Biomedical subjects

F E Herring-Gillam

Publications and source records attributed to F E Herring-Gillam.

8 recordsLinked to original sources

PubMed2000

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 Crohn's 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.

Adult↗

PubMed1999

Expression of functionally distinct variants of the beta(4)A 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 alpha(6)beta(4), which is thought to be ubiquitously expressed by intestinal cells. However, in this study, a re-evaluation of the beta(4) subunit expression with different antibodies revealed that two forms of beta(4) exist in the human intestinal epithelium. Furthermore, we show that differentiated enterocytes express a full-length 205-kDa beta(4)A subunit, whereas undifferentiated crypt cells express a novel beta(4)A subunit that does not contain the COOH-terminal segment of the cytoplasmic domain (beta(4)A(ctd-)). This new form was not found to arise from alternative beta(4) mRNA splicing. Moreover, we found that these two beta(4)A forms can associate into alpha(6)beta(4)A complexes; however, the beta(4)A(ctd-) integrin expressed by the undifferentiated crypt cells is not functional for adhesion to laminin-5. Hence, these studies identify a novel alpha(6)beta(4)A(ctd-) integrin expressed in undifferentiated intestinal crypt cells that is functionally distinct.

Adult↗

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

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

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

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↗

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↗

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↗