Carole Siret

Early adhesion induces interaction of FAK and Fyn in lipid domains and activates raft-dependent Akt signaling in SW480 colon cancer cells

Integrin-dependent interaction of epithelial tumor cells with extracellular matrix (ECM) is critical for their migration, but also for hematogenous dissemination. Elevated expression and activity of Src family kinases (SFKs) in colon cancer cells is often required in the disease progression. In this work, we highlighted how focal adhesion kinase (FAK) and SFKs interacted and we analyzed how PI3K/Akt and MAPK/Erk1/2 signaling pathways were activated in early stages of colon cancer cell adhesion. During the first hour, integrin engagement triggered FAK-Y397 phosphorylation and a fraction of FAK was located in lipid rafts/caveolae domains where it interacted with Fyn. The FAK-Y861 and/or -Y925 phosphorylations led to a subsequently FAK translocation out of lipid domains. In parallel, a PI3K/Akt pathway dependent of lipid microdomain integrity was activated. In contrast, the MAPK/Erk1/2 signaling triggered by adhesion increased during at least 4 h and was independent of cholesterol disturbing. Thus, FAK/Fyn interaction in lipid microdomains and a Akt-1 activation occurred at the same time during early contact with ECM suggesting a specific signaling dependent of lipid rafts/caveolae domains.

Saccharomyces boulardii Improves Intestinal Cell Restitution through Activation of the α2β1 Integrin Collagen Receptor

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of inflammatory bowel diseases such as ulcerative colitis or Crohns disease. Complete remission of these diseases requires both the cessation of inflammation and the migration of enterocytes to repair the damaged epithelium. Lyophilized Saccharomyces boulardii (Sb, Biocodex) is a nonpathogenic yeast widely used as a therapeutic agent for the treatment and prevention of diarrhea and other gastrointestinal disorders. In this study, we determined whether Sb could accelerate enterocyte migration. Cell migration was determined in Sb force-fed C57BL6J mice and in an in vitro wound model. The impact on α2β1 integrin activity was assessed using adhesion assays and the analysis of α2β1 mediated signaling pathways both in vitro and in vivo. We demonstrated that Sb secretes compounds that enhance the migration of enterocytes independently of cell proliferation. This enhanced migration was associated with the ability of Sb to favor cell-extracellular matrix interaction. Indeed, the yeast activates α2β1 integrin collagen receptors. This leads to an increase in tyrosine phosphorylation of cytoplasmic molecules, including focal adhesion kinase and paxillin, involved in the integrin signaling pathway. These changes are associated with the reorganization of focal adhesion structures. In conclusion Sb secretes motogenic factors that enhance cell restitution through the dynamic regulation of α2β1 integrin activity. This could be of major importance in the development of novel therapies targeting diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Mature DC from skin and skin-draining LN retain the ability to acquire and efficiently present targeted antigen

Skin‐draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody‐opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag‐specific CD4+ T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild‐type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross‐presenting it to CD8+ T cells. Induction of additional maturation in vivo by LPS or treatment with double‐stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin‐draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor‐mediated endocytosis.

Saccharomyces boulardii Improves Intestinal Epithelial Cell Restitution by Inhibiting αvβ5 Integrin Activation State

Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohns disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.

Lebectin increases N-cadherin-mediated adhesion through PI3K/AKT pathway

Cell adhesion molecules, including cadherins and integrins, play an essential role during tumor progression and represent potential targets for the development of new therapeutic agents. We previously showed that lebectin, a C-type lectin protein (CLP) issued from Macrovipera lebectina snake venom, inhibits integrin-mediated migration of IGR39 melanoma cells. Here we assessed whether lebectin modulates cell-cell adhesion. We demonstrated that lebectin promotes N-cadherin/catenin complex reorganization at cell-cell contacts, inducing a strengthening of intercellular adhesion. This reorganization is associated to phosphorylation of beta-catenin on tyrosine 142 residue. Interestingly, lebectin acts on N-cadherin-mediated cell-cell contacts through PI3K/Akt pathway. This effect could contribute to the blockage of tumor cell migration previously observed.

Interplay between cadherins and α2β1 integrin differentially regulates melanoma cell invasion.

Background:Malignant transformation of melanocytes frequently coincides with an alteration in the expression of cell–cell adhesion molecules (cadherins) and cell-extracellular matrix proteins (integrins). How these two adhesion systems interplay to impact on cell invasion remains to be described in melanoma.Methods:Cell adhesion networks were localised by immunofluorescence in human primary cutaneous melanoma, metastatic melanoma in the lymph nodes, and melanoma cell lines. The role of these cell adhesion networks was assessed both in vivo, by analysing their impact on tumour growth in mice, and in vitro, with the use of functional tests including cell aggregation and cell migration.Results:We found that α2β1 integrin associates with both E-cadherin and N-cadherin to form two adhesive networks, distinguishable by the interaction—or not—of α2β1 integrin with type I collagen. N-cadherin/α2β1 integrin and E-cadherin/α2β1 integrin networks differently participated towards tumour growth in mice. The N-cadherin/α2β1 integrin network showed specific involvement in melanoma cell invasion and migration towards type I collagen. On the other hand, the E-cadherin/α2β1 network regulated cell–cell adhesion.Conclusions:This suggests that different signalling environments can be generated, depending on the type and/or local concentration of cadherin present in the adhesion complex, which potentially leads to differential cell responses. Further clarification of how these adhesive networks are regulated is fundamental to understanding important physiological and pathological processes such as morphogenesis, wound healing, tumour invasion and metastasis.

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial.

Saccharomyces boulardii CNCM I-745 Restores intestinal Barrier Integrity by Regulation of E-cadherin Recycling

Background and Aims Alteration in intestinal permeability is the main factor underlying the pathogenesis of many diseases affecting the gut, such as inflammatory bowel disease [IBD]. Characterization of molecules targeting the restoration of intestinal barrier integrity is therefore vital for the development of alternative therapies. The yeast Saccharomyces boulardii CNCM I-745 [Sb], used to prevent and treat antibiotic-associated infectious and functional diarrhea, may have a beneficial effect in the treatment of IBD. Methods We analyzed the impact of Sb supernatant on tissue integrity and components of adherens junctions using cultured explants of colon from both IBD and healthy patients. To evaluate the pathways by which Sb regulates the expression of E-cadherin at the cell surface, we developed in vitro assays using human colonic cell lines, including cell aggregation, a calcium switch assay, real-time measurement of transepithelial electrical resistance [TEER] and pulse-chase experiments. Results We showed that Sb supernatant treatment of colonic explants protects the epithelial morphology and maintains E-cadherin expression at the cell surface. In vitro experiments revealed that Sb supernatant enhances E-cadherin delivery to the cell surface by re-routing endocytosed E-cadherin back to the plasma membrane. This process, involving Rab11A-dependent recycling endosome, leads to restoration of enterocyte adherens junctions, in addition to the overall restoration and strengthening of intestinal barrier function. Conclusion These findings open new possibilities of discovering novel options for prevention and therapy of diseases that affect intestinal permeability.

Cadherin-1 and cadherin-3 cooperation determines the aggressiveness of pancreatic ductal adenocarcinoma

Background:Pancreatic ductal adenocarcinoma (PDAC) is characterised by an extensive tissue invasion and an early formation of metastasis. Alterations in the expression of cadherins have been reported in PDAC. Yet, how these changes contribute to tumour progression is poorly understood. Here, we investigated the relationship between cadherins expression and PDAC development.Methods:Cadherins expression was assessed by immunostaining in both human and murine tissue specimens. We have generated pancreatic cancer cell lines expressing both cadherin-1 and cadherin-3 or only one of these cadherins. Functional implications of such genetic alterations were analysed both in vitro and in vivo.Results:Cadherin-3 is detected early at the plasma membrane during progression of pancreatic intraepithelial neoplasia 1 (PanIN-1) to PDAC. Despite tumoural cells turn on cadherin-3, a significant amount of cadherin-1 remains expressed at the cell surface during tumourigenesis. We found that cadherin-3 regulates tumour growth, while cadherin-1 drives type I collagen organisation in the tumour. In vitro assays showed that cadherins differentially participate to PDAC aggressiveness. Cadherin-3 regulates cell migration, whereas cadherin-1 takes part in the invadopodia activity.Conclusions:Our results show differential, but complementary, roles for cadherins during PDAC carcinogenesis and illustrate how their expression conditions the PDAC aggressiveness.