TISSUE-SPECIFIC EXPRESSION OF THE VASOACTIVE-INTESTINAL-PEPTIDE GENE REQUIRES BOTH AN UPSTREAM TISSUE SPECIFIER ELEMENT AND THE 5' PROXIMALCYCLIC AMP-RESPONSIVE ELEMENT

An upstream enhancer element [tissue specifier element (TSE)] located between 4.66 and 4.02 kb from the transcription start site is importan t for cell type-specific expression and phorbol ester induction of the vasoactive intestinal peptide (VIP) gene. An element located within 1 00 bases of the VIP promoter [the VIP cyclic AMP-responsive element (V IP-CRE)] confers cyclic AMP and phorbol ester responsiveness to hetero logous promoters. The possibility that these two regions of the VIP ge ne function cooperatively to determine tissue-specific and second mess enger-dependent expression of the VIP gene was addressed by assaying t ranscription from a VIP-luciferase reporter gene with progressive dele tions from the 5' flanking sequence of the gene, with or without inact ivation of the proximal VIP-CRE. Basal expression of the reporter gene in both SH-EP and SK-N-SH human neuroblastoma cells, which express en dogenous VIP mRNA, was absolutely dependent on the presence of the ups tream TSE. Full constitutive expression was also dependent on the inta ct VIP-CRE. Forskolin-mediated induction of the reporter gene in SH-EP and SK-N-SH cells was completely abolished by mutations in the VIP-CR E but not by deletion of the upstream sequence, indicating that the VI P-CRE alone determines cyclic AMP responsiveness. In contrast to repor ts that the VIP-CRE imparts 12-O-teaatradecanoylphorbol 13-acetate (ph orbol 12-myristate 13-acetate; PMA) responsiveness to heterologous pro moters, PMA stimulation in SK-N-SH cells was independent of an intact VIP-CRE but dependent on a region between -2.5 kb and the VIP-CRE. Seq uencing of the entire 5.2-kb VIP 5' flank revealed a consensus PMA-res ponsive element (TGACTCA) 2.25 kb upstream of the transcription start site that may represent the site imparting PMA responsiveness to the V IP gene.