Hepatocyte nuclear factor 4alpha, a key factor for homeostasis, cell architecture, and barrier function of the adult intestinal epithelium.

Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a transcription factor which is highly expressed in the intestinal epithelium from duodenum to colon and from crypt to villus. The homeostasis of this constantly renewing epithelium relies on an integrated control of proliferation, differentiation, and apoptosis, as well as on the functional architecture of the epithelial cells. In order to determine the consequences of HNF-4alpha loss in the adult intestinal epithelium, we used a tamoxifen-inducible Cre-loxP system to inactivate the Hnf-4a gene. In the intestines of adult mice, loss of HNF-4alpha led to an increased proliferation in crypts and to an increased expression of several genes controlled by the Wnt/beta-catenin system. This control of the Wnt/beta-catenin signaling pathway by HNF-4alpha was confirmed in vitro. Cell lineage was affected, as indicated by an increased number of goblet cells and an impairment of enterocyte and enteroendocrine cell maturation. In the absence of HNF-4alpha, cell-cell junctions were destabilized and paracellular intestinal permeability increased. Our results showed that HNF-4alpha modulates Wnt/beta-catenin signaling and controls intestinal epithelium homeostasis, cell function, and cell architecture. This study indicates that HNF-4alpha regulates the intestinal balance between proliferation and differentiation, and we hypothesize that it might act as a tumor suppressor.

Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions.

Enterocytes of the intestinal epithelium are continually regenerated. They arise from precursor cells in crypts, migrate along villi, and finally die, 3-4 days later, when they reach the villus apex. Their death is thought to occur by anoikis, a form of apoptosis induced by cell detachment, but the mechanism of this process remains poorly understood. We have previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin (Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. J Biol Chem 279: 43061-43069, 2004). Here we have further investigated the mechanisms underlying this disassembly of the adherens junctions. We show that disruption of the junctions occurs through endocytosis of E-cadherin and that this process depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 maintained E-cadherin and its cytoplasmic partners beta- and alpha-catenin at cell-cell contacts and decreased anoikis. Finally, EGFR activation was evidenced in the intestinal epithelium in vivo, in rare individual cells, which were shown to lose their interactions with the basal lamina. We conclude that EGFR is activated as enterocytes become detached from the basal lamina, and that this mechanism contributes to the disruption of E-cadherin-dependent junctions leading to anoikis. This suggests that EGFR participates in the physiological elimination of the enterocytes.

The cellular prion protein PrP(c) is involved in the proliferation of epithelial cells and in the distribution of junction-associated proteins.

BACKGROUND: The physiological function of the ubiquitous cellular prion protein, PrP(c), is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrP(c) is targeted to cell-cell junctions of polarized epithelial cells, where it interacts with c-Src. METHODOLOGY/FINDINGS: We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrP(c) is differentially targeted either to the nucleus in dividing cells or to cell-cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrP(c) interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrP(c), desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrP(c) is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrP(c) knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi. CONCLUSIONS/SIGNIFICANCE: From these results, PrP(c) could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.

E-cadherin-dependent transcriptional control of apolipoprotein A-IV gene expression in intestinal epithelial cells: a role for the hepatic nuclear factor 4.

Cell-matrix and cell-cell adhesion play a central role in the control of cell proliferation, differentiation, and gene expression. Integrins and E-cadherin are the key components involved in these processes in epithelial cells. We recently showed that integrin-dependent adhesion to the extracellular matrix reinforces the formation of E-cadherin-actin complexes inducing the polarization of Caco-2 enterocytes and increases the expression of a marker of enterocyte differentiation, the apolipoprotein A-IV (apoA-IV) gene. By impairing or enhancing E-cadherin-dependent cell adhesion, we demonstrate in the present study its involvement in the transcriptional activation of the apoA-IV gene in Caco-2 cells. This control requires the regulatory sequence that we have previously identified as necessary and sufficient to drive and restrict apoA-IV gene expression in enterocytes in vivo. Furthermore, using chimeric E-cadherin-Fc homophilic ligand-coated surfaces, we show that a direct activation of E-cadherin triggers the transcriptional activation of the apoA-IV promoter. Finally, E-cadherin-dependent cell-cell adhesion controls the nuclear abundance of the transcription factor hepatic nuclear factor 4alpha, which is involved in the enterocyte-specific expression of apoA-IV gene. Altogether, our results suggest that E-cadherin controls enterocyte-specific expression of genes, such as the apoA-IV gene, through the control of hepatic nuclear factor 4alpha nuclear abundance.

