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La PrPc et ses partenaires desmosomaux et nucléaires : rôle dans la régulation de l’adhérence cellulaire, l’homéostasie et la fonction de barrière de l’épithélium intestinal – PREDESTINE

Submission summary

The functional maintenance of the intestinal epithelium homeostasis involves a fine coordination between proliferation in crypts, differentiation, during migration along the villi and apoptosis. Enterocytes represent the major differentiated cell population of this epithelium. Their absorptive function strictly depends on their high polarization. Understanding of the processes for maintenance and functions of intestinal epithelium is based on the comprehension of the onset and stability of tissue architecture, in which cell-cell adhesion plays a central role. This process relies upon the assembly of numerous proteins within junctional complexes that can activate signalling pathways involved in the onset and maintenance of epithelial cell polarisation. We identified the cellular prion protein (PrPc) as a new actor of cell-cell junction. In intestinal epithelial cells, PrPc is localized in the junctional complexes, where it interacts with Src kinase and desmosomal proteins, while, in dividing cells, PrPc is localized in the nucleus. We demonstrated that the invalidation of PrPc induces an arrest of cell multiplication and disorganisation of desmosomes. Our aims are to analyze the role of PrPc in the organization and functional properties of desmosomes in the intestinal epithelium, to characterize the mechanisms through which PrPc and its partners control cell-cell adherence and the proliferation/differentiation processes and to determine whether dysfunctions of desmosome-associated PrPc affect the barrier properties of the epithelium. To determine how PrPc is involved in the onset and/or in the maintenance of intestinal epithelial cell architecture, but also in the proliferation/ differentiation processes, we created a model of Caco-2/TC7 enterocytes allowing the dissociation of these intimately linked effects through an inducible expression of shRNA-PrPc in exponentially growing, pre-confluent or post-confluent cells. In parallel, we possess PrPc -/- mice for the studies in vivo of the physiological function of intestinal barrier. Our project will be organized into three tasks: Task 1: We will determine the role of PrPc in the organization and functional properties of desmosomes. The impacts of PrPc down regulation on cell morphology and polarization, on the localization of the desmosomal PrPc partners in membrane raft domains and sub-cellular compartments and on the organization of the cytoskeleton networks will be characterized along with modifications of hyper-adhesion capacity and resistance to stretch of desmosomes. Task 2: We will decipher the signalling pathways through which PrPc and its partners control cell-cell adherence and the proliferation/differentiation processes. The signalling protein Src kinase interacting with PrPc in junctions and being present in nucleus, along with PrPc in dividing cells, we will check whether PrPc and Src interact in the nucleus and analyze the phosphorylation state of c-Src when complexed to PrPc in each compartment. We will determine if Src kinase and PKCa, which is involved in the regulation of desmosome hyper-adhesion, relay PrPc in the regulation of cell-cell adhesion. Nuclear partners of PrPc in dividing epithelial cells will be characterized to precise the potential signalling pathways involving PrPc. The desmosomal protein plakoglobin (g catenin) being a nuclear PrPc partner in dividing cells, we will determine whether both proteins are involved in the Wnt signalling pathway to control the balance between proliferation and differentiation. Task 3: We will examine the contribution of desmosomes-associated PrPc to epithelium homeostasis and efficiency of barrier function. The impact of the invalidation of PrPc, in vitro and in vivo on the integrity of the epithelial barrier will be analyzed. Permeability and trans-epithelial resistance will be measured. The resistance to bacterial infection and the control of induced inflammation will be characterized. In conclusion, PREDESTINE (Prion-Enterocytes-DESmosomes-inteSTINE) project will allow, through analyses in the intestinal epithelium, to bring important knowledge not only on the contribution of PrPc but more generally on that of desmosomes, whose role in intestine is still unknown, to the physiology of renewing epithelia, from the mechanisms involved in the balance between proliferation and differentiation and the regulation of cell polarisation to the onset of architecture and barrier functions.

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The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

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Beginning and duration of the scientific project: - 0 Months

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