The human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1) promoter: characterization and role in receptor expression during enterocytic differentiation of the colon cancer cell line Caco-2Cl.20.
The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5'-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5'-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5'-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2 cells.
Couvineau
A
A
Laboratoire de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, INSERM U-410, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris Cedex 18, France. coucou@bichat.inserm.fr
Maoret
J J
JJ
Rouyer-Fessard
C
C
Carrero
I
I
Laburthe
M
M
D001483:Base Sequence; D018938:Caco-2 Cells; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D020895:Enterocytes / Q000166:cytology* / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide / Q000235:genetics*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013394:Sucrase-Isomaltase Complex / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D004247:DNA; D013394:Sucrase-Isomaltase Complex; D018819:Dipeptidyl Peptidase 4