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 fami ly, Previous work had localized expression of ITF primarily within gob let cells in the small and large bowel, making it a candidate gene for the study of the molecular basis of intestinal and goblet cell-specif ic 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 tra nscriptional start site, Various lengths of the 5'-flanking region wer e linked to the reporter gene luciferase and transfected into the colo n cancer cell lines LS174T and Caco-2, representing, respectively, cel ls with and without goblet cell-like phenotype, Expression in the gobl et 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 transcript ion 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 gobl et cell phenotype as assessed by expression of immuno-reactive mucin, These initial studies of the 5' flanking region of the ITF gene demons trate the presence of cis-regulatory elements capable of directing gob let cell specific expression.