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.

Intestinal apolipoprotein A-IV gene transcription is controlled by two hormone-responsive elements: a role for hepatic nuclear factor-4 isoforms.

In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4alpha and gamma. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4alpha repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4alpha and gamma functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

Early loss of E-cadherin from cell-cell contacts is involved in the onset of Anoikis in enterocytes.

Anoikis, i.e. apoptosis induced by detachment from the extracellular matrix, is thought to be involved in the shedding of enterocytes at the tip of intestinal villi. Mechanisms controlling enterocyte survival are poorly understood. We investigated the role of E-cadherin, a key protein of cell-cell adhesion, in the control of anoikis of normal intestinal epithelial cells, by detaching murine villus epithelial cells from the underlying basement membrane while preserving cell-cell interactions. We show that upon the loss of anchorage, normal enterocytes execute a program of apoptosis within minutes, via a Bcl-2-regulated and caspase-9-dependent pathway. E-cadherin is lost early from cell-cell contacts. This process precedes the execution phase of detachment-induced apoptosis as it is only weakly modulated by Bcl-2 overexpression or caspase inhibition. E-cadherin loss, however, is efficiently prevented by lysosome and proteasome inhibitors. We also found that a blocking anti-E-cadherin antibody increases the rate of anoikis, whereas the activation of E-cadherin using E-cadherin-Fc chimera proteins reduces anoikis. In conclusion, our results stress the striking sensitivity of normal enterocytes to the loss of anchorage and the contribution of E-cadherin to the control of their survival/apoptosis balance. They open new perspectives on the key role of this protein, which is dysregulated in the intestinal epithelium in both inflammatory bowel disease and cancer.

[Cadherins: structure and signalisation protein of cell-cell junctions. Introduction and conclusions]. / Les cadhérines: des protéines de structure et de signalisation des jonctions cellule-cellule. introduction et conclusions.

Cadherin is a super family of genes, with at least 80 members. These members include classic cadherins, desmogleins, desmocollins, protocadherins, CNRs, Fats, seven-pass transmembrane cadherins and Ret tyrosine kinase. The repeated EC extracellular domains (N-terminal domain) are common to the family members and ensure cell adherence in a calcium dependant mechanism. The cadherins are expressed from amoebae to mammals. The biological complexity of cadherins is expressed at different levels, multigenic family and multiple functions in different tissues leading to use different methodological approaches. All the talks in this session broach in a promising aspect in the field of the basic comprehension of cell adhesion (R. M. Mège), at the molecular level (H. Feracci), physiological homeostasis of gut (S. Thenet), cell lineage (V. Delmas) or cancer transformation (L. Larue).

[Control of the survival/apoptosis balance by E-cadherin: role in enterocyte anoikis]. / Le contrôle de la balance survie/apoptose par la E-cadhérine: implication dans l'anoïkis des entérocytes.

Cadherins are transmembrane glycoproteins involved in cell-cell adherence. Recent developments indicate that classical cadherins may act as adherence-activated signaling receptors. Here, we review recent data from the literature concerning the role of classical cadherins in the control of cell survival and the signaling pathways involved. We focus on the fate and the role of E-cadherin, the main classical cadherin expressed in epithelial cells, in the cell death program triggered in enterocytes by loss of anchorage from the extracellular matrix (anoikis). These data open new perspectives on the key role of this protein, which is dysregulated in most carcinoma and is considered as a tumour-suppressor.

The cellular prion protein PrPc is expressed in human enterocytes in cell-cell junctional domains.

The physiological function of PrPc, the cellular isoform of prion protein, still remains unclear, although it has been established, in vitro or by using nerve cells, that it can homodimerize, bind copper, or interact with other proteins. Expression of PrPc was demonstrated as necessary for prion infection propagation. Considering the importance of the intestinal barrier in the process of oral prion infectivity, we have analyzed the expression of PrPc in enterocytes, which represent the major cell population of the intestinal epithelium. Our study, conducted both on normal human intestinal tissues and on the enterocytic cell line Caco-2/TC7, shows for the first time that PrPc is present in enterocytes. Interestingly, we found that this glycosylphosphatidylinositol-anchored glycoprotein was localized in cholesterol-dependent raft domains of the upper lateral membranes of enterocytes, beneath tight junctions, in cell-cell junctional domains. We observed that PrPc, E-cadherin, and Src co-localized in adherens junctions and that PrPc was co-immunoprecipitated with Src kinase but not with E-cadherin. Alteration of cell polarity after cholesterol depletion or loosening of the cell-cell junctions after EGTA treatment rapidly impaired membrane targeting of PrPc. Overall, our results point out the signaling of cell-cell contacts as a putative role for PrPc in epithelial cells.

Apoptotic neuronal cell death induced by the non-fibrillar amyloid-beta peptide proceeds through an early reactive oxygen species-dependent cytoskeleton perturbation.

In the present study, we have determined the nature and the kinetics of the cellular events triggered by the exposure of cells to non-fibrillar amyloid-beta peptide (A beta). When cortical neurons were treated with low concentrations of soluble A beta (1-40), an early reactive oxygen species (ROS)-dependent cytoskeleton disruption precedes caspase activation. Indeed, caspase activation and neuronal cell death were prevented by the microtubule-stabilizing drug taxol. A perturbation of the microtubule network was noticeable after being exposed to A beta for 1 h, as revealed by electron microscopy and immunocytochemistry. Microtubule disruption and neuronal cell death induced by A beta were inhibited in the presence of antioxidant molecules, such as probucol. These data highlight the critical role of ROS production in A beta-mediated cytoskeleton disruption and neuronal cell death. Finally, using FRAP (fluorescence recovery after photo bleaching) analysis, we observed a time-dependent biphasic modification of plasma membrane fluidity, as early as microtubule disorganization. Interestingly, molecules that inhibited neurotubule perturbation and cell death did not affect the membrane destabilizing properties of A beta, suggesting that the lipid phase of the plasma membrane might represent the earliest target for A beta. Altogether our results convey the idea that upon interaction with the plasma membrane, the non-fibrillar A beta induces a rapid ROS-dependent disorganization of the cytoskeleton, which results in apoptosis.

Restriction of apolipoprotein A-IV gene expression to the intestine villus depends on a hormone-responsive element and parallels differential expression of the hepatic nuclear factor 4alpha and gamma isoforms.

The apoA-I/C-III/A-IV gene cluster, like most intestine-specific genes, displays a specific pattern of expression along the intestinal cephalocaudal and crypt-to-villus axes. We have shown that this specific pattern of expression requires the distal apoA-IV promoter and the apoC-III enhancer. Using a new set of transgenic mice, we demonstrate here that the restriction of apoA-IV gene transcription to villus enterocytes requires a hormone-responsive element (HRE) located within the apoA-IV distal promoter. We showed, using nuclear extracts from villus or crypt epithelial cells, that this HRE bound the transcription factor hepatic nuclear factor 4 (HNF-4). We also found that the HNF-4gamma isoform was produced only in the villus, whereas the HNF-4alpha isoform was produced along the entire length of the crypt-to-villus axis. Our results demonstrate that the HRE of the distal apoA-IV promoter is responsible for the restriction of gene expression to villus epithelial cells and that this HRE binds HNF-4 isoforms. The in vivo observation of parallel gradients for apoA-IV and HNF-4gamma gene expression raises questions concerning whether this transcription factor plays a specific role in the control of enterocyte differentiation.

Integrin-mediated functional polarization of Caco-2 cells through E-cadherin--actin complexes.

Enterocyte differentiation is a dynamic process during which reinforcement of cell-cell adhesion favours migration along the crypt-to-villus axis. Functional polarization of Caco-2 cells, the most commonly used model to study intestinal differentiation, is assessed by dome formation and tightness of the monolayer and is under the control of the extracellular matrix (ECM). Furthermore, our biochemical and confocal microscopy data demonstrate that the ECM dramatically reinforces E-cadherin targeting to the upper lateral membrane, formation of the apical actin cytoskeleton and its colocalization with E-cadherin in functional complexes. In our model, these effects were produced by native laminin-5-enriched ECM as well as by type IV collagen or laminin 2, which suggests a common pathway of induction through integrin receptors. Indeed, these effects were antagonized by blocking anti-beta1- and anti-alpha6-integrin antibodies and directly induced by a stimulating anti-beta1-integrin antibody. These results demonstrate that integrin-dependent cell to ECM adhesion reinforces E-cadherin-dependent cell-cell adhesion in Caco-2 cells and further support the notion that enterocyte differentiation is supported by a molecular crosstalk between the two adhesion systems of the cell.