Abstract. The expression of the constituent o~1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the ~1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of otl-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the etl chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the 131/~/1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the otl chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin odchain gene may be a regulatory element in determining cell differentiation.ASEMENT membranes (BM) l are specialized, sheetlike extracellular matrices dividing tissues into compartments; they form the supporting structure on which epithelial cells lie. BM consists of ubiquitous components: type IV collagen, laminin, nidogen (also known as entactin), and heparan sulfate proteoglycan (perlecan) that are secreted locally by epithelial or parenchymal as well as fibroblastic cells. The BM functions as a dynamic structure in tissular morphogenesis, differentiation, and maintenance of the mature structural and functional steady states; its constituent molecules are able to regulate different types of cell behavior such as adhesion, proliferation, and maintenance of cell polarity either directly or via the delivery of growth/migration signals.Laminin-1, a major constituent of the basement membranes, is the earliest molecule produced in embryogenesis; its potential importance has been largely stressed in the last decade (Timpl and Brown, 1994). Indeed, laminin-1, as well as type IV collagen, has been shown to self-assemble,

Rat ggnomic DNA was isohtted by homology with Cdvl, a routine homeogene seleettvely expressed in intestinal eclh ot'endodermal origin. Southern blot analys~s indtcated that the rat genome contains a single or a small number of closely related Cdx gene(s). A major 1.7 kb Cdr mRNA was detected in neonate, suckling and adult rats whereas a 6 5 kb mRNA was restricted to sucklings and adults. Both transcripts showed decreasing concentration from the colon toward~ the proximal part of the small intestine. No obvtous correlation could be established with the patterns of expression of transcripts corresponding to markers of cell proliferation and cell differentmtton during postnatM development.

No abstract

No abstract

We have shown by electrophoretic mobility shift assays that the nucleotide sequence CE-LPHl, centred at position -49 with respect to the transcription start site of the rat gene encoding intestinal lactase-phlorizin hydrolase, interacts in vitro with nuclear proteins present in the jejunum of suckling animals. Proteins binding to this element were also found in organs of endodennal origin that do not (or no longer) express lactase-phlorizin hydrolase, i.e. the colon, lung and the liver, but not in the brain. However, a DNA-protein interaction was hardly detected with nuclear extracts prepared from adult tissues, although typical factors binding to the Spl binding site were detected at the adult stage as in the sucklings. Southwestern blotting experiments conducted with nuclear extracts prepared from the tissues of suckling rats indicated that CE-LPHl interacts with several factors in the jejunum, colon, lung and the liver. Some of these DNA-binding proteins are specifically expressed in the jejunum or in the liver, whereas others seem to be shared with the colon and the lung. Hence, the c&element CE-LPHl located in close vicinity to the pseudo-TATA-box of the intestinal lactase-phlorizin hydrolase gene promoter interacts in vitro with a family of nuclear proteins which may represent markers of the endodermal lineage predominantly expressed prior to weaning.

We have determined the sequence of a 2784 bp rat genomic fragment originating from the 5' region of the gene coding for intestinal lactase-phlorizin hydrolase. The fragment overlaps the gene exon 1, part of the intron 1 and the 5'-upstream segment including a TATA-like box. Over 155 bp, the upstream segment shows 72% similarity with the corresponding sequence in human. Far upstream, the rat sequence exhibits a Calcium Responsive Element and putative binding sites for AP2, C/EBP, and CTF/NF. The intron contains a T-rich sequence that may cause DNA helix distortion.