Characterization of Promoter Elements Required for Cell-Specific Expression of the Neurotensin/Neuromedin N Gene in a Human Endocrine Cell Line

Abstract

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.

Molecular Characterization and Differential Gene Induction of the Neuroendocrine‐Specific Genes Neurotensin, Neurotensin Receptor, PC1, PC2, and 7B2 in the Human Ocular Ciliary Epithelium

“…A great deal of information has been accumulated on the induction of NT mRNA expression in neuroendocrine cell lines, including the rat medullary thyroid carcinoma (rMTC), the rat pheochromocytoma (PC12), and human carcinoid BON cells (Tischler et al, 1986;Kislauskis and Dobner, 1990;Evers et al, 1995;Scarcériaux et al, 1995). Semiquantitative RT- in the human CB and human ciliary NPE cell line, ODM-2.…”

Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene

“…The procedures for 32 P oligonucleotide labeling and electrophoretic mobility shift assay were as described previously (24).…”

“…Human NT/N promoter-luciferase gene constructs (3 g) were co-transfected with 0.2 g of the plasmid p110 (40), which contains the ␤-galactosidase gene fused to the SV40 early promoter, by calcium phosphate co-precipitation as described previously (24). The ␤-galactosidase assays were carried out as described previously (41).…”

“…The extracts were quick-frozen, stored in aliquots at Ϫ80°C, and used within 2 months of extraction. Whole-cell extracts from COS-1 cells transfected with vectors pMT2-HNF4 (from Dr. Frances M. Sladek, Riverside, CA), pTM2-ARP1, pMT2-ARP⌬A1, pMT2-ARP⌬A6, pMT2-ARP⌬A7, pMT2-RXR␣, pMT2-RAR␣, pMT2-RAR␤, and pMT2-null (all kindly provided by Dr. Karathanasis, Pfizer Global Research and Development) were prepared as described previously (24). In vitro DNase I footprinting was performed using the hNT/N promoter fragment (Ϫ373 to ϩ26) as described previously (24).…”

“…In the GI tract, the NT/neuromedin N (NT/N) gene is developmentally regulated in the human and rat intestines in a temporospatial manner (21)(22)(23). The promoter proximal 5Ј-flanking region of the rat NT/N gene has previously been shown to integrate multiple environmental stimuli in neuroendocrine PC12 cells and to be sufficient for cell-specific expression in enteroendocrine BON cells (24,25). These results indicated that an array of distinct cis-regulatory motifs localized in the proximal 120 bp of the 5Ј-flanking sequence are required for both inducible and high level, constitutive NT/N expression, depending on the cell type.…”