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J F Beaulieu

Publications and source records attributed to J F Beaulieu.

At least 37 records · Page 2Linked to original sources
PubMed1998

Regulated expression of the integrin alpha9beta1 in the epithelium of the developing human gut and in intestinal cell lines: relation with cell proliferation.

The integrin alpha9beta1 is one of the recently identified integrins whose expression is restricted to specialized tissues. Its exact function is still unknown. In the present study, we have analyzed the expression of the alpha9 subunit in human fetal and adult small intestinal and colonic epithelia as well as in intestinal cell lines by indirect immunofluorescence, immunoprecipitation, Western blot, and Northern blot. In intact tissues, the antigen was restricted to the basolateral domain of epithelial cells in intestinal crypts at the fetal stage and was absent in the adult. The alpha9beta1 integrin was also detected in the intestinal cell lines HIEC-6 and Caco-2/15. The presence of alpha9beta1 in HIEC-6 was found to be consistent with their proliferative crypt-like status. In Caco-2/15 cells, the integrin was present at high levels in proliferating cells but was downregulated when cells cease to grow and undertake their differentiation. EGF treatment, which is known to maintain Caco-2/15 cells in a proliferative state, resulted in higher levels of alpha9 as compared to control cells. Taken together, these observations suggest a relation between integrin alpha9beta1 expression and proliferation in human intestinal cells.

Blotting, Northern↗
PubMed1998

Primary cultures of fully differentiated and pure human intestinal epithelial cells.

The epithelium of the small intestinal mucosa is a highly dynamic system particularly well suited for analyzing key biological phenomena such as cell differentiation and migration, cell-matrix interactions, and apoptosis. However, in vitro models of fully differentiated normal human enterocytes are still lacking. The objective of the present study was to investigate the possibility of generating such differentiated intestinal cell cultures from the fetal small intestine. For this purpose, various dissociation methods were tested in order to obtain pure, viable, and functional enterocytes. One of these methods, based on the procedure to recover epithelial cells grown on Matrigel and involving the use of Matrisperse, a nonenzymatic dissociating solution, was found to allow the isolation of the integral epithelial lining from the mesenchyme. In culture, these epithelial fractions plated on collagen I spread rapidly and reached confluence after 3-4 days. When tested after 5-7 days, these primary cultures of differentiated enterocytes (PCDE) remained well preserved. Both goblet and absorptive cells exhibit all the main characteristics of intact villus intestinal cells as assessed by electron microscopy. Indirect immunofluorescence and Western blot analyses confirmed the purity of PCDE. The functional status of these cells was demonstrated by the presence of uniformly distributed tight junction, zonula adherens, and desmosomal components at the region of cell to cell contact as well as expression of various brush border enzymes, namely sucrase-isomaltase and lactase, and goblet cell mucins. As expected, cell proliferation was found to be negligible as assessed by DNA synthesis. Taken together, these data show that primary cultures of pure and viable differentiated enterocytes can be generated from the human fetal small intestine.

Cell Culture Techniques↗
PubMed1998

Apolipoproteins in human fetal colon: immunolocalization, biogenesis, and hormonal regulation.

The present investigation aimed at defining the localization of apolipoproteins (apo) A-I, A-IV, B-48, and B-100 along the crypt-villus axis of the human fetal colon, their biogenesis during gestation, and their hormonal regulation. Using immunofluorescence, the distribution of apo A-I and A-IV appeared as a gradient, increasing from the developing crypt to the tip of the villus. On the other hand, apo B-100 staining was found in the crypt and the lower mid-villus region with varying intensities in the upper villus cells, while the 2D8 antibody which recognizes both apo B-100 and B-48, revealed uniform staining along the crypt-villus axis. Apolipoprotein synthesis, determined by [35S] methionine labeling, immunoprecipitation, and SDS-PAGE showed a predominance of apo A-IV (53%), followed by apo A-I (23.9%), apo B-48 (13.4%), and apo B-100 (9.7%). The synthesis of each apolipoprotein was significantly modulated by hydrocortisone, insulin and epidermal growth factor (EGF). Apart from a decrease in apo B-100 exerted by EGF and a reduction in apo A-I resulting from the addition of insulin, the other apolipoproteins were all enhanced. Our data confirm that the fetal colon has the capacity to synthesize apolipoprotein A-I, A-IV, B-48, and B-100 and establish that their synthesis are modulated by hormonal and growth factors known to be involved in the regulatory mechanism of the functional development of human jejunum.

