Abstract
Vasoactive intestinal peptide (VIP) gene expression is highly restricted throughout the neuroaxis and regulated by extracellular factors that activate tyrosine- or serine/threonine-directed protein kinase pathways. Cytokine, cyclic AMP, and tissue-specific response elements on the VIP gene have been characterized. Those mediating responsiveness to protein kinase C have not. The endogenous VIP gene and a 5.2-kilobase pair (kb) VIP-luciferase reporter gene, are up-regulated by phorbol 12-myristate 13-acetate (PMA) in SK-N-SH neuroblastoma cells. PMA stimulation was abolished by deletion of sequences at -1.37 to -1.28 or -1.28 to -0.904 kb, but not by removal of the single phorbol ester response element (TRE; TGACTCA) located at -2.25 kb. Mutation of sites at -1.32 or -1.20 that mediate neurotrophin responsiveness of the VIP gene (Symes, A., Lewis, S., Corpus, L., Rajan, P., Hyman, S. E., and Fink, J. S. (1994) Mol. Endocrinol. 8, 1750-1763) each reduced PMA induction in SK-N-SH cells by >50%, and double mutation abolished it. The two mutations also reduced basal VIP reporter gene transcription in SH-EP neuroblastoma cells expressing VIP constitutively. Both cis-active elements bound pre-existing AP-1 proteins in SH-EP- or PMA-stimulated SK-N-SH cell nuclear extracts. The AP-1 complex at both sites contained a Fos-related protein with c-Jun in SH-EP cells and c-Fos with a Jun-related protein in SK-N-SH cells. Recruitment of combinatorially distinct AP-1 complexes to these elements may underlie cell type-specific regulation of the VIP gene.
Highlights
Tern of expression in cortex, limbic system, hypothalamus, and the remainder of the suprachiasmatic nucleus postnatally (2, 4 –7)
We have previously reported that domains c and dЈ, acting in combination with other elements in the gene, separately contribute positive and negative regulatory functions required for neuroendocrine cell-specific expression of the Vasoactive intestinal peptide (VIP) gene and its silencing in non-VIPergic cells [18]
AP-1 binds to a cis-active element present on many genes (TRE; TGA(C/G)TCA) defined by its ability to mediate phorbol ester-dependent induction of transcription [25]
Summary
Vol 274, No 36, Issue of September 3, pp. 25588 –25593, 1999 Printed in U.S.A. Two Separate Cis-active Elements of the Vasoactive Intestinal Peptide Gene Mediate Constitutive and Inducible Transcription by Binding Different Sets of AP-1 Proteins*. The two mutations reduced basal VIP reporter gene transcription in SH-EP neuroblastoma cells expressing VIP constitutively Both cis-active elements bound pre-existing AP-1 proteins in SH-EP- or PMA-stimulated SK-N-SH cell nuclear extracts. Induction of VIP transcription by neurotrophic factors that activate the Jak-STAT tyrosine kinase signaling pathway requires a 180-bp cytokine-responsive element (CyRE) located at Ϫ1.33 kb in the VIP 5Ј-flank, containing binding sites for STAT and AP-1 proteins (19 –21). Candidate domains for regulation of VIP gene transcription by activation of protein kinase C have been tested using deletional and mutational analysis in the context of a VIP reporter gene that supports constitutive, tyrosine kinasemediated, and serine/threonine kinase-mediated transcriptional regulation Both the STAT and ncAP-1 sites of the VIPCyRE bound AP-1-related protein complexes and were required for phorbol ester-stimulated transcription in SK-N-SH neuroblastoma cells and for constitutive VIP gene transcription in SH-EP cells. The role of these two noncanonical AP-1 sites may be to recruit multiple, unique AP-1-related complexes to the transcriptional platform of the gene depending on first messenger signaling and cellular context
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