Jean-François Beaulieu

Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line

To gain further insight on the mosaic expression of specific functional intestinal markers (such as sucrase-isomaltase) in postconfluent Caco-2 cells, a human colon cancer cell line unique in its property to differentiate in vitro into a mature enterocyte-like cell type, a comparative study was undertaken to examine the morphological and functional differentiation of Caco-2 cells at various culture stages. The observations clearly indicate that Caco-2 cells can exist only in three different states in culture: homogeneously undifferentiated (at subconfluence), heterogeneously polarized and differentiated (between 0 and 20 days after confluence), and homogeneously polarized and differentiated (after 30 days). Indeed, in the intermediate state, a strong discrepancy is found among adjacent differentiating cells throughout the monolayer relative to sucrase-isomaltase expression as well as to cell morphology and brush border organization. Back-scattered electron imaging analysis showed a lack of correlation between these parameters at the cellular level. These observations indicate that morphological and functional differentiations of Caco-2 cells progress concomitantly according to a transient mosaic pattern, thus providing evidence that these two processes are not coupled.

Human cell models to study small intestinal functions: Recapitulation of the crypt-villus axis

The intestinal epithelium is continuously and rapidly renewed by a process involving cell generation, migration, and differentiation, from the stem cell population located at the bottom of the crypt to the extrusion of the terminally differentiated cells at the tip of the villus. Because of the lack of normal human intestinal cell models, most of our knowledge about the regulation of human intestinal cell functions has been derived from studies conducted on cell cultures generated from experimental animals and human colon cancers. However, important advances have been achieved over recent years in the generation of normal human intestinal cell models. These models include (a) intestinal cell lines with typical crypt cell proliferative noncommitted characteristics, (b) conditionally immortalized intestinal cell lines that can be induced to differentiate, and (c) primary cultures of differentiated villuslike cells that can be maintained in culture for up to 10 days. Each of these models should help in the investigation of the specific aspects of human intestinal function and regulation. Furthermore, taken together, these models provide an integrated system that allows an in vitro recapitulation of the entire crypt‐villus axis of the normal human small intestine. Microsc. Res. Tech. 49:394–406, 2000.

Differential expression of claudin‐2 along the human intestine: Implication of GATA‐4 in the maintenance of claudin‐2 in differentiating cells

Claudins, and particularly claudin‐2, are important regulatory components of tight junction permeability. A better understanding of the involvement of claudin‐2 in intestinal barrier functions requires the characterization of its distribution and regulation in the intestine. Interestingly, the claudin‐2 gene promoter harbors a number of similarities to that of sucrase‐isomaltase, a marker of enterocyte differentiation. We thus investigated the expression of claudin‐2 in relation to the transcription factors CDX2, HNF‐1α, and GATA‐4 in the human intestine. The characterization of claudin‐2 and the expression of the above transcription factors were performed by immunofluorescence, Western blot, and RT‐PCR in the developing human intestinal epithelium. The functional role of CDX2, HNF‐1α, and GATA‐4 on claudin‐2 regulation was also examined by ectopic expression studies in intestinal cell models. Claudin‐2 was detected in both crypt and villus cells of the small intestine but restricted to undifferentiated crypt cells in the colon. CDX2 and HNF‐1α were expressed along the entire intestine whereas GATA‐4 was undetectable in the colon. Accordingly, in the colonic Caco‐2 cell model, claudin‐2 was found to be present only in undifferentiated cells. Like in the colonic epithelium, GATA‐4 was found to be also lacking in Caco‐2 cells while CDX2 and HNF‐1α were present at significant levels. Cotransfection experiments showed that the claudin‐2 promoter was activated by CDX2, HNF‐1α, and GATA‐4 in a cooperative manner. Furthermore, forced GATA‐4 expression in Caco‐2 cells enhances maintenance of claudin‐2 expression during differentiation. These observations suggest that optimal claudin‐2 expression in the gut relies on the presence of GATA‐4, suggesting a role for this factor in intestinal regionalization.