Antibodies, Monoclonal↗
PubMed1998

Epithelial vs mesenchymal contribution to the extracellular matrix in the human intestine.

The basement membrane (BM) underlying the epithelium of the intestine is generally believed to be of both epithelial and mesenchymal origin but the exact contribution of each tissue has not been directly examined in the human. In this study, we have used a newly described procedure to dissociate the human intestine into pure epithelial and corresponding mesenchymal fractions. Northern blot and RT-PCR analyses of the fractions for the presence of transcripts encoding extracellular matrix molecules revealed that the epithelium produces the formal BM molecules such as the alpha 1, alpha 2, and beta 1 chains of laminin-1 and laminin-2 and the alpha 5(IV) and alpha 6(IV) chains of collagen as well as fibronectin, a BM-associated molecule. Interestingly, the alpha 1(IV) chain of collagen, which associates with the alpha 2(IV) chain to form the main BM collagen network, as well as tenascin-C and decorin, two BM-associated molecules, was found to be exclusively of mesenchymal origin. Taken together, these data support the concept that in the human, as in experimental animals, the intestinal BM is composed of components produced from both the epithelium and the mesenchyme.

Basement Membrane↗
PubMed1998

Expression of the alpha9beta1 integrin in human colonic epithelial cells: resurgence of the fetal phenotype in a subset of colon cancers and adenocarcinoma cell lines.

Cell-matrix interactions are thought to be of critical importance in the regulation of various cell functions, including proliferation, migration and control of gene expression. The integrins, a large family of specific receptors for the macromolecules of the extracellular matrix, are important mediators of these interactions. The integrin alpha9beta1 is one of the integrins whose expression is restricted to specialized tissues. Its exact function is unknown. In the present study, we have analyzed expression of the alpha9 subunit in human colonic epithelial cells by indirect immuno-fluorescence and Western and Northern blots. In normal intact tissues, the antigen was detected at the basolateral domain of epithelial cells in colonic glands at the fetal stage but was absent in adults. Strong staining was detected constitutively in contractile cells at both stages. In adenocarcinomas, the alpha9 subunit was detected at the basolateral domain of epithelial cells in 6 of the 10 tumors tested, while a reduction of the staining was observed in the sub-epithelial myofibroblasts in parallel with peri-glandular stroma disorganization. The potential for colon adenocarcinoma cells to express the integrin alpha9 subunit was confirmed at both the protein and transcript levels in Caco-2 and T84 cell lines, 2 well-characterized cell lines known to exhibit polarization features. The 5 other cell lines tested were negative for expression of the alpha9 subunit. Taken together, our observations suggest that the alpha9 integrin subunit is subject to an onco-fetal pattern of expression in human colonic epithelium.

Adenocarcinoma↗
PubMed1998

Identification, distribution, and tissular origin of the alpha5(IV) and alpha6(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, alpha1-alpha6. The alpha1(IV) and alpha2(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 alpha5(IV) and alpha6(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, alpha(IV) and alpha6(IV) are present in the epithelial BM and, in contrast to alpha1(IV) and alpha2(IV), are produced by both epithelial and mesenchymal cells. A distinct tissular origin for the alpha1/alpha2(IV) and alpha5/alpha6(IV) chains suggests that alpha5(IV) and alpha6(IV) associate as a heterotrimer in this organ. We have also found that a particular situation of alpha5(IV)/alpha6(IV) chain expression occurs in the adult intestine. Indeed, as compared with the fetal intestine, alpha6(IV) chain production is maintained while the expression of the alpha5(IV) chain is substantially reduced. Altered expression of the alpha5(IV) chain was also observed in the differentiating enterocytic-like Caco-2/15 cells, suggesting that in the intestinal model, the alpha5(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 alpha1(IV)/alpha2(IV) chains, which originate from mesenchymal cells, and the alpha5(IV)/alpha6(IV) chains, which are of both epithelial and mesenchymal origin and have their expression regulated throughout development.

Caco-2 Cells↗
PubMed1997

Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells.

BACKGROUND & AIMS: Intestinal epithelial cell lines capable of differentiating in monolayer culture have been difficult to obtain, prompting the use of novel approaches including immortalization with oncogenes or tumor viruses. The aim of this study was to obtain conditionally immortalized human intestinal epithelial cells (temperature-sensitive fetal human intestinal [tsFHI] cells) and study their growth and differentiation capabilities. METHODS: Intestinal cells from human fetuses were transformed with a temperature-sensitive simian virus 40 large tumor antigen, cloned, and screened for expression of intestinal markers. RESULTS: Establishment of tsFHI cells was crucially dependent on a growth medium based on OptiMEM. At 32 degrees C, the tsFHI cells proliferate and display crypt cell markers. Only a few scattered differentiated cells are observed together with cells displaying the morphological signs of apoptosis. A shift to 39 degrees C results in irreversible growth arrest and acquisition of an enterocyte-like phenotype. Expression of the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 is induced within hours of a shift to 39 degrees C 1-2 days before corresponding increases in brush border enzymes, suggesting a role in the induction rather than in the maintenance of a differentiated phenotype. CONCLUSIONS: The tsFHI cells recapitulate key aspects of intestinal cell dynamics in vitro and seem particularly suited for the study of early events in cell differentiation.

Analysis of Variance↗
PubMed1997

Relation between integrin alpha7Bbeta1 expression in human intestinal cells and enterocytic differentiation.

BACKGROUND & AIMS: Cell-laminin interactions are principally mediated by specific membrane receptors of the integrin family. The integrin alpha7beta1 is one of them. Its expression in the intestine has not yet been investigated although it appears to be a key element in muscle cell differentiation. In this study, the expression of its three known isoforms has been analyzed in developing and adult small intestine and in intestinal cell lines. METHODS: The expression of the integrin alpha7beta1 was analyzed by indirect immunofluorescence, Western blotting, immunoprecipitation, and reverse-transcription polymerase chain reaction. RESULTS: The alpha7B isoform, but not the alpha7A and C isoforms, was detected in intestinal epithelial cells. In vivo, the presence of the alpha7B subunit was closely paralleled with (1) acquisition of differentiation characteristics during development and along the crypt-villus axis in the adult small intestine and (2) loss of enterocytic functions in the re-differentiated colonic epithelium. In vitro, the expression of alpha7B was also shown to correlate with the acquisition of enterocytic functions. In Caco-2 cells, the alpha7Bbeta1 integrin was found transiently up-regulated at the onset of sucrase-isomaltase expression. CONCLUSIONS: Taken together, these results suggest that alpha7Bbeta1 expression is correlated with human intestinal cell differentiation.

Amino Acid Sequence↗
PubMed1996

Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures.

The regulation of intestinal cell proliferation, migration, and differentiation has been the subject of numerous studies. However, in human, progress in this field has been traditionally hampered by the lack of normal epithelial cell models. The aim of the present study was to define conditions in order to isolate, and more importantly to grow in a continuous manner, human small intestinal epithelial cells. A number of mechanical and/or enzymatic dissociation methods have been tested to isolate viable epithelial cells from the fetal small intestine. Cultured cells were characterized by indirect immunofluorescence and Western blot analysis. It was found that the use of thermolysin (50 microgram/ml, 2-3 h at 37 degrees C) can be advantageously applied to the isolation of viable epithelial cells free from contaminating fibroblasts when obtained from the 17- to 19-week fetal ileum. Furthermore, this procedure allowed the generation of continuously growing human intestinal epithelial cell cultures, which retain the ability to express specific cytokeratins as well as intestinal cell markers over a number of passages. This study shows that normal epithelial cell cultures can be relatively easily and reproducibly generated from the human fetal small intestine.

Acetyltransferases↗
PubMed1996

Triiodothyronine stimulates the expression of sucrase-isomaltase in Caco-2 cells cultured in serum-free medium.

