Dysregulation of laminins in intestinal inflammation.

Laminins are structural components of basement membranes that regulate and control many cellular functions. Changes in basement membrane composition play significant roles in etiology of diseases. Inflammatory bowel diseases are conditions that lead to defects in the mucosal barrier which includes the basement membrane underlying the epithelium. This review will summarize the main findings related to the involvement of laminins and of the laminin-binding receptors in inflammatory conditions such as Crohn's disease and ulcerative colitis. We will review the current literature devoted to studies in humans (immunolocalisation, genetic factors, microarray data), as well as experimental cell models that show that laminins contribute to the inflammation process probably linked to the deregulation of proinflammatory cytokines.

The role of the basement membrane as a modulator of intestinal epithelial-mesenchymal interactions.

Intestinal development is a process of continuous dynamic bidirectional crosstalk between epithelial and underlying mesenchymal cells. This crosstalk is mediated by well-dissected signaling pathways. Another crucial actor in the epithelio-mesenchymal interactions is the stromal microenvironment, which is composed of extracellular matrix molecules. Among them, the basement membrane (BM) molecules are secreted by the epithelium and mesenchyme in a complementary manner. These molecules signal back to the cells via the integrins or other specific receptors. In this review, we mainly focus on the BM molecules, particularly laminins. The major BM molecules are organized in a complex molecular network, which is highly variable among organs. Cell culture, coculture, and grafting models have been of great interest in understanding the importance of these molecules. Mouse gene ablation of laminin chains are interesting models, which often lead to embryonic death and are frequently accompanied by compensatory processes. Overall, the BM molecules have a crucial role in the careful maintenance of intestinal homeostasis.

Basement membrane component laminin-5 is a target of the tumor suppressor Smad4.

The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.

In vitro models of intestinal epithelial cell differentiation.

The intestinal epithelium is a particularly interesting tissue as (1) it is in a constant cell renewal from a stem cell pool located in the crypts which form, with the underlying fibroblasts, a stem cell niche and (2) the pluripotent stem cells give rise to four main cell types: enterocytes, mucus, endocrine, and Paneth cells. The mechanisms leading to the determination of phenotype commitment and cell-specific expressions are still poorly understood. Although transgenic mouse models are powerful tools for elucidating the molecular cascades implicated in these processes, cell culture approaches bring easy and elegant ways to study cellular behavior, cell interactions, and cell signaling pathways for example. In the present review, we will describe the major tissue culture technologies that allow differentiation of epithelial cells from undifferentiated embryonic or crypt cells. We will point to the necessity of the re-creation of a complex microenvironment that allows full differentiation process to occur. We will also summarize the characteristics and interesting properties of the cell lines established from human colorectal tumors.

Generation of a conditionally null allele of the laminin alpha1 gene.

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (alpha1, beta1, gamma1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin alpha1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed-allele-carrying mice (Lama1(flox/flox)) display no overt phenotype. Lama1(flox/flox) mice were crossed with transgenic deleter mice (CMV-Cre) to generate Lama1-deficient mice (Lama1(Delta/Delta)). Lama1(Delta/Delta) embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin alpha1 is normally highly expressed. In parallel, Lama1(Delta/Delta) embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time- and tissue-specific inactivation of the Lama1 gene in various organs.

Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.

Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.

Tenascin and laminin function in target recognition and central synaptic differentiation.

The influence of the target cell-issued extracellular molecules tenascin-C and laminin on synaptogenesis was studied in mixed primary cultures of pituitary melanotrophs and hypothalamic neurons. We could demonstrate in this neuron-target co-culture system a new role for tenascin-C, which appeared to be expressed as an early and transitory signal of target recognition for selective afferent fibers. Tenascin-C expression disappeared from the melanotrophs soon after the establishment of neural contacts. Concomitantly, the melanotrophs became immunoreactive for laminins, and more specifically for the synaptic isoform beta2 chain-containing laminin. The laminin signal appeared to be involved in the induction of synaptic differentiation, selectively with fibers containing both dopamine and GABA, like those innervating the melanotrophs in situ.

Adhesion complexes implicated in intestinal epithelial cell-matrix interactions.

