Overexpression of NeuroD in PC12 cells alters morphology and enhances expression of the adenylate kinase isozyme 1 gene

NeuroD, a basic helix–loop–helix transcription factor, plays an important role in neuronal differentiation. A rat NeuroD cDNA was obtained by the aid of reverse transcription-polymerase chain reaction (RT-PCR) and ligated to an expression vector having a CMV promoter. Transfection of the NeuroD-expression plasmid into PC12 cells, a rat pheochromocytoma cell line, induced morphological changes featured by neurite-like processes and synapse-like structures without a differentiation-inducing reagent such as NGF. In the transfected cells, the overproduced NeuroD was detected by Western blot analysis, and the expression of the gene encoding mid-sized neurofilaments, a neuron-specific marker, was demonstrated by RT-PCR. Adenylate kinase isozyme 1 (AK1) is an enzyme involved in the homeostasis of energy metabolism and appears specifically in neuronal cells during differentiation. The CAT reporter assay of the 5′-flanking region of the AK1 gene suggests that NeuroD activates the AK1 expression through E-boxes in the promoter region. RT-PCR analysis indicated the enhanced level of AK1 mRNA in NeuroD-producing PC12 cells. Electrophoretic mobility shift assays demonstrated that NeuroD was able to interact with a proximal E-box element of the AK1 promoter. The results indicated that NeuroD promoted the PC12 cells to differentiate into neuron-like cells with concomitant activation of the target genes including the AK1 and the neurofilament genes.