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.
Noma
T
T
Department of Biochemistry, Yamaguchi University, School of Medicine, Ube, Yamaguchi 755-8505, Japan. tnoma@po.cc.yamagucyhi-u.ac.jp
Yoon
Y S
YS
Nakazawa
A
A
D000263:Adenylate Kinase / Q000235:genetics*; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D015152:Blotting, Northern; D017931:DNA Primers; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016501:Neurites / Q000201:enzymology; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors
D000263:Adenylate Kinase; D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C490555:Neurod1 protein, rat; C093564:Neurogenic differentiation factor 1; C110939:adenylate kinase 1