Pierre H. Vachon

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.

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders.

Merosin-deficient congenital muscular dystrophy. Partial genetic correction in two mouse models.

Humans and mice with deficiency of the alpha2 subunit of the basement membrane protein laminin-2/merosin suffer from merosin-deficient congenital muscular dystrophy (MCMD). We have expressed a human laminin alpha2 chain transgene under the regulation of a muscle-specific creatine kinase promoter in mice with complete or partial deficiency of merosin. The transgene restores the synthesis and localization of merosin in skeletal muscle, and greatly improves muscle morphology and integrity and the health and longevity of the mice. However, the transgenic mice share with the nontransgenic dystrophic mice a progressive lameness of hind legs, suggestive of a nerve defect. These results indicate that the absence of merosin in tissues other than the muscle, such as nervous tissue, is a critical component of MCMD. Future gene therapies of human MCMD, and perhaps of other forms of muscular dystrophy, may require restoration of the defective gene product in multiple tissues.

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.

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.

Merosin‐integrin promotion of skeletal myofiber cell survival: Differentiation state‐distinct involvement of p60Fyn tyrosine kinase and p38α stress‐activated MAP kinase

Myofiber survival and suppression of anoikis depend in large part on the merosin (laminin‐2/‐4)‐integrin α7β1D cell adhesion system; however, the question remains as to the nature of the signaling molecules/pathways involved. In the present study, we investigated this question using the C2C12 cell model of myogenic differentiation and its merosin‐ and laminin‐deficient derivatives. Herein, we report that: 1) of four members of the Src family of tyrosine kinases studied (p60Src, p53/56Lyn, p59Yes, or p60Fyn), the expression and activity of p60Fyn are found in myotubes exclusively; 2) a severe decrease of p60Fyn activity correlates with myotube apoptosis/anoikis induced by pharmocological compounds (herbimycin A or PP2) which inhibit tyrosine kinases of the Src family, by merosin deficiency and by β1 integrin inhibition; 3) myoblast survival depends on Fak and the MEK/Erk pathway, in contrast to myotubes; 4) the PI3‐K pathway is not involved in either myoblast or myotube survival; and 5) p38α SAPK stimulation and activity (but not that of p38β) are required in the progression of myotube apoptosis/anoikis induced by p60Fyn inhibition, merosin deficiency or β1 integrin‐inhibition; however, p38 is not involved in myoblast apoptosis. Taken together, these results suggest that the promotion of myotube survival by the merosin‐α7β1D adhesion system involves p60Fyn, and that disruptions in this cell adhesion system induce myotube apoptosis/anoikis through a p38α SAPK‐dependent pathway. J. Cell. Physiol. 191: 69–81, 2002.

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.

Identification, distribution, and tissular origin of the α5(IV) and α6(IV) collagen chains in the developing human intestine

The basement membrane type IV collagen is a family composed of six genetically distinct but structurally similar polypeptide chains, α1–α6. The α1(IV) and α2(IV) chains are ubiquitous components of all BMs whereas the other four have a restricted tissue distribution. In the present study, we have analyzed the expression, distribution, and cellular origin of the α5(IV) and α6(IV) chains in the developing and adult human small intestine and in well‐characterized in vitro models by indirect immunofluorescence, Western blot, and RT‐PCR. We have found that in the fetal small intestine, α5(IV) and α6(IV) are present in the epithelial BM and, in contrast to α1(IV) and α2(IV), are produced by both epithelial and mesenchymal cells. A distinct tissular origin for the α1/α2(IV) and α5/α6(IV) chains suggests that α5(IV) and α6(IV) associate as a heterotrimer in this organ. We have also found that a particular situation of α5(IV)/α6(IV) chain expression occurs in the adult intestine. Indeed, as compared with the fetal intestine, α6(IV) chain production is maintained while the expression of the α5(IV) chain is substantially reduced. Altered expression of the α5(IV) chain was also observed in the differentiating enterocytic‐like Caco‐2/15 cells, suggesting that in the intestinal model, the α5(IV) chain is subject to a regulated expression. Taken together, these observations indicate that the human intestinal epithelial BM contains up to four type IV collagen chains: the classical α1(IV)/α2(IV) chains, which originate from mesenchymal cells, and the α5(IV)/α6(IV) chains, which are of both epithelial and mesenchymal origin and have their expression regulated throughout development. Dev. Dyn. 1998; 212:437–447.

