Valérie Vouret-Craviari

cDNA cloning and expression of a hamster α‐thrombin receptor coupled to Ca2+ mobilization

The serine protease α‐thrombin (thrombin) potently stimulates G‐protein‐coupled signaling pathways and DNA synthesis in CCL39 hamster lung fibroblasts. To clone a thrombin receptor cDNA, selective amplification of mRNA sequences displaying homology to the transmembrane domains of G‐protein‐coupled receptor genes was performed by polymerase chain reaction. Using reverse transcribed poly(A)+ RNA from CCL39 cells and degenerate primers corresponding to conserved regions of several phospholipase C‐coupled receptors, three novel putative receptor sequences were identified. One corresponds to an mRNA transcript of 3.4 kb in CCL39 cells and a relatively abundant cDNA. Microinjection of RNA transcribed in vitro from this cDNA in Xenopus oocytes leads to the expression of a functional thrombin receptor. The hamster thrombin receptor consists of 427 amino acid residues with 8 hydrophobic domains, including one at the extreme N‐terminus that is likely to represent a signal peptide. A thrombin consensus cleavage site is present in the N‐terminal extracellular region of the receptor sequence followed by a negatively charged cluster of residues present in a number of proteins that interact with the anion‐binding exosite of thrombin.

A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease

Susceptibility to Crohns disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohns disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohns disease–associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohns disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.

EphrinA1 inactivates integrin-mediated vascular smooth muscle cell spreading via the Rac/PAK pathway.

Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA1 is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA1 in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA1-induced integrin inactivation. EphrinA1 did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA1 effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA1-mediated inhibition of spreading in VSMCs. The ephrinA1-mediated morphological alterations correlated with inhibition of Rac1 and p21-activated kinase 1 (PAK1) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover, repression of Rac1 synthesis with siRNA amplifies the ephrinA1-induced inhibition of spreading. Finally, sphingosine-1-phosphate (S1P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA1 effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA1-mediated inhibition of spreading. In this way, ephrinA1, alone or in synergy with S1P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.

Angiogenesis and G-protein-coupled receptors: signals that bridge the gap

Angiogenesis is a mechanism that has repercussions in a number of physiological and pathological situations. Vascular endothelial growth factor and basic fibroblast growth factor have understandably received enormous research coverage for being the major mediators of new blood vessel growth, often overshadowing other agonist that also have strong angiogenic potential. We wish to put the spotlight on GPCR agonists that undoubtedly have their word to say on the subject of angiogenesis. In this short review, we will discuss our findings along with the work from other groups on the mechanisms by which GPCR agonists, like thrombin and angiotensin II, control a number of angiogenic signals. A complete understanding of these mechanisms could, by the design of new therapeutic strategies, have a strong impact in clinical oncology.

ILK is required for the assembly of matrix-forming adhesions and capillary morphogenesis in endothelial cells

Integrins play a key role in regulating endothelial cell survival, migration and differentiated function during angiogenic blood-vessel remodeling. Integrin-linked kinase (ILK) is a multidomain protein that interacts with the cytoplasmic tail of integrin β subunits and is thought to participate in integrin-mediated signal transduction. We report here that attenuation of ILK expression in cultured bovine aortic endothelial cells by RNA interference had marked effects on surface distribution of α5β1 integrin and the organization of cell-matrix adhesions characterized by the disappearance of fibrillar (3D-like) adhesions that are rich in α5β1 and paxillin, and associated fibrillar fibronectin matrix. This defect was not caused by a decrease in fibronectin mRNA levels or by intracellular retention of the protein. Adhesion to surface-adsorbed matrix proteins based on β1 and β3 integrin was enhanced following ILK depletion, whereas cell spreading, migration and multilayer alignment into capillary-like structures on Matrigel were impaired. We conclude that ILK is an important regulator of the endothelial phenotype and vascular network formation by directing the assembly and/or maturation of α5β1-competent matrix-forming adhesions.

Autophagy and Crohn's Disease: At the Crossroads of Infection, Inflammation, Immunity, and Cancer

Inflammatory bowel diseases (IBD) are common inflammatory disorders of the gastrointestinal tract that include ulcerative colitis (UC) and Crohns disease (CD). The incidences of IBD are high in North America and Europe, affecting as many as one in 500 people. These diseases are associated with high morbidity and mortality. Colorectal cancer risk is also increased in IBD, correlating with inflammation severity and duration. IBD are now recognized as complex multigenetic disorders involving at least 32 different risk loci. In 2007, two different autophagy-related genes, ATG16L1 (autophagy-related gene 16-like 1) and IRGM (immunity-related GTPase M) were shown to be specifically involved in CD susceptibility by three independent genome-wide association studies. Soon afterwards, more than forty studies confirmed the involvement of ATG16L1 and IRGM variants in CD susceptibility and gave new information on the importance of macroautophagy (hereafter referred to as autophagy) in the control of infection, inflammation, immunity and cancer. In this review, we discuss how such findings have undoubtedly changed our understanding of CD pathogenesis. A unifying autophagy model then emerges that may help in understanding the development of CD from bacterial infection, to inflammation and finally cancer. The Pandoras box is now open, releasing a wave of hope for new therapeutic strategies in treating Crohns disease.

