Tissue-specific expression of the vasoactive intestinal peptide gene requires both an upstream tissue specifier...

An upstream enhancer element [tissue specifier element (TSE)] located between 4.66 and 4.02 kb from the transcription start site is important for cell type-specific expression and phorbol ester induction of the vasoactive intestinal peptide (VIP) gene. An element located within 100 bases of the VIP promoter [the VIP cyclic AMP-responsive element (VIP-CRE)] confers cyclic AMP and phorbol ester responsiveness to heterologous promoters. The possibility that these two regions of the VIP gene function cooperatively to determine tissue-specific and second messenger-dependent expression of the VIP gene was addressed by assaying transcription from a VIP-luciferase reporter gene with progressive deletions from the 5' flanking sequence of the gene, with or without inactivation of the proximal VIP-CRE. Basal expression of the reporter gene in both SH-EP and SK-N-SH human neuroblastoma cells, which express endogenous VIP mRNA, was absolutely dependent on the presence of the upstream TSE. Full constitutive expression was also dependent on the intact 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-CRE but not by deletion of the upstream sequence, indicating that the VIP-CRE alone determines cyclic AMP responsiveness. In contrast to reports that the VIP-CRE imparts 12-O-tetradecanoylphorbol 13-acetate (phorbol 12-myristate 13-acetate; PMA) responsiveness to heterologous promoters, 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. Sequencing of the entire 5.2-kb VIP 5' flank revealed a consensus PMA-responsive element (TGACTCA) 2.25 kb upstream of the transcription start site that may represent the site imparting PMA responsiveness to the VIP gene.