In a previous study we have shown that triiodothyronine (T3) added to a serum-free medium supplemented with insulin, transferrin, and selenous acid (ITS) can stimulate Caco-2 cell differentiation. In this study we have focused on the effects of T3 on sucrase activity. The results obtained demonstrate that T3 (50 nM) does not change Caco-2 cell proliferation but enhances sucrase activity from 50 to 80%. Similar increases were observed whether or not insulin was present in the culture medium, showing that there was no synergistic effect between T3 and insulin on sucrase activity. Moreover, T3 acts specifically during the differentiation period since addition of T3 to the defined TS medium before confluency is reached does not stimulate sucrase activity. Sucrase kinetic parameters were evaluated for the first time in Caco-2 cells under various culture conditions. The presence of a single enzyme was verified, with a Km of about 7 mM and a Vmax around 20 nmol of substrate hydrolyzed min-1 mg-1 of protein. Our results showed that T3 did not change the enzyme's affinity for sucrose but doubled the Vmax. Moreover, immunoblotting using anti-sucrase-isomaltase (SI) antibodies revealed an approximately twofold increase in the relative amount of SI immunoreactive protein in T3-stimulated cells compared to untreated cells. Results obtained by both Northern hybridization and RT-PCR amplification showed a significant increase in SI mRNA contents. These results suggest that T3 acts primarily on sucrase expression at the mRNA level.

Base Sequence↗
PubMed1996

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 heterogeneous ("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 (> or = 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 KI-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.

Alkaline Phosphatase↗
PubMed1995

CCAAT/enhancer binding protein isoforms expression in the colon of neonatal mice.

Development of murine proximal colon follows a complex pattern of morphological and functional differentiation. Molecular mechanisms and factors responsible for colon-specific gene expression remain to be established. In an attempt to identify some of these factors, we examined the expression of the alpha, beta, and delta isoforms of the CCAAT/enhancer binding protein (C/EBP) transcription factor gene family during murine colon development. Whereas C/EBP alpha mRNA levels are reduced during the third post-natal week, C/EBP alpha 42 and 30 kD proteins levels decrease between post-natal days 8 and 21. C/EBP beta mRNA levels increase between post-natal days 4 and 8 and remain constant subsequently, in contrast to a decrease in C/EBP beta protein levels between post-natal days 11 and 15. C/EBP delta mRNA levels increase gradually while C/EBP delta protein levels show variations during post-natal development. Changes in C/EBP DNA binding activity coincides with modifications in C/EBP isoforms expression. By indirect immunofluorescence, we show restriction of C/EBP alpha expression to differentiated surface epithelial cells during crypt formation. C/EBP alpha is predominantly nuclear with some cytoplasmic staining at all developmental stages. C/EBP beta and delta are both predominantly nuclear in crypt and differentiated surface epithelial cells, as well as in various cells of the lamina propria and muscular layers. Thus, specific C/EBP isoforms are differentially regulated during murine colon post-natal development. Differential C/EBP isoforms pattern of expression suggests a role for these transcription factors in colon-specific gene expression during development.

Animals↗
PubMed1995

Appearance and distribution of laminin A chain isoforms and integrin alpha 2, alpha 3, alpha 6, beta 1, and beta 4 subunits in the developing human small intestinal mucosa.

BACKGROUND: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. METHODS: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4, four potential receptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. RESULTS: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins alpha 6 beta 1 and alpha 6 beta 4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the alpha 2 beta 1 and alpha 3 beta 1 integrins was found time- and site-restricted. The alpha 2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the alpha 3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19-20-week-old crypts. CONCLUSIONS: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development.

Antigens, CD↗
PubMed1995

Cellular fibronectin expression is down-regulated at the mRNA level in differentiating human intestinal epithelial cells.

The expression of human cellular fibronectin (HFN) was analyzed in relation to the differentiation process of the human enterocyte-like cell line Caco-2. A single approximately 290-kDa form of HFN, corresponding to a 10.0-kb mRNA species, was observed in Caco-2 cells. The expression levels of both protein and mRNA were found to decrease to undetectable levels concomitantly to the differentiation process of these cells. This decline in HFN expression was correlated with the expression of sucrase-isomaltase (a specific marker of intestinal cell differentiation). These results indicate that intestinal epithelial expression of HFN is down-regulated through its mRNA levels and is correlated with the acquisition of the differentiated enterocytic phenotype.

Biomarkers↗
PubMed1995

Extracellular heterotrimeric laminin promotes differentiation in human enterocytes.

