Id3 modulates cellular localization of bHLH Ptf1-p48 protein.

International audiencePtf1-p48 is a pancreas-specific bHLH transcriptional protein, which, in the normal adult pancreas, shows a restricted expression in acinar cells where it is predominantly localized in the nucleus and activates the transcription of exocrine-specific genes. Ptf1-p48 partners with two proteins to form the PTF1 active complex: a bHLH E-protein and suppressor of hairless RBP-J. Cytoplasmic mislocalization of Ptf1-p48 has been reported in pancreatic pathologies, suggesting its contribution in the early steps of pancreatic carcinogenesis. The aim of the our work was to elucidate the mechanisms regulating Ptf1-p48 subcellular localization. We hypothesized a role of Id proteins acting in a dominant-negative fashion by heterodimerizing with bHLH proteins. We reproduced Ptf1-p48 cytoplasmic mislocalization in acinar AR4-2J cells and demonstrated that a proliferative signal elicited by gastrin leads to increases in Id3 protein expression and levels of Id3/E47 and Id3/Ptf1-p48 interactions, and a decrease in the level of E47/Ptf1-p48 interaction. By contrast, Id3 silencing reversed the cytoplasmic mislocalization of Ptf1-p48 induced by gastrin. As E47 is responsible for the nuclear import of the PTF1 complex, disruption of this complex via Id3 interactions with both E47 and Ptf1-p48 appears to induce cytoplasmic mislocalization of Ptf1-p48. We then found that Ptf1-p48 is either absent or mislocalized in the cytoplasm and Id3 is overexpressed in human and murine pancreatic preneoplastic lesions. Our data provide novel insight into the regulation of Ptf1-p48 function and provide evidence that Ptf1-p48 cytoplasmic mislocalization and Id3 overexpression are early events in pancreatic cancer progression

Impact of Salmonella Typhimurium on intestinal stem cells and progenitors.

During infection, Salmonella Typhimurium is able to hijack signaling pathways, controlling the cellular growth and proliferation. However, the precise mechanism by which Salmonella Typhimurium infection impacts stem cells and progenitors, responsible for the ability of the intestinal epithelium to renew and repair itself remains unclear. In our study, we used in vivo systemic mouse model and in vitro intestinal organoid model to characterize the changes in the epithelium growth after Salmonella Typhimurium infection. Our data present evidence that compared to other segments (ileum and colon), the cecal crypt is preferentially targeted by the bacteria. Analysis of mRNA expression profile, protein phosphorylation and immunohistochemistry of infected in situ cecal crypts and in vitro organoids, point out an upregulated proliferation, leading to an increase in size. Moreover, EGFR pathways, crucial to primitive cells proliferation, are taken over by the bacteria. As Salmonella express the outer membrane invasin called Rck, able to interact with EGFR, its potential role in the deregulation of intestinal stem cells and progenitors has been investigated.This new mechanism targeting the proliferation of intestinal primitive cells raises questions about the long-term consequences of repeated Salmonella infections on the crypt integrity and its dysregulation in pathologies such as cancer

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells

International audienceBackground: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions.Methods: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression.Results: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance.Conclusions: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt

Sexual dimorphism in PAR(2)-dependent regulation of primitive colonic cells

BACKGROUND: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.status: publishe

Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation

International audienceImbalance between proteases and their inhibitors plays a crucial role in the development of Inflammatory Bowel Diseases (IBD). Increased elastolytic activity is observed in the colon of patients suffering from IBD. Here, we aimed at identifying the players involved in elastolytic hyperactivity associated with IBD and their contribution to the disease. We revealed that epithelial cells are a major source of elastolytic activity in healthy human colonic tissues and this activity is greatly increased in IBD patients, both in diseased and distant sites of inflammation. This study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. We demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier. Epithelial ELA2A hyperactivity also modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcripts, while reducing the expression of pro-resolving and repair factor genes. ELA2A thus appears as a novel actor produced by intestinal epithelial cells, which can drive inflammation and loss of barrier function, two essentials pathophysiological hallmarks of IBD. Targeting ELA2A hyperactivity should thus be considered as a potential target for IBD treatment

Chymotrypsin activity signals to intestinal epithelium by protease‐activated receptor‐dependent mechanisms

International audienceAbstract Background and Purpose Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease‐activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. Experimental Approach The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N‐terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC–MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. Key Results We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin‐10 (IL‐10) up‐regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. Conclusion and Implications Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis

Active thrombin produced by the intestinal epithelium controls mucosal biofilms

International audienceProteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin