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Biomedical subjects

Elena Vigorito

Publications and source records attributed to Elena Vigorito.

At least 19 recordsLinked to original sources
PubMed2007

Vav family proteins are required for optimal regulation of PLCgamma2 by integrin alphaIIbbeta3.

Vav proteins belong to the family of guanine-nucleotide-exchange factors for the Rho/Rac family of small G-proteins. In addition, they serve as important adapter proteins for the activation of PLCgamma (phospholipase Cgamma) isoforms by ITAM (immunoreceptor tyrosine-based activation motif) receptors, including the platelet collagen receptor GPVI (glycoprotein VI). Vav proteins are also regulated downstream of integrins, including the major platelet integrin alphaIIbbeta3, which has recently been shown to regulate PLCgamma2. In the present study, we have investigated the role of Vav family proteins in filopodia and lamellipodia formation on fibrinogen using platelets deficient in Vav1 and Vav3. Wild-type mouse platelets undergo a limited degree of spreading on fibrinogen, characterized by the formation of numerous filopodia and limited lamellipodia structures. Platelets deficient in Vav1 and Vav3 exhibit reduced filopodia and lamellipodia formation during spreading on fibrinogen. This is accompanied by reduced alphaIIbbeta3-mediated PLCgamma2 tyrosine phosphorylation and reduced Ca(2+) mobilization. In contrast, the G-protein agonist thrombin stimulates full spreading of control and Vav1/3-deficient platelets. Consistent with this, stimulation of F-actin (filamentous actin) formation and Rac activation by thrombin is not altered in Vav-deficient cells. These results demonstrate that Vav1 and Vav3 are required for optimal spreading and regulation of PLCgamma2 by integrin alphaIIbbeta3, but that their requirement is by-passed upon G-protein receptor activation.

Animals↗
PubMed2006

RhoG regulates the neutrophil NADPH oxidase.

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG(-/-) neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG(-/-) neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.

Animals↗
PubMed2005

Synergistic activation of PKD by the B cell antigen receptor and CD19 requires PI3K, Vav1 and PLCgamma.

Antigens coated with complement fragments coligate the B cell receptor (BCR) with the CD21/CD19 complex which results in synergistic activation of B cells. Previous studies identified PI3K, Vav proteins and PLCgamma as important components of this synergy. We now show that protein kinase D (also known as PKCmu) is also a point of convergence of these signalling pathways. We found that PKD activation upon BCR engagement or coligation of the BCR with CD19 is entirely dependent on PI3K and PLCgamma but differ in the requirement for Vav proteins. Whereas PKD activation is Vav1 and Vav2 dependent in response to BCR cross-linking, PKD activation is sensitive to the lack of Vav1 under synergistic stimulation of BCR and CD19. These findings show that Vav proteins and PI3K regulation of PLCgamma contributes to the activation of PKD in response to BCR and or CD19 cross-linking.

Animals↗
PubMed2005

Cutting edge: T cell development requires the combined activities of the p110gamma and p110delta catalytic isoforms of phosphatidylinositol 3-kinase.

The role of PI3K activity in T lymphocyte development is obscure because mice deficient in single PI3K catalytic subunits either die before birth (p110alpha-/- and p110beta-/-) or lack a significant T cell developmental phenotype (p110gamma-/- and p110delta-/-). We have generated mice deficient in both p110gamma and p110delta and show that p110gamma/delta-/- mice have a profound block in T cell development that occurs at the beta-selection checkpoint. We show that pre-TCR-induced signaling is significantly reduced in p110gamma/delta-/- thymocytes and that this results in a concomitant lack of proliferative expansion and increased apoptosis. The survival defect in p110gamma/delta-/- thymocytes is associated with increased levels of the pro-apoptotic molecule Bcl2 interacting mediator of cell death. This work demonstrates that PI3K activity is critical for T cell development and depends on the combined function of p110gamma and p110delta.

Animals↗
PubMed2005

Vav1 and Vav2 play different roles in macrophage migration and cytoskeletal organization.

Vav family proteins act as guanine nucleotide exchange factors for Rho family proteins, which are known to orchestrate cytoskeletal changes and cell migration in response to extracellular stimuli. Using mice deficient for Vav1, Vav2 and/or Vav3, overlapping and isoform-specific functions of the three Vav proteins have been described in various hematopoietic cell types, but their roles in regulating cell morphology and migration have not been studied in detail. To investigate whether Vav isoforms have redundant or unique functions in regulating adhesion and migration, we investigated the properties of Vav1-deficient and Vav2-deficient macrophages. Both Vav1-deficient and Vav2-deficient cells have a smaller adhesive area; yet, only Vav1-deficient cells have a reduced migration speed, which coincides with a lower level of microtubules. Vav2-deficient macrophages display a high level of constitutive membrane ruffling, but neither Vav1 nor Vav2 is required for colony stimulating factor-1-induced membrane ruffling and cell spreading. Our results suggest that the migration speed of macrophages is regulated independently of spread area or membrane ruffling and that Vav1 is selectively required to maintain a normal migration speed.

Animals↗
PubMed2005

Vav proteins regulate peripheral B-cell survival.

Mice lacking all 3 Vav proteins fail to produce significant numbers of recirculating follicular or marginal zone B cells. Those B cells that do mature have shortened lifespans. The constitutive nuclear factor-kappaB (NF-kappaB) activity of resting naive B cells required Vav function and expression of cellular reticuloendotheliosis (c-Rel). Rel-A was reduced in Vav-deficient B cells. Furthermore, expression of the NF-kappaB-regulated antiapoptotic genes A1 and Bcl-2 was reduced in mature Vav-deficient B cells. Overexpression of Bcl-2 restored the number of mature follicular B cells in the spleens of Vav-deficient mice. When activated by B-cell receptor (BCR) cross-linking, Vav-deficient B cells failed to activate NF-kappaB. Vav proteins thus regulate an NF-kappaB-dependent survival signal in naive B cells and are required for NF-kappaB function after BCR cross-linking.

Animals↗
PubMed2005

Vav proteins are required for B-lymphocyte responses to LPS.

B lymphocytes respond to bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) and CD180 (previously called RP105). We show here that the responses of B lymphocytes to LPS require the function of the Vav family of guanine nucleotide exchange factors. Vav1-mutant mice generate defective humoral immunoglobulin G (IgG) responses following administration of low doses of LPS but respond normally to higher doses, while mice lacking both Vav1 and Vav2 manifest defective responses even after a high dose of LPS. Vav1/2-mutant B cells fail to divide extensively in vitro in response to LPS or CD180, while deficiency of Vav1 alone impairs CD180-but not LPS-driven proliferation. Likewise, activation of Akt (a PI3K [phosphatidylinositol 3-kinase] target) and phosphorylation of IkappaBalpha in response to CD180 or LPS required Vav1 and Vav2, while Vav1 deficiency led to defective responses to CD180. In addition, activation of ERK (extracellular signal regulated kinase) required Vav1 and Vav2 in response to CD180 but was Vav1 and vav2 independent in response to LPS. Induction of CD86 and CD25 by anti-CD180 also required Vav function, as did the induction of the anti-apoptotic protein Bcl-xL (B-cell leukemia XL). These data provide evidence for the function for the Vav proteins in regulating the responses