To investigate the role of laminin (Ln) and merosin (a Ln variant) in the modulation of intestinal epithelial cell differentiation, we have analyzed their functional expression as well as their potential influence during the differentiation process of Caco-2 cells, a human cell line unique in its property to differentiate into a mature enterocyte-like cell type in vitro. By indirect immunofluorescence and Western blotting, Caco-2 cells have been found to express the B1 and B2 chains of Ln, as well as a heavy approximately 350- to 370-kDa chain related to the human A chain, but not the M chain, of merosin. A gradual deposition of this A-like chain-containing Ln molecule has been observed as Caco-2 cells undergo differentiation. At the cellular level, a clear relationship between basal staining for the A chain and apical staining for the brush-border membrane enzyme sucrase-isomaltase (SI) has been observed. Human Ln, used as substratum, has been found to increase significantly the expression of SI and lactase in Caco-2 cells, whereas both Ln and human merosin have been shown to stimulate aminopeptidase N and alkaline phosphatase expression. Taken together, these data indicate that enterocytic differentiation-related gene expression is promoted by an extracellular deposition of Ln and may be susceptible to a differential modulation by variant forms of the molecule.

Cell Differentiation↗
PubMed1994

Immunolocalization of a mesenchymal antigen specific to the gastrointestinal tract.

BACKGROUND: The aim of the present study was to localize, at the fine structural level, a protein found by indirect immunofluorescence to be associated with the mesenchymal tissue 1) closely applied to the intervillus epithelium before the formation of intestinal crypts in the mouse fetus and 2) around intestinal crypts during and after their formation. METHODS: We used a pre-embedding immunolabeling technique for extracellular matrix molecules, and a monoclonal antibody (Mab) directed against antigen MIM-1/130. RESULTS: Immunofluorescence disclosed the presence of antigen 1/130 in the connective tissue closely applied to the epithelium of the gallbladder, pyloric glands, and intestinal and colonic crypts in adult mice. The antigen was absent in all salivary glands, kidney, liver, lung, spleen, and pancreas. At the fine structural level, gold particles in positive organs were associated with the interstitial matrix around collagen fibrils underneath the epithelia; gold particles were completely absent in the basement membranes. In the small intestine, labeling was seen only around crypts from cell position 1 up to the crypt-villus junction; it was totally absent under the villus epithelium. In order to confirm this particular localization in vivo, Mab 1/130 was administered orogastrically to 9-day-old mice: after 3 hours the antibody was found lining the immediate periphery of duodenal crypts as seen by indirect immunofluorescence. In control animals, an anti-mouse laminin Mab of the same subclass as Mab 1/130 was orogastrically fed using the same protocol: basal laminae were labeled under the epithelium of duodenal villi and crypts and also in the lamina propria, with a decreasing gradient from the top of the villi to the bottom of the crypts. CONCLUSION: These observations indicate that the extracellular matrix associated with the epithelium of pyloric glands, of intestinal and colonic crypts, and of gallbladder contains a new antigen whose function remains to be determined. The neonatal mouse hence constitutes a good model to study the role of extracellular matrix components in determining organ differentiation in vivo.

Animals↗
PubMed1994

Expression of the alpha-5(IV) collagen chain in the fetal human small intestine.

BACKGROUND/AIMS: The basement membrane type IV collagen is a family composed of at least five genetically distinct but structurally similar polypeptide chains, alpha 1-alpha 5. The alpha 1(IV) and alpha 2(IV) chains are ubiquitous components of basement membranes, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains have a restricted tissue distribution. The aim of this study was to analyze the presence of these minor type IV collagen chains in the small intestinal mucosa. METHODS: The expression of type IV collagen chains in the developing and adult human small intestine was determined by indirect immunofluorescence with monoclonal and polyclonal antibodies. Western blotting and Northern hybridization analysis were also used to additionally investigate the expression of the alpha 1(IV) and alpha 5(IV) chains. RESULTS: The alpha 3-alpha 5(IV) chains were absent from the adult epithelium, but, surprisingly, the alpha 5(IV) chain was consistently detected in the fetal mucosa. Its expression was confirmed by Western blotting, complementary DNA polymerase chain-reaction amplification, and Northern hybridization analysis. CONCLUSIONS: The alpha 5(IV) chain of collagen is expressed in the fetal but not adult human intestinal epithelium. Its position at the basolateral domain of epithelial cells suggests a potential role for this molecule during development.

Aging↗