Biomedical subjects

Violaine Moreau

Publications and source records attributed to Violaine Moreau.

7 recordsLinked to original sources

PubMed2006

TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA.

TGFbeta is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFbeta1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFbeta1 on the activation state of members of the RhoGTPase family. TGFbeta1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFbeta1-mediated change of phenotype. Although TGFbeta1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFbeta1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFbeta1 in the process of endothelial tube formation in collagen I gels.

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PubMed2006

A signalling cascade involving PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in response to phorbol ester.

The involvement of Src, Cdc42, RhoA and PKC in the regulation of podosome assembly has been identified in various cell models. In endothelial cells, the ectopic expression of constitutively active mutants of Src or Cdc42, but not RhoA, induced the formation of podosomes. Short-term exposure to phorbol-12-myristate-13-acetate (PMA) induced the appearance of podosomes and rosettes after initial disruption of stress fibres. Molecular analysis of PMA-induced podosomes and rosettes revealed that their composition was identical to that of podosomes described in other models. Pharmacological inhibition and siRNA knock-down experiments revealed that both PKCalpha and PKCdelta isotypes were necessary for podosome assembly. However, only constitutively active PKCalpha could mimic PMA in podosome formation. Src, Cdc42 and RhoA were required downstream of PKCs in this process. Src could be positioned between PKC and Cdc42 in a linear cascade leading to podosome assembly. Using in vitro matrix degradation assays, we demonstrated that PMA-induced podosomes are endowed with proteolytic activities involving MT1-MMP-mediated activation of MMP2. Endothelial podosomes may be involved in subendothelial matrix degradation during endothelium remodelling in pathophysiological processes.

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PubMed2006

Discontinuous epitope prediction based on mimotope analysis.

MOTIVATION: Phage display is a widespread technique used to obtain peptide mimotopes selected by binding to a given monoclonal antibody in a similar way as the native epitope. However, the localization of the interaction site mimicked by the mimotopes on the surface of the antigen is not always a straightforward task. MIMOP is a computational tool developed with the aim of helping experimentalists to analyze a set of mimotope sequences and guide them in the identification of the mimicked region. RESULTS: To predict potential epitopic regions, MIMOP integrates two different approaches combining two- and three-dimensional analyses: MimAlign starts from degenerated alignment analyses, and MimCons is based on consensus identification. The relevance and usefulness of the tool are illustrated by four use cases corresponding to real-life situations.

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PubMed2006

Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells.

Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.

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PubMed2005

Cdc42-driven podosome formation in endothelial cells.

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.

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PubMed2003

Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA.

Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology. To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator. Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin. Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction. Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein. Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites. Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity. Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery. These data provide the first description of podosomes in endothelial cells. The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

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PubMed2002

Grb2 and Nck act cooperatively to promote actin-based motility of vaccinia virus.

The Wiskott-Aldrich syndrome protein family member N-WASP is a key integrator of the multiple signalling pathways that regulate actin polymerization via the Arp2/3 complex. Our previous studies have shown that N-WASP is required for the actin-based motility of vaccinia virus and is recruited via Nck and WIP. We now show that Grb2 is an additional component of the vaccinia actin tail-forming complex. Recruitment of Nck and Grb2 to viral particles requires phosphorylation of tyrosine residues 112 and 132 of A36R, the vaccinia actin tail nucleator, respectively. The presence of Grb2 on the virus is also dependent on the polyproline-rich region of N-WASP. The Grb2 pathway alone is therefore unable to nucleate actin tails, as its recruitment requires the prior recruitment of N-WASP by Nck. However, Grb2 does play an important role in actin-based motility of vaccinia, as in its absence, the mean number of actin tails per cell is reduced 2.6-fold. Thus, both Nck and Grb2 act in a cooperative manner to stabilize and/or activate the vaccinia actin-nucleating complex. We suggest that such cooperativity between "primary" and "secondary" adaptor proteins is likely to be a general feature of receptor-mediated signalling.

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