorylated CREB and MSY-1 bound the Glut 3 gene trans-activating the expression in neurons, whereas Sp1/Sp3 bound the repressor elements. We speculate that phosphorylated CREB and Sp3 also interacted to bring about GLUT 3 expression in response to development/ cell differentiation and neurotransmission.