Transcriptional regulation of the human secretin gene in duodenal and neuronal cells

Abstract

Secretin is a member of the glucagon/secretin/vasoactive intestinal peptide (VIP) family. To unravel the mechanism that regulates the human secretin gene expression in various cell lineages, 5 kb of the 5V flanking region of the human secretin gene were investigated. As secretin is expressed in the brain and gut, a human duodenal adenocarcinoma cell (HuTu-80) and a human neuroblastoma cell (SH-SY5Y) were employed in the present study. In duodenal cells, an E-box and two GC-boxes were found to be essential for the human secretin promoter activity. In vivo (chromatin immunoprecipitation, ChIP) and in vitro binding studies (gel mobility shift) showed that these motifs were binding sites for transcription factors, namely, NeuroD, E2A, Sp1 and Sp3. Expression of various ratios of Sp1 and Sp3 in Drosophila SL2 cells and HuTu-80 cells reflected the importance of the stoichiometric ratio of Sp1 and Sp3 on the control of secretin gene expression. The GC-boxes are located within a putative CpG island suggesting a role of CpG methylation in human secretin gene regulation. Hyper-methylated promoter regions were observed in secretin non-expressing cells (PANC-1 and HepG2). Also, there was an augmentation of the secretin transcript levels when the cells were treated 5V-Aza-2V deoxycytidine (5V-Aza-dC). These evidences suggested that promoter methylation was a key regulator for the cell-specific expression of secretin. As a neuropeptide, secretin is expressed in distinct central neurons. However, there is no information regarding how secretin gene is regulated in neuronal cells. For this reason, a well established neuronal differentiation cell model, SH-SY5Y, was used. Not just that we found high secretin transcript and peptide levels in this cell, the secretin gene expression and its promoter activity were up-regulated upon all-trans retinoic acid (RA) treatment. Within the promoter, a functional GC-box was found to be regulated by a brain-specific Sp protein, Sp4, and ubiquitous factors Sp1 and Sp3. The RA-induced activation is a partial result of a decreased of Sp3. In addition to the GC-box, an N1 motif in the close proximity was also responsible for the RA-induced secretin gene activation. Competitive gel mobility shift and Southwestern blot studies revealed the binding of Nuclear Factor I (NFI) with the N1 motif. Consistent with this observation, NFI-C transcript levels are augmented after RA-treatment. We conclude that the RA-induction of secretin gene in neuronal cells is regulated by the combined actions of reducing Sp3 and increasing NFI-C expressions.