Upregulation of a functional form of the |[beta]|4 integrin subunit in colorectal cancers correlates with c-Myc expression

The integrin β4 subunit has been shown to be involved in various aspects of cancer progression. The aim of the present work was to evaluate the expression of β4 in primary colon cancers and to investigate the occurrence of a previously identified intestinal nonfunctional variant of β4 (β4ctd−) for adhesion to laminin. Immunodetection of β4 using a panel of antibodies and RT–PCR analyses were performed on series of paired primary colon tumors and corresponding resection margins. The β4 subunit was found to be significantly overexpressed in cancer specimens at both the protein and transcript levels. Surprisingly, β4 levels of expression were closely correlated with those of the oncogene c-Myc in individual specimens. In vitro studies of c-Myc overexpression showed an upregulation of β4 promoter activity. Finally, the β4ctd− form was identified in the normal proliferative colonic cells but was found to be predominantly absent in colon cancer cells, both in situ and in vitro. We concluded that the β4ctd− form is lost from colon cancer cells, while the level of the wild-type form of β4, which is functional for adhesion to laminin, is increased in primary tumors in relation with the expression of c-Myc.

Differential sensitivity to apoptosis between the human small and large intestinal mucosae: linkage with segment-specific regulation of BCL-2 homologs and involvement of signaling pathways.

The small and large intestines differ in their expression profiles of Bcl‐2 homologs. Intestinal segment‐specific Bcl‐2 homolog expression profiles are acquired as early as by mid‐gestation (18–20 weeks) in man. In the present study, we examined the question whether such distinctions underlie segment‐specific control mechanisms of intestinal cell survival. Using mid‐gestation human jejunum and colon organotypic cultures, we analyzed the impact of growth factors (namely insulin; 10 μg/ml) and pharmacological compounds that inhibit signal transduction molecules/pathways (namely tyrosine kinases, Fak, PI3‐K/Akt, and MEK/Erk) on cell survival and Bcl‐2 homolog expression (anti‐apoptotic: Bcl‐2, Bcl‐XL, Mcl‐1; pro‐apoptotic: Bax, Bak, Bad). The relative activation levels of p125Fak, p42Erk‐2, and p57Akt were analyzed as well. Herein, we report that (1) the inhibition of signal transduction molecules/pathways revealed striking differences in their impact on cell survival in the jejunum and colon (e.g., the inhibition of p125Fak induced apoptosis with a significantly greater extent in the jejunum [∼43%] than in the colon [∼24%]); (2) sharp distinctions between the two segments were noted in the modulatory effects of the various treatments on Bcl‐2 homolog steady‐state levels (e.g., inhibition of tyrosine kinase activities in the jejunum down‐regulated all anti‐apoptotics analyzed while increasing Bax, whereas the same treatment in the colon down‐regulated Bcl‐XL only and increased all pro‐apoptotics); and (3) in addition to their differential impact on cell survival and Bcl‐2 homolog expression, the MEK/Erk and PI3‐K/Akt pathways were found to be distinctively regulated in the jejunum and colon mucosae (e.g., insulin in the jejunum increased p42Erk‐2 activation without affecting that of p57Akt, whereas the same treatment in the colon decreased p42Erk‐2 activation while increasing that of p57Akt). Altogether, these data show that intestinal cell survival is characterized by segment‐specific susceptibilities to apoptosis, which are in turn linked with segmental distinctions in the involvement of signaling pathways and the regulation of Bcl‐2 homolog steady‐state levels. Therefore, these indicate that cell survival is subject to segment‐specific control mechanisms along the proximal‐distal axis of the intestine. J. Cell. Biochem. 82: 339–355, 2001.