Reciprocal expression of laminin A-chain isoforms along the crypt-villus axis in the human small intestine

BACKGROUND/AIMSnIntestinal epithelial cell proliferation, migration, and differentiation are susceptible to various influences along the crypt-villus axis including compositional changes in the basement membrane and differential expression of receptors for these components. However, laminin has been consistently detected at the epithelial basement membrane without significant variation in relation to the crypt-villus functional unit. The aim of this study was to examine the distribution of individual laminin chains.nnnMETHODSnThe patterns of distribution of laminin A, M (an A-chain variant), B1, and B2 chains as well as the integrins alpha 6 beta 1 and alpha 6 beta 4, two laminin receptors, were determined by indirect immunofluorescence in the adult human small intestine.nnnRESULTSnMarkedly distinct patterns of laminin chain expression were observed along the crypt-villus axis. The A chain was found predominantly associated with the differentiated epithelial cells on the villus whereas the M chain was restricted to the basement membrane of crypts. However, both B1 and B2 chains were detected all along the intestinal basement membrane. Furthermore, alpha 6 beta 1 and alpha 6 beta 4 integrins were found to be expressed by all enterocytes.nnnCONCLUSIONSnThese observations show a reciprocal expression of laminin isoforms along the crypt-villus axis and support the concept that the intestinal epithelial basement membrane is subjected to intricate compositional changes.

Interactions between laminin and epithelial cells in intestinal health and disease.

Laminins are a multigene family of extracellular matrix molecules. Quantitatively, they are one of the most abundant glycoproteins present in basement membranes. Functionally, they can modulate several key biological activities, including cell adhesion and migration, gene expression and cell survival. Variability in the spatial and temporal expression of laminins, as well as of their specific receptors of the integrin family, in various tissues and organs, suggests that different laminins perform distinct functions. This article focuses on the human intestinal epithelium as a paradigm to illustrate the potential relationship between laminin-cell interactions and the cell state. This rapidly renewing epithelium consists of spatially separated proliferative and differentiated cell populations located in the crypts and on the villi, respectively. Differential distributions of the various laminins and laminin-binding integrins have been observed along the crypt-villus axis in both the developing and the adult intestine, and important alterations in the pattern of laminin expression have been reported in various intestinal pathologies, such as tufting enteropathy, Crohns disease and ulcerative colitis, and colorectal cancer. More-direct approaches, including experimentation with in vitro and in vivo models, have provided evidence in support of a role for laminins in intestinal cell functions. Although further work is still needed, laminins emerge more and more as key regulators of specific cell functions important in both intestinal health and intestinal disease.

Integrins as mediators of epithelial cell‐matrix interactions in the human small intestinal mucosa

The intestinal epithelium is a highly dynamic tissue, which depends on a variety of factors for the regulation of its rapid renewal and expression of digestive functions. Over the last 10 years, it has become evident that among these factors are cell interactions with the extracellular matrix, more specifically with the underlying basement membrane, through a series of specific cell membrane receptors, many of which are integrins. Integrins regulate the assembly of adhesive junctions as well as the activation of various signaling pathways, leading to the modulation of gene expression. The analysis of the integrin repertoire along the crypt‐villus axis in the human small intestinal epithelium identifies a number of β1 and β4 integrins, showing differential patterns of expression relative to its two functional compartments. Among them are the integrins α3β1, α7Bβ1 and the functional form of α6β4 that appear to be related, in concert with the distribution of their ligands, to the process of intestinal cell differentiation, and the integrins α2β1, α1β1, α5β1, and the non‐functional form of α6β4 that seem to be coupled with the undifferentiated/proliferative status of crypt cells. These observations delineate the potential complexity of the organization of epithelial cell‐matrix interactions involved in the maintenance of the human intestinal crypt‐villus axis. Microsc. Res. Tech. 51:169–178, 2000.

Immunolocalization of extracellular matrix components during organogenesis in the human small intestine

SummaryThe expression and distribution of several major extracellular matrix macromolecules were investigated at the epithelial-mesenchymal interface of the human fetal small intestine from 8 to 20 weeks of gestation. Localization of heparan sulfate proteoglycan, type-IV collagen and laminin, three basement membrane components, as well as fibronectin and tenascin, were assessed by indirect immunofluorescence staining on cryostat sections, and correlated to morphogenesis and epithelial cell differentiation. Basement membrane components and fibronectin were all detected as early as 8 weeks (a time when the epithelium is still stratified and does not express sucrase-isomaltase). Tenascin appeared only after short villi had developed (around 10 weeks) and was restricted to the connective tissue at the tip of villus rudiments. At 18 weeks, well-formed villi and crypts were apparent. The antibody against heparan sulfate proteoglycan stained exclusively the epithelial basement membrane. Anti-type-IV collagen and anti-laminin anti-bodies stained the epithelial basement membrane and also cellular and fibrillar structures in the lamina propria. Fibronectin was found uniformly distributed over the lamina propria except in the upper third position of the villus core. On the contrary tenascin was mainly localized in the stroma at the tip of the villi. Staining for tenascin was also detected at the epithelial-mesenchymal interface of the villus and in the mesenchyme immediately surrounding budding crypts. These results provide basic data concerning the development of the human gut, and suggest that extracellular matrix components could be involved in the remodelling process of the intestinal mucosa.

