Activation of the human NPY gene during neuroblastoma cell differentiation: induced transcriptional activities of AP-1 and AP-2.
During functional neuronal differentiation of human SH-SY5Y neuroblastoma cells, induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), the mRNA expression of c-fos and c-jun displayed a synchronous and biphasic type of induction for both mRNAs, with an early transient (30 to 120 min) and a later (> 8 h) more persistent increase. This was coupled to increased in vitro DNA binding activity of cFos/cJun AP-1 heterodimers in SH-SY5Y nuclear extracts using the electrophoretic mobility shift assay. Functional AP-1 activity was demonstrated in differentiating SH-SY5Y cells by transient transfection assays using a TPA-responsive reporter plasmid. The second expression phase of these protooncogenes was paralleled by a sustained induction of neuronal differentiation markers, as exemplified by growth-associated protein 43 and neuropeptide tyrosine (NPY) mRNAs. DNA-protein interaction between an evolutionarily conserved region (-73 to -45) of the human NPY promoter, containing potential binding sites for AP-1, AP-2, and Sp1, and nuclear extracts prepared from untreated and TPA-treated SH-SY5Y cells revealed one complex (CI) that was unaffected and three complexes (CII to CIV) that were induced by TPA treatment. Competition for DNA binding using AP-1, AP-2, and Sp1 consensus sequences and an anti-cJun antibody, respectively, revealed cooperative interactions between AP-1, AP-2, and Sp1 transcription factors and the NPY promoter. In addition, TPA-mediated induction of AP-2 DNA binding activity to the NPY promoter was not dependent on increased AP-2 mRNA expression. This high degree of complexity presumably involved in NPY gene expression during neuronal differentiation of SH-SY5Y cells suggests productive cooperative interactions between multiple transcription factors.
Andersson
G
G
Department of Cell Research, Uppsala Genetic Center, Swedish University of Agricultural Sciences.
Påhlman
S
S
Parrow
V
V
Johansson
I
I
Hammerling
U
U
D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016762:Genes, fos / Q000235:genetics*; D016758:Genes, jun / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D010641:Phenotype; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate