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Tissue‐Specific Expression of the Vasoactive Intestinal Peptide Gene Requires Both an Upstream Tissue Specifier Element and the 5′ Proximal Cyclic AMP‐Responsive Element

Abstract: 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.