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 beta1 and beta4 integrins, showing differential patterns of expression relative to its two functional compartments. Among them are the integrins alpha3beta1, alpha7Bbeta1 and the functional form of alpha6beta4 that appear to be related, in concert with the distribution of their ligands, to the process of intestinal cell differentiation, and the integrins alpha2beta1, alpha1beta1, alpha5beta1, and the non-functional form of alpha6beta4 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.

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.

The human polycystic kidney disease 2-like (PKDL) gene: exon/intron structure and evidence for a novel splicing mechanism.

Polycystin-L is a member of the expanding family of polycystins. Mutations in polycystin-1 or -2 cause human autosomal dominant polycystic kidney disease (ADPKD). The mouse ortholog of PKDL, Pkdl, is deleted in a mouse line with renal and retinal defects. We recently have shown that polycystin-L has calcium channel properties. In the current study, we determined the exon/intron organization of the PKDL gene and its alternative splicing. We show that PKDL has 16 exons. All splice acceptor/donor sites for these exons conform to the GT-AG rule. The positions of introns and the sizes of exons in the PKDL gene are very similar to those of PKD2, except for the last two 3' end exons. RT-PCR demonstrates the existence of at least three polycystin-L splice variants: PKDL(Delta5), PKDL(Delta456), and PKDL(Delta15) that are expressed in a tissue-specific manner. In addition, we have localized polymorphic marker D10S603 to intron 4 and exon 5 of PKDL. Elucidation of the gene structure, exact location, and alternative splicing patterns of PKDL will facilitate its evaluation as a candidate gene in cystic or other genetic disorders.

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.

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.

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.

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.

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.

Transport function of the naturally occurring pathogenic polycystin-2 mutant, R742X.

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations truncating polycystin-1 (PC1) or polycystin-2 (PC2), products of the PKD1 and PKD2 genes, respectively. A third member of the polycystin family, polycystin-L (PCL), was recently shown to function as a Ca(2+)-modulated nonselective cation channel. More recently, PC2 was also shown to be a nonselective cation channel with comparable properties to PCL, though the membrane targeting of PC2 likely varies with cell types. Here we show that PC2 expressed heterologously in Xenopus oocytes is targeted to intracellular compartments. By contrast, a truncated form of mouse PC2 corresponding to a naturally occurring human mutation R742X is targeted predominantly to the plasma membrane where it mediates K(+), Na(+), and Ca(2+) currents. Unlike PCL, the truncated form does not display Ca(2+)-activated transport activities, possibly due to loss of an EF-hand at the C-terminus. We propose that PC2 forms ion channels utilizing structural components which are preserved in the R742X form of the protein. Implications for epithelial cell signaling are discussed.

Polycystin-L is a calcium-regulated cation channel permeable to calcium ions.

Polycystic kidney diseases are genetic disorders in which the renal parenchyma is progressively replaced by fluid-filled cysts. Two members of the polycystin family (polycystin-1 and -2) are mutated in autosomal dominant polycystic kidney disease (ADPKD), and polycystin-L is deleted in mice with renal and retinal defects. Polycystins are membrane proteins that share significant sequence homology, especially polycystin-2 and -L (50% identity and 71% similarity). The functions of the polycystins remain unknown. Here we show that polycystin-L is a calcium-modulated nonselective cation channel that is permeable to sodium, potassium and calcium ions. Patch-clamp experiments revealed single-channel activity with a unitary conductance of 137 pS. Channel activity was substantially increased when either the extracellular or intracellular calcium-ion concentration was raised, indicating that polycystin-L may act as a transducer of calcium-mediated signalling in vivo. Its large single-channel conductance and regulation by calcium ions distinguish it from other structurally related cation channels.

Polycystin-2 is a novel cation channel implicated in defective intracellular Ca(2+) homeostasis in polycystic kidney disease.

Mutations in polycystins-1 and -2 (PC1 and PC2) cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by progressive development of epithelial renal cysts, ultimately leading to renal failure. The functions of these polycystins remain elusive. Here we show that PC2 is a Ca(2+)-permeable cation channel with properties distinct from any known intracellular channels. Its kinetic behavior is characterized by frequent transitions between closed and open states over a wide voltage range. The activity of the PC2 channel is transiently increased by elevating cytosolic Ca(2+). Given the predominant endoplasmic reticulum (ER) location of PC2 and its unresponsiveness to the known modulators of mediating Ca(2+) release from the ER, inositol-trisphosphate (IP(3)) and ryanodine, these results suggest that PC2 represents a novel type of channel with properties distinct from those of the other Ca(2+)-release channels. Our data also show that the PC2 channel can be translocated to the plasma membranes by defined chemical chaperones and proteasome modulators, suggesting that in vivo, it may also function in the plasma membrane under specific conditions. The sensitivity of the PC2 channel to changes of intracellular Ca(2+) concentration is deficient in a mutant found in ADPKD patients. The dysfunction of such mutants may result in defective coupling of PC2 to intracellular Ca(2+) homeostasis associated with the pathogenesis of ADPKD.