Lactase and sucrase-isomaltase gene expression during Caco-2 cell differentiation.
The Caco-2 cell line is derived from a human colon adenocarcinoma and differentiates in vitro into small-intestinal enterocyte-like cells, expressing the hydrolases lactase and sucrase-isomaltase. We cultured Caco-2 cells on permeable supports from 0 to 37 days after plating to study endogenous lactase and sucrase-isomaltase gene expression in relation to cell differentiation. Profiles of lactase and sucrase-isomaltase mRNA, protein and enzyme activity were analysed on a per-cell basis, using immunocytochemistry, RNase protection assays, metabolic polypeptide labelling and enzyme activity assays. Tight-junction formation was complete 6 days after plating. Immunocytochemistry of Caco-2 cross-sections showed lactase and sucrase-isomaltase predominantly in the microvillar membrane of polarized cells. mRNA, protein and enzyme activity of lactase appeared consecutively, reaching maximum levels 8-11 days after plating. Whereas lactase mRNA and protein biosynthesis showed a sharp decline after peak levels, lactase activity remained high until 37 days after plating. In contrast, mRNA and protein biosynthesis and activity of sucrase-isomaltase peaked successively 11-21 days after plating, and exhibited comparable levels throughout the entire experiment. The following conclusions were reached. (1) In Caco-2 cells, biosynthesis of lactase and sucrase-isomaltase is regulated by the amount of their mRNAs, indicating transcriptional control. (2) Sucrase-isomaltase activity is most probably transcriptionally controlled at all time points. (3) In contrast, lactase activity is initially regulated by its level of biosynthesis. After its peak at 8 days, the slow decline in activity compared with its biosynthesis indicates high stability. (4) Different mRNA profiles for lactase and sucrase-isomaltase indicate different mechanisms of transcriptional regulation of these genes.
Van Beers
E H
EH
Department of Pediatrics G8-260, Academic Medical Centre, Amsterdam, The Netherlands.
Al
R H
RH
Rings
E H
EH
Einerhand
A W
AW
Dekker
J
J
Büller
H A
HA
D002452:Cell Count; D002454:Cell Differentiation / Q000235:genetics; D003110:Colonic Neoplasms / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D043322:Lactase; D011233:Precipitin Tests; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D015347:RNA Probes / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013394:Sucrase-Isomaltase Complex / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D012313:RNA; D012260:Ribonucleases; D013394:Sucrase-Isomaltase Complex; D043322:Lactase; D001616:beta-Galactosidase