CHARACTERIZATION OF PHORBOLESTER-INDUCIBLE HUMAN NEURONAL FACTORS INVOLVED IN TRANSACTIVATION OF THE GALANIN GENE

The expression of the neuropeptide galanin (GAL) is elevated in vivo u pon nerve stimulation, injury, and in vitro by phorbol 12-myristate-13 -acetate (PMA), suggesting that a signal pathway involving protein kin ase C activation may be involved in GAL-gene activation. When plasmids containing a different length of the bovine GAL-promoter fused to luc iferase were transfected into the human neuroblastoma cell line (SK-N- SH subclone SH-SY5Y), a PMA-responsive element was identified in the p romoter-region -68 to -46 base pairs (bp). Co-transfection experiments with plasmids expressing cJun and cFos revealed that they could act a lone, as well as synergistically with PMA to induce luciferase activit y. Electrical mobility shift assays revealed that a cAMP response elem ent (CRE)-like sequence (TGACGCGG; -59 to -52 bp) bound PMA-inducible nuclear proteins present in SH-SY5Y cells. These proteins appear to bi nd mainly as RE-binding-protein/activating-transcription-factor (CREB/ ATF) and Jun/ATF heterodimers. In addition, an apparent PMA-inducible protein(s) not recognized by CREB/ATF and Jun antibodies bound to the CRE-like containing probe. (C) 1998 Academic Press.