Molecular cloning of the rat intestinal trefoil factor gene. Characterization of an intestinal goblet cell-associated promoter.
Intestinal trefoil factor (ITF) is a small peptide bearing the unique motif of intrachain disulfide bonds characteristic of the trefoil family. Previous work had localized expression of ITF primarily within goblet cells in the small and large bowel, making it a candidate gene for the study of the molecular basis of intestinal and goblet cell-specific gene expression. In order to study the regulation of ITF expression, we have cloned the rat ITF gene and sequenced 1.7 kilobases of the 5'-flanking region. RNase protection analysis demonstrated a single transcriptional start site. Various lengths of the 5'-flanking region were linked to the reporter gene luciferase and transfected into the colon cancer cell lines LS174T and Caco-2, representing, respectively, cells with and without goblet cell-like phenotype. Expression in the goblet cell-like LS174T colon cancer cell line was nearly 10-fold greater than expression in Caco-2 cells which exhibit columnar enterocyte-like phenotype. The pattern of goblet cell-associated selective transcription required only 153 base pairs of the rat ITF 5'-flanking sequence. Transfection of a construct of human growth hormone under the control of the rat ITF promoter in the N2 subclone of HT-29 cells demonstrated expression of the reporter gene only in those cells exhibiting a goblet cell phenotype as assessed by expression of immunoreactive mucin. These initial studies of the 5'-flanking region of the ITF gene demonstrate the presence of cis-regulatory elements capable of directing goblet cell specific expression.
Sands
B E
BE
Gastrointestinal Unit, Massachusetts General Hospital, Boston 02114, USA.
Ogata
H
H
Lynch-Devaney
K
K
deBeaumont
M
M
Ezzell
R M
RM
Podolsky
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D013006:Growth Hormone / Q000096:biosynthesis; D006133:Growth Substances / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009077:Mucins / Q000096:biosynthesis; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3
D006133:Growth Substances; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides; C099037:TFF3 protein, rat; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D013006:Growth Hormone; D008156:Luciferases