Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation.

Abstract

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,

Laminin-5-integrin interaction signals through PI 3-kinase and Rac1b to promote assembly of adherens junctions in HT-29 cells

“…However, in HT-29 cells, laminin 5, which activates PI 3-kinase, is unable to induce full cell differentiation (data not shown). This discrepancy might be explained by the fact that contrary to HT-29 cells, Caco-2 cells at confluence produce laminin 1, which has been previously described to induce both enterocytic differentiation (Basson et al, 1996;De Arcangelis et al, 1996;Vachon and Beaulieu, 1995) and PI 3-kinase activation (Shaw et al, 1997). The HT-29 cell model indicates that, although necessary, PI 3-kinase signaling is not sufficient for full differentiation, which requires additional factors.…”

“…Laminins represent the most abundant glycoproteins of the basement membrane and display the highest variability in their spatial and temporal expression either during intestinal development or in the adult (Simon-Assmann et al, 1994;Simon-Assmann et al, 1998;Teller and Beaulieu, 2001). Beside their fundamental role in organizing the basement membrane network, laminins promote several cellular processes such as adhesion, growth, polarization, differentiation and gene expression (Baker et al, 1996;Colognato and Yurchenco, 2000;De Arcangelis et al, 1996;Lampe et al, 1998;Vachon and Beaulieu, 1995). The distinct biological activities of laminins depend on both the isoform type and the repertoire of laminin receptors expressed.…”

Overexpression of laminin ?1 chain in colonic cancer cells induces an increase in tumor growth

“…These data are complementary to those observed by implanting the formerly described laminin ␣1 antisense targeted Caco2 cells. 11 Indeed the disruption of the laminin ␣1 mR⌵〈 accompanied by a decrease in laminin-10 staining 11 blocked the tumor growth in vivo (Fig. 8a, b) without significant modification in the histologic grade of the tumor.…”

“…Several major functions have been assigned to the constitutive ␣1 chain of laminin-1, among which are the polarization of developing kidney tubules, 6,7 lung alveolar formation, 8 development of submandibular glands 9 and polarization and functional differentiation of mammary and intestinal cells. 10,11 Conversely, alterations in cell-matrix and cell-cell interactions have been shown in a number of pathologic situations, such as inflammatory processes, wound healing and malignant transformation. In colorectal cancer, perturbations in the production, deposition and degradation of matrix molecules, among which are laminins, have been observed.…”

Epithelial organization and cyst lumen expansion require efficient Sec13–Sec31-driven secretion

“…The laminin is predominantly laminin-1, including the a1, b1 and c1 subunits, i.e. the essential form of laminin for correct Caco-2 polarity and organization (De Arcangelis et al, 1996). Caco-2 cells form cysts when embedded in 3D EHS-derived matrix such as Matrigel or Geltrex (Ivanov et al, 2008;Jaffe et al, 2008).…”