Differential expression and regulation of ADAM17 and TIMP3 in acute inflamed intestinal epithelia.

The acute phase of Crohns disease (CD) is characterized by a large afflux of polymorphonuclear leukocytes (PMNL) into the mucosa and by the release of TNF-alpha. Conversion of inactive TNF-alpha into an active form requires the cleavage of a transmembrane TNF-alpha precursor by the TNF-alpha-converting enzyme (ADAM17), a protease mainly regulated by the tissue inhibitor of metalloproteinase 3 (TIMP3). The aim of the present study was to investigate in an in vitro model of PMNL transepithelial migration and in the intestinal mucosa of patients with CD the expression and regulation of ADAM17 and TIMP3 in intestinal epithelial cells (IEC). ADAM17 and TIMP3 expression was analyzed by Western blotting, RT-PCR, confocal microscopy, and immunohistochemistry by using the T84 model and digestive biopsies. ADAM17 expression in IEC was increased at a posttranscriptional level during the early phase (from 2 to 4 h) of PMNL transepithelial migration whereas TIMP3 was only increased 24 h later. TNF-alpha induced an early upregulation of ADAM17 in T84 cells, whereas PMNL adhesion, H(2)O(2), or epithelial tight junction opening alone did not affect the amount of ADAM17. Immunohistochemistry of intestinal biopsies revealed that strong expression of ADAM17 was associated with a high activity of CD. In contrast, TIMP3 was very poorly expressed in these biopsies. ADAM17 and TIMP3 profiling did not correlated with the NOD2/CARD15 status. The ADAM17 activity was higher both in the early phase of PMNL transepithelial migration and in active CD. These results showed early posttranscriptional upregulation of ADAM17 in IEC linked to PMNL transepithelial migration and a high activity of CD.

Modulation of Rho GTPase activity in endothelial cells by selective proteinase-activated receptor (PAR) agonists

Summary.  The proteinase‐activated receptors (PAR) PAR1 and PAR2 mediate responses to thrombin and trypsin‐like proteases, respectively. Both receptors are expressed on endothelial cells where they have been reported to transduce a similar set of intracellular responses. In cultured human umbilical vein endothelial cells (HUVEC), we observed a marked difference in shape changes induced by PAR‐activating peptides (PAR‐APs); unlike PAR1‐AP, PAR2‐AP failed to stimulate cell rounding. Objectives were to shed light on the mechanisms underlying PAR‐mediated cytoskeletal responses. We examined the activation of the Rho family GTPases in HUVEC using highly selective PAR1‐ and PAR2‐APs to do this. Both peptides induced a robust and transient activation of RhoA, with the time course of activation being more sustained for the PAR1‐AP. Interestingly, divergent effects on Rac activity were observed. Addition of PAR1‐AP inhibited basal Rac activity as well as the phosphorylation of the Rac effector, p21‐activated kinase (PAK). In contrast, PAR2‐AP induced a modest activation of Rac, phosphorylation of PAK and translocation of cortactin from the cytosol to membrane ruffles, a Rac‐dependent event. In vivo, only PAR1‐AP rapidly enhanced vascular permeability in a mouse skin assay. We conclude that the differential regulation of the Rac/PAK pathway by PAR1 and PAR2 agonists in endothelial cells points toward distinct roles for these receptors in the control of vascular permeability and blood vessel remodeling.

Autophagy Plays a Critical Role in the Degradation of Active RHOA, the Control of Cell Cytokinesis, and Genomic Stability

Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance.

HIF‐1α mediates the induction of IL‐8 and VEGF expression on infection with Afa/Dr diffusely adhering E. coli and promotes EMT‐like behaviour

Microbes regulate a large panel of intracellular signalling events that can promote inflammation and/or enhance tumour progression. Indeed, it has been shown that infection of human intestinal cells with the Afa/Dr diffusely adhering E. coli C1845 strain induces expression of pro‐angiogenic and pro‐inflammatory genes. Here, we demonstrate that exposure of cryptic‐like intestinal epithelial cells to C1845 bacteria induces HIF‐1α protein levels. This effect depends on the binding of F1845 adhesin to the membrane‐associated DAF receptor that initiates signalling cascades promoting translational mechanisms. Indeed, inhibition of MAPK and PI‐3K decreases HIF‐1α protein levels and blocks C1845‐induced phosphorylation of the ribosomal S6 protein. Using RNA interference we show that bacteria‐induced HIF‐1α regulates the expression of IL‐8, VEGF and Twist1, thereby pointing to a role for HIF‐1 in angiogenesis and inflammation. In addition, infection correlates with a loss of E‐cadherin and cytokeratin 18 and a rise in fibronectin, suggesting that bacteria may induce an epithelial to mesenchymal transition‐like phenotype. Since HIF‐1α silencing results in reversion of bacteria‐induced EMT markers, we speculate that HIF‐1α plays a key role linking bacterial infection to angiogenesis, inflammation and some aspects of cancer initiation.