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Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line.

ABSTRACT: The characteristics of the transport of the dipeptide glycylsarcosine were studied in the human colon carcinoma cell line Caco-2 grown as a monolayer on impermeable plastic support. Transport of glycylsarcosine in these cells was found to be Na(+)-independent, but was stimulated by an inwardly directed H+ gradient. This H(+)-dependent transport of glycylsarcosine was inhibited by di- and tri-peptides and also by the beta-lactam antibiotic cephalexin, but was unaffected by the amino acids glycine and leucine. The transport system exhibited a Michaelis-Menten constant (Kt) of 1.1 +/- 0.1 mM for glycylsarcosine. The specific activity of the transport system in this cell line was found to be maximal when the cultures were confluent. Treatment of the cells with phorbol esters which activate protein kinase C resulted in a significant inhibition of the transport system. This inhibition was specific and could be blocked if treatment was done in the presence of staurosporine, an inhibitor of protein kinase C. Kinetic analysis revealed that the inhibition was associated with a decrease in the maximal velocity, the Kt remaining unaffected. The phorbol-ester-induced inhibition of the peptide-transport system was not prevented by co-treatment with cycloheximide, an inhibitor of cellular protein synthesis. In addition, there was no change in the intracellular pH following treatment with the phorbol ester, suggesting that the effect was not due to alterations in the transmembrane pH gradient. It is concluded that the peptide/H+ co-transport system, which is known to exist in the normal intestine, is expressed in Caco-2 cells and that the function of the transport system is under the regulatory control of protein kinase C.

SUBMITTER: Brandsch M

PROVIDER: S-EPMC1138049 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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Project description: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.

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Expression and protein kinase C-dependent regulation of peptide/H+ co-transport system in the Caco-2 human colon carcinoma cell line.

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