Altered Expression of Laminins in Crohn's Disease Small Intestinal Mucosa

Laminins are a large family of heterotrimeric basement membrane molecules that mediate crucial cell functions such as adhesion, proliferation, migration, and differentiation. Up to now, three distinct laminins have been identified in the normal human small intestinal epithelium. Laminin-1 (alpha1beta1gamma1) and laminin-5 (alpha3beta3gamma2) are mainly expressed at the base of villus cells, whereas laminin-2 (alpha2beta1gamma1) is restricted to the bottom of the crypts. The expression of these molecules has not yet been studied in Crohns disease (CD), but it could be altered, in light of the important changes occurring in the architecture of the crypt-villus axis under the active state of the disease. To test this hypothesis, the expression of laminin alpha1, alpha2, and alpha3 subunits was analyzed in control, inflamed, and corresponding uninflamed CD small intestinal specimens by indirect immunofluorescence and reverse transcriptase-polymerase chain reaction. Surprisingly, alpha1 and alpha3 remained strongly expressed by all villus cells, whereas alpha2, normally expressed in the bottom of the crypts in control and uninflamed CD specimens, was lacking in inflamed CD specimens. However, this loss of alpha2 expression was associated with a significant up-regulation of both alpha1 and alpha3 expression in the crypts of inflamed CD specimens. A significant up-regulation of the alpha1 subunit was also observed in the crypts of uninflamed CD specimens. At the transcript levels, alpha1 was found significantly higher in inflamed than uninflamed CD specimens. Taken together, these observations identify important alterations in laminin expression in the small intestine with CD and suggest that compositional changes in the epithelial basement membrane may play a role in this disease.

Expression of Functionally Distinct Variants of the β4A Integrin Subunit in Relation to the Differentiation State in Human Intestinal Cells

Integrins are important mediators of cell-laminin interactions. In the small intestinal epithelium, which consists of spatially separated proliferative and differentiated cell populations located, respectively, in the crypt and on the villus, laminins and laminin-binding integrins are differentially expressed along the crypt-villus axis. One exception to this is the integrin α6β4, which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the β4 subunit expression with different antibodies revealed that two forms of β4 exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa β4A subunit, whereas undifferentiated crypt cells express a novel β4A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (β4Actd−). This new form was not found to arise from alternative β4 mRNA splicing. Moreover, we found that these two β4A forms can associate into α6β4A complexes; however, the β4Actd− integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel α6β4Actd− integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct.

Uncoordinated, transient mosaic patterns of intestinal hydrolase expression in differentiating human enterocytes.

The heterogenous expression of brush border membrane hydrolases by the human enterocyte‐like Caco‐2 cell line during morphological and functional differentiation in vitro was investigated at the cellular level. Indirect immunofluorescence revealed that the heretogenous (“mosaic”) expression of sucrase‐isomaltase, lactase, aminopeptidase N, and alkaline phosphatase was, in fact, transient in nature. The labeling indexes for each hydrolase gradually increased during culture at postconfluence in order to reach a maximum (≥90%) after 30 days, concomitant with an upregulation of their respective protein expression levels. In contrast, dipeptidylpeptidase IV labeling remained relatively constant. Backscattered electron imaging analysis in midstage (12 days postconfluence) monolayers demonstrated a lack of correlation between brush border membrane development and expression of each enzyme studied. Moreover, double immunostaining revealed that none of the other four hydrolases correlated directly with sucrase‐isomaltase expression. Finally, immunodetection for the proliferation‐associated antigen Kl‐67 revealed a transient mosaic pattern of proliferation which was inversely related to Caco‐2 cell differentiation. These data indicate that enterocytic differentiation‐related (as well as proliferation‐related) gene expression in Caco‐2 cells is regulated but uncoordinated at the cellular level, suggesting that an overall control mechanism is lacking.