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 lifera tive sympathetic neuron-like cells. neuroblast-like cells originate fr om the parental cell line SK-N-SH from which two phenotypically distin ct major cell types have been subcloned: the neuroblast-like SH-SY5Y c ells 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-acet ate). Interestingly, the effects of TPA are mimicked by the protein ki nase inhibitor, staurosporine, which induces the expression of TPA tar get genes such as the neuronal differentiation-associated gene NPY in SH-SY5Y cells. Following activation of PKC, the effects of TPA are kno wn to act through the transcription factor AP-1. To study transcriptio nal regulation during sympathetic differentiation of human neuroblasto ma cells by TPA as well as by staurosporine, we focussed on protein co mplexes at an evolutionarily conserved AP-1 like motif located at nucl eotide positions -70 to -65 within the 5'-flanking region of the NPY g ene. We show that both c-Jun and c-Fos are part of the protein complex es 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 abse nce of these complexes in the SH-EP subclone was associated with the a bsence 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 el ement 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.