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-SYSY neuroblast oma cells, induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), the mRNA expression of c-fos and c-jun displayed a synchronous and biphasi c 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 heterodimer s in SH-SY5Y nuclear extracts using the electrophoretic mobility shift assay. Functional AP-1 activity was demonstrated in differentiating S H-SY5Y cells by transient transfection assays using a TPA-responsive r eporter plasmid. The second expression phase of these protooncogenes w as paralleled by a sustained induction of neuronal differentiation mar kers, as exemplified by growth-associated protein 43 and neuropeptide tyrosine (NPY) mRNAs. DNA-protein interaction between an evolutionaril y 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 com plex (CI) that was unaffected and three complexes (CII to CIV) that we re induced by TPA treatment. Competition for DNA binding using AP-1, A P-2, and Spl consensus sequences and an anti-cJun antibody, respective ly, revealed cooperative interactions between AP-1, AP-2, and Spl tran scription factors and the NPY promoter. In addition, TPA-mediated indu ction of AP-2 DNA binding activity to the NPY promoter was not depende nt on increased AP-2 mRNA expression. This high degree of complexity p resumably involved in NPY gene expression during neuronal differentiat ion of SH-SY5Y cells suggests productive cooperative interactions betw een multiple transcription factors.