Sophie Thenet

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.

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.

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 4α, 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 4α nuclear abundance.

Requirement of Cellular Prion Protein for Intestinal Barrier Function and Mislocalization in Patients With Inflammatory Bowel Disease

BACKGROUND & AIMS Cell adhesion is one function regulated by cellular prion protein (PrP(c)), a ubiquitous, glycosylphosphatidylinositol-anchored glycoprotein. PrP(c) is located in cell-cell junctions and interacts with desmosome proteins in the intestinal epithelium. We investigated its role in intestinal barrier function. METHODS We analyzed permeability and structure of cell-cell junctions in intestine tissues from PrP(c) knockout (PrP(c-/-)) and wild-type mice. PrP(c) expression was knocked down in cultured human Caco-2/TC7 enterocytes using small hairpin RNAs. We analyzed colon samples from 24 patients with inflammatory bowel disease (IBD). RESULTS Intestine tissues from PrP(c-/-) mice had greater paracellular permeability than from wild-type mice (105.9 ± 13.4 vs 59.6 ± 10.1 mg/mL fluorescein isothiocyanate-dextran flux; P < .05) and impaired intercellular junctions. PrP(c-/-) mice did not develop spontaneous disease but were more sensitive than wild-type mice to induction of colitis with dextran sulfate (32% mortality vs 4%, respectively; P = .0033). Such barrier defects were observed also in Caco-2/TC7 enterocytes following PrP(c) knockdown; the cells had increased paracellular permeability (1.5-fold over 48 hours; P < .001) and reduced transepithelial electrical resistance (281.1 ± 4.9 vs 370.6 ± 5.7 Ω.cm(2); P < .001). Monolayer shape and cell-cell junctions were altered in cultures of PrP(c) knockdown cells; levels of E-cadherin, desmoplakin, plakoglobin, claudin-4, occludin, zonula occludens 1, and tricellulin were decreased at cell contacts. Cell shape and junctions were restored on PrP(c) re-expression. Levels of PrP(c) were decreased at cell-cell junctions in colonic epithelia from patients with Crohns disease or ulcerative colitis. CONCLUSIONS PrP(c) regulates intestinal epithelial cell-cell junctions and barrier function. Its localization is altered in colonic epithelia from patients with IBD, supporting the concept that disrupted barrier function contributes to this disorder.

Yersinia pseudotuberculosis disrupts intestinal barrier integrity through hematopoietic TLR-2 signaling

Intestinal barrier function requires intricate cooperation between intestinal epithelial cells and immune cells. Enteropathogens are able to invade the intestinal lymphoid tissue known as Peyers patches (PPs) and disrupt the integrity of the intestinal barrier. However, the underlying molecular mechanisms of this process are poorly understood. In mice infected with Yersinia pseudotuberculosis, we found that PP barrier dysfunction is dependent on the Yersinia virulence plasmid and the expression of TLR-2 by hematopoietic cells, but not by intestinal epithelial cells. Upon TLR-2 stimulation, Y. pseudotuberculosis-infected monocytes activated caspase-1 and produced IL-1β. In turn, IL-1β increased NF-κB and myosin light chain kinase activation in intestinal epithelial cells, thus disrupting the intestinal barrier by opening the tight junctions. Therefore, Y. pseudotuberculosis subverts intestinal barrier function by altering the interplay between immune and epithelial cells during infection.

Roles of the cellular prion protein in the regulation of cell-cell junctions and barrier function

The cellular prion protein was historically characterized owing to its misfolding in prion disease. Although its physiological role remains incompletely understood, PrPC has emerged as an evolutionary conserved, multifaceted protein involved in a wide-range of biological processes. PrPC is a GPI-anchored protein targeted to the plasma membrane, in raft microdomains, where its interaction with a repertoire of binding partners, which differ depending on cell models, mediates its functions. Among identified PrPC partners are cell adhesion molecules. This review will focus on the multiple implications of PrPC in cell adhesion processes, mainly the regulation of cell-cell junctions in epithelial and endothelial cells and the consequences on barrier properties. We will show how recent findings argue for a role of PrPC in the recruitment of signaling molecules, which in turn control the targeting or the stability of adhesion complexes at the plasma membrane.

The cellular prion protein PrPc is a partner of the Wnt pathway in intestinal epithelial cells

The cellular prion protein (PrPc), a component of desmosomes, contributes to the intestinal barrier function. A nuclear pool of PrPc interacts with γ-catenin; up-regulates the transcriptional activity of the β-catenin/TCF7L2 complex, the main effector of the canonical Wnt pathway; and promotes intestinal cell proliferation.

PrPc deficiency and dasatinib protect mouse intestines against radiation injury by inhibiting of c-Src

BACKGROUND & AIM Despite extensive study of the contribution of cell death and apoptosis to radiation-induced acute intestinal injury, our knowledge of the signaling mechanisms involved in epithelial barrier dysfunction remains inadequate. Because PrP(c) plays a key role in intestinal homeostasis by renewing epithelia, we sought to study its role in epithelial barrier function after irradiation. DESIGN Histology, morphometry and plasma FD-4 levels were used to examine ileal architecture, wound healing, and intestinal leakage in PrP(c)-deficient (KO) and wild-type (WT) mice after total-body irradiation. Impairment of the PrP(c) Src pathway after irradiation was explored by immunofluorescence and confocal microscopy, with Caco-2/Tc7 cells. Lastly, dasatinib treatment was used to switch off the Src pathway in vitro and in vivo. RESULTS The decrease in radiation-induced lethality, improved intestinal wound healing, and reduced intestinal leakage promoted by PrP(c) deficiency demonstrate its involvement in acute intestinal damage. Irradiation of Cacao2/Tc7 cells induced PrP(c) to target the nuclei associated with Src activation. Finally, the protective effect triggered by dasatinib confirmed Src involvement in radiation-induced acute intestinal toxicity. CONCLUSION Our data are the first to show a role for the PrP(c)-Src pathway in acute intestinal response to radiation injury and offer a novel therapeutic opportunity.

The nucleo-junctional interplay of the cellular prion protein: A new partner in cancer-related signaling pathways?

ABSTRACT The cellular prion protein PrPc plays important roles in proliferation, cell death and survival, differentiation and adhesion. The participation of PrPc in tumor growth and metastasis was pointed out, but the underlying mechanisms were not deciphered completely. In the constantly renewing intestinal epithelium, our group demonstrated a dual localization of PrPc, which is targeted to cell-cell junctions in interaction with Src kinase and desmosomal proteins in differentiated enterocytes, but is predominantly nuclear in dividing cells. While the role of PrPc in the dynamics of intercellular junctions was confirmed in other biological systems, we unraveled its function in the nucleus only recently. We identified several nuclear PrPc partners, which comprise γ-catenin, one of its desmosomal partners, β-catenin and TCF7L2, the main effectors of the canonical Wnt pathway, and YAP, one effector of the Hippo pathway. PrPc up-regulates the activity of the β-catenin/TCF7L2 complex and its invalidation impairs the proliferation of intestinal progenitors. We discuss how PrPc could participate to oncogenic processes through its interaction with Wnt and Hippo pathway effectors, which are controlled by cell-cell junctions and Src family kinases and dysregulated during tumorigenesis. This highlights new potential mechanisms that connect PrPc expression and subcellular redistribution to cancer.

Effects of retinoic acid on the growth of cultured rabbit articular chondrocytes: Relation with alkaline phosphatase activity and beta receptor.

The effects of retinoic acid (RA) on rabbit articular cartilage cells were studied for concentrations ranging from 5.10-5 M to 10-7 M; the treatment with RA over three days resulted in dose dependent inhibition of chondrocyte proliferation between 5.10-5 and 10-5 M with persistence of the inhibitory effect until 10-6 M. RA until 10-7 M induced a slight, but significant, enhancement of cell proliferation. This growth stimulating effect seems to be related to the Beta receptor system because Beta blockers, such as sotalol and DL propranolol, were able to suppress the stimulating action of agonist type isoprenaline. The activity of alkaline phosphatase (AP) was also determined. The highest dose of RA (5.10-5 M) induced an increase (x 3) of AP activity, and 10-7 M RA decreased it (x 0.4).