This article review summarizes data on cell-substratum adhesion complexes involved in the regulation of cellular functions in the intestine. We first focus on the molecular composition of the two main adhesion structures-the beta1 integrin-adhesion complex and the hemidesmosome-found in vivo and in two human intestinal cell lines. We also report the key findings on the cellular behavior and response to the extracellular matrix that involve integrins, the main transmembrane anchors of these complexes. How the dynamics of cell/extracellular matrix interactions contribute to cell migration, proliferation, differentiation, and tumorigenicity is discussed in the light of the data provided by the human intestinal cells.

Transcriptional regulation of laminin gene expression.

Laminins are the most abundant structural non-collagenous glycoproteins ubiquitously present in basement membranes. They are multidomain molecules consisting of of alpha, beta, and gamma chains. Although the precise functional differences between the laminin variants are not well understood, the diversity of laminin isoforms may reflect the formation of distinct basement membranes. The laminins display a remarkable restricted expression profile, suggesting a fine regulation of their genes. In this review, we focus on the most recent developments of laminin biology, centering on transcriptional and posttranscriptional controls. We discuss only those laminin chains whose gene organization and promoter elements have been characterized and proved to be functional. When possible, we correlate the effects of growth factors, cytokines, retinoids, and transcription factors on laminin gene expression with the identity of cis-acting elements in their genomic control regions.

Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF.

Talin concentrates to the midbody region during mammalian cell cytokinesis.

In this study we investigated the cellular distribution of talin, a cytoskeletal protein, during mammalian cell cytokinesis. Immunohistochemical experiments on various carcinoma cell lines and mesenchyme-derived cells reveal that talin displays a cell cycle-dependent cellular localization. During metaphase, talin is located in the centromeric region of the chromosome, like the TD-60 protein and intrinsic centromere components detected by a CREST serum. From anaphase to telophase, talin is present in the cleavage furrow. As the cells progress to cytokinesis, when the furrow is complete, talin is concentrated in the midbody structures, as assessed by immunofluorescence and confirmed by Western blot experiments on purified midbodies. Double staining experiments reveal that alpha-tubulin, TD-60 protein, and talin co-localize in the midbodies. These results suggest that talin, in addition to its implication in focal adhesion organization and signaling, may play a critical role in cytokinesis. (J Histochem Cytochem 47:1357-1367, 1999)

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.

Characterization of human intestinal stromal cell lines: response to cytokines and interactions with epithelial cells.

The maintenance of the physiological homeostasis of the gut mucosa characterized by continuous proliferation and differentiation processes results from epithelial-mesenchymal cell cross-talk. To set out stable and homogeneous models for the study of the (dys)regulation of various morphofunctional aspects, we established and characterized three clonal cell lines (C9, C11, and C20) derived from human duodenal mucosal connective tissue. We defined the expression of (i) cytoskeletal proteins; (ii) basement membrane molecules (laminins, collagen IV, nidogen) which have been shown formerly to be deposited at the epithelial/mesenchymal interface in situ by the mesenchymal compartment; and (iii) soluble factors, HGF, and TGFbeta1. The three cell lines display common but also specific proliferative responses to cytokines (IL1beta, IL2, IL8, TNFalpha, IFNgamma, TGFbeta1, and HGF). When cocultured with embryonic intestinal endoderms or with human colonic Caco2 or HT29 cancer cells, C9 versus C11 and C20 cell lines induced limited versus extensive growth of the associated epithelial cells. In addition C20 cells allowed spreading of HT29 cells with the formation of a basement membrane at the heterologous interface. Morphogenesis obtained by intracoelomic grafts of associations comprising the mesenchymal cell lines and intestinal endoderms was also different among those composed of C9 cells or of C11 or C20 cells. In conclusion, these data indicate that the mucosal connective tissue is heterogeneous and comprises several phenotypically different mesenchyme-derived cells whose equilibrium may be important in the gut homeostasis. These cells can now be used to define tissue-specific factors which may be involved in the physiopathology of the intestinal epithelium.

Cellular and molecular partners involved in gut morphogenesis and differentiation.

The intestinal mucosa represents an interesting model to study the cellular and molecular basis of epithelial-mesenchymal cross-talk participating in the development and maintenance of the digestive function. This cross-talk involves extracellular matrix molecules, cell-cell and cell-matrix adhesion molecules as well as paracrine factors and their receptors. The cellular and molecular unit is additionally regulated by hormonal, immune and neural inputs. Such integrated cell interactions are involved in pattern formation, in proximodistal regionalization, in maintenance of a gradient of epithelial proliferation and differentiation, and in epithelial cell migration. We focus predominantly on two aspects of these integrated interactions in this paper: (i) the role of basement membrane molecules, namely laminins, in the developmental and spatial epithelial behaviour; and (ii) the importance of the mesenchymal cell compartment in these processes.

