Fos and Jun form cell specific protein complexes at the neuropeptide tyrosine promoter.
In this study we have investigated DNA-protein interactions at an AP1-like motif of the neuropeptide tyrosine (NPY) promoter during in vitro differentiation of human neuroblastoma cells SH-SY5Y to mature nonproliferative sympathetic neuron-like cells. These neuroblast-like cells originate from the parental cell line SK-N-SH from which two phenotypically distinct major cell types have been subcloned: the neuroblast-like SH-SY5Y cells and the epithelial-like SH-EP cells. SH-SY5Y cells can be induced to differentiate towards mature noradrenergic ganglion-like cells by the protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate). Interestingly, the effects of TPA are mimicked by the protein kinase inhibitor, staurosporine, which induces the expression of TPA target genes such as the neuronal differentiation-associated gene NPY in SH-SY5Y cells. Following activation of PKC, the effects of TPA are known to act through the transcription factor AP-1. To study transcriptional regulation during sympathetic differentiation of human neuroblastoma cells by TPA as well as by staurosporine, we focussed on protein complexes at an evolutionarily conserved AP-1 like motif located at nucleotide positions -70 to -65 within the 5'-flanking region of the NPY gene. We show that both c-Jun and c-Fos are part of the protein complexes that bind to this sequence in SH-SY5Y cells. Both staurosporine and TPA enhanced and modulated the binding of these DNA-protein complexes concomitant with the NPY mRNA expression. On the other hand, the absence of these complexes in the SH-EP subclone was associated with the absence of NPY mRNA expression and a lack of differentiation-associated morphological changes. The data suggest that Fos and Jun heterodimers are part of the protein complexes that bind to the AP-1 regulatory element of the NPY promoter in the neuroblast-like SH-SY5Y cells. These protein complexes appear to contribute to the cell specific expression of the NPY gene and seem to be required during differentiation of SH-SY5Y human neuroblastoma cells further along the sympathetic neuronal lineage induced by either TPA or staurosporine.
Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland.
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D000470:Alkaloids / Q000494:pharmacology; D001483:Base Sequence; D002454:Cell Differentiation; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009478:Neuropeptide Y / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014443:Tyrosine
D000470:Alkaloids; D009478:Neuropeptide Y; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D014443:Tyrosine; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate