The Cellular Prion Protein PrPc 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

La protéine cellulaire du Prion, nouvel acteur de l'homéostasie de l'épithélium intestinal, joue un rôle essentiel dans la fonction de barrière de l'intestin

La fonction physiologique de la protéine cellulaire du prion (PrPc) est encore mal comprise et a été majoritairement étudiée dans les cellules neuronales. Dans l intestin, notre équipe a montré que la PrPc est adressée soit aux complexes jonctionnels des cellules différenciées où elle interagit avec des protéines desmosomales et la kinase Src, soit au noyau dans les cellules en division. L objectif de mon travail de thèse a donc été de comprendre le rôle de la PrPc dans l épithélium intestinal. En caractérisant l épithélium intestinal de souris KO pour la PrPc, j ai mis en évidence une diminution de la taille des desmosomes, ainsi qu une réduction de la taille des villosités associée à une augmentation du nombre de cellules en mitose, suggérant que la PrPc est impliquée à la fois dans l organisation des desmosomes et dans des mécanismes qui contrôlent la prolifération des cellules. En combinant des approches in vivo et in vitro, j ai également montré que la PrPc participe à la régulation de la fonction de barrière de l intestin, son absence s accompagnant d une augmentation de la perméabilité paracellulaire de l épithélium intestinal et de la susceptibilité à l inflammation dans un modèle de colite expérimentale. Dans la lignée entérocytaire Caco-2/TC7, l absence de PrPc suffit à perturber l organisation des trois jonctions intercellulaires impliquées dans l adhérence et la fonction de barrière, indépendamment des acteurs cellulaires et moléculaires de l inflammation. Enfin, nous avons montré que la PrPc présente une localisation perturbée dans l épithélium colique de sujets atteints de la maladie de Crohn. L ensemble de ces travaux présentent pour la première fois la PrPc, et potentiellement les desmosomes, comme des acteurs essentiels de la fonction de barrière de l intestin. Ils permettent également de proposer une fonction importante de la PrPc dans la régulation de l homéostasie de l épithélium intestinal, dont les mécanismes restent à explorerPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Special Issue on the “Regulation and Physiopathology of the Gut Barrier”

The importance of gut barrier integrity in intestinal homeostasis and the consequences of its alteration in the etiology of human pathologies have been subjects of exponentially growing interest during the last decade [...

Special Issue on the “Regulation and Physiopathology of the Gut Barrier”

The importance of gut barrier integrity in intestinal homeostasis and the consequences of its alteration in the etiology of human pathologies have been subjects of exponentially growing interest during the last decade [...

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

International audienceThe 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

Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine

International audienceAn increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls

Rapid Evaluation of Intestinal Paracellular Permeability Using the Human Enterocytic-Like Caco-2/TC7 Cell Line

International audienc

Use of Ussing Chambers to Measure Paracellular Permeability to Macromolecules in Mouse Intestine

International audienceAn increased intestinal permeability has been described in many diseases including inflammatory bowel disease and metabolic disorders, and a better understanding of the contribution of intestinal barrier impairment to pathogenesis is needed. In recent years, attention has been paid to the leak pathway, which is the route of paracellular transport allowing the diffusion of macromolecules through the tight junctions of the intestinal epithelial lining. While the passage of macromolecules by this pathway is very restricted under physiological conditions, its amplification is thought to promote an excessive immune activation in the intestinal mucosa. The Ussing chambers have been widely used to measure both active and passive transepithelial fluxes in intact tissues. In this chapter we present how this simple device can be used to measure paracellular permeability to macromolecules in the mouse intestine. We propose a detailed protocol and describe how to best exploit all the possibilities of this technique, correctly interpret the results, and avoid the main pitfalls

Rapid Evaluation of Intestinal Paracellular Permeability Using the Human Enterocytic-Like Caco-2/TC7 Cell Line

International audienc

Enterocytes'tight junctions play a protective role in limiting invasion of <em>Candida albicans</em> into intestinal cells

National audienceC. albicans is a commensal yeast of the mucous membranes in healthy humans that can also be responsible for disseminated candidiasis, mainly originating from the digestive tract in vulnerable patients. Deciphering the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes is necessary to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In intestinal epithelia, E-cadherin is constitutive of the Adherens Junctions localized just below the Tight Junctions (TJs) which ensure impermeability of the intestinal barrier. We hypothesized the absence of endocytosis of C. albicans in enterocytes could result from inaccessibility of C. albicans to E-cadherin blocked by TJs. We conducted experiments aimed at studying the kinetics of TJs formation in parallel with invasion of the intestinal cell line Caco-2 by C. albicans. Invasion of C. albicans in either non differentiated enterocytes (i.e. without TJs) or differentiated Caco-2 cells with chemically altered TJs was also investigated. Enterocytes were further treated with endocytosis inhibitors in order to determine whether accessibility of C. albicans to E-cadherin in Caco-2 cells could trigger endocytosis of the yeast. Our results reported an increase in invasion of C. albicans in both non differentiated Caco-2 cells and differentiated Caco-2 cells with chemically altered TJs, suggesting that TJs play a crucial role in limiting invasion of the enterocytes by the fungus. Moreover, using pharmacological inhibitors of endocytosis, we observed a decrease in the invasion of Caco-2 cells with chemically altered TJs by C. albicans, suggesting that facilitating access of the yeast to the basolateral side of the epithelial cells promote endocytosis of C. albicans. These data were confirmed with SEM observations of C. albicans interacting with differentiated Caco-2 cells with chemically altered TJs, showing membranes’ protrusions suggestive of endocytic internalization of C. albicans. Involvement of E-cadherin in endocytosis of C. albicans by intestinal cells is currently under investigations using mAbs anti-E-cadherin aimed at blocking E-cadherin at different stages of differentiation of Caco-2 cells. In addition, experiments using KO mutants of C. albicans will be conducted aimed at specifying the molecules of the fungus involved in these processes