Human colonic cancer cells synthesize and adhere to laminin-5. Their adhesion to laminin-5 involves multiple receptors among which is integrin alpha2beta1.

In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observations, these data provide a baseline knowledge for the understanding of the regulation of laminin-5 in normal and pathological intestine.

The laminins: role in intestinal morphogenesis and differentiation.

Dynamic and reciprocal heterotypic cell interactions are crucial for intestinal morphogenesis and differentiation. This paper emphasizes the role of basement membrane molecules and in particular of laminins as potent mediators in this intercellular cross talk. Changes in the expression or localization of laminin isoforms or of integrins during development and cell migration strengthen the concept that heterogeneity in cell-matrix interactions could mediate distinct cell responses. A combination of genetic or biochemical approaches associated with in vitro models allows us to study the potential role of each laminin isoform in basement membrane assembly, cell migration, or cell differentiation.

Cytokines modulate fibroblast phenotype and epithelial-stroma interactions in rat intestine.

BACKGROUND & AIMS: Homeostasis of the intestinal epithelium depends on interactions with the underlying connective tissue that may be altered during the pathogenesis of disease. The aim of this study was to establish whether fibroblast phenotype can influence morphogenesis and differentiation of the gut epithelium. METHODS: Permanently growing, nontumorigenic, homogenous fibroblast lines were established from postnatal rat intestinal mucosa. Their phenotypic characterization included their growth response to cytokines and expression of cytoskeletal and membrane markers, as well as expression of basement membrane components, laminin 1 and collagen IV. Their influence on epithelial growth, functional polarization, and morphogenesis was analyzed using coculture and tissue grafting of the fibroblast lines with fetal gut endoderm. RESULTS: Two intestinal fibroblast lines are described, one that supports normal intestinal morphogenesis and differentiation, and one that induces growth of fetal epithelial cells. Among the phenotypic differences between the two lines, the former differentiates into myofibroblasts in response to transforming growth factor beta1 and, in basal conditions, expresses twice as much laminin than the latter. The growth of the two lines is also affected differentially by transforming growth factor beta1 and interleukin 2. CONCLUSIONS: Cytokines, which are expressed in association with inflammation, regulate fibroblast differentiation. Fibroblasts may modify the function and organization of the overlying intestinal epithelium.

Effect of prenatal exposure to ethanol on intestinal development of rat fetuses.

BACKGROUND: Chronic alcoholism in pregnant animals and humans lead to general growth impairment in their offspring, which show multiple birth defects and delayed grown (fetal alcohol syndrome). Here we study the maturation of the intestine under the effect of chronic exposure to ethanol in utero together with associated malnutrition. METHODS: Lactase, acid beta-galactosidase, maltase, and alkaline phosphatase activity profiles were monitored in 18-, 19-, 20-, and 21-day-old fetuses from rats kept under three nutritional treatments before and during gestation: alcohol-treated (25% ethanol in drinking water), fiber-treated (50% cellulose-diluted diet) as a control of the malnutrition associated with chronic alcoholism, and control or normal diet. Serum corticosterone determination and lactase immunolocalization were carried out. To detect possible direct effects of ethanol during the period of mucosa development, intestinal explants from 18-, 19-, and 20-day-old control fetuses were cultured either in the basal medium alone or in a medium containing 25 mM ethanol for 72, 48, and 24 h of incubation, respectively. RESULTS: Following chronic ethanol exposure in utero, intestinal weight and brush-border protein content and the specific activities of lactase, acid beta-galactosidase, maltase, and alkaline phosphatase were significantly lower than those of nutritional controls. Organ culture results, under the assay conditions stated, did not show a direct effect of ethanol 25 mM on prenatal mucosal functionality. CONCLUSIONS: All these results suggest that maternal malnutrition is not primarily responsible for the impaired intestinal maturation in rat fetuses from alcohol-treated mothers; indirect effects of ethanol and/or its derivatives throughout embryo-fetal development could be necessary to promote this intestinal delay.

Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit, laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-alpha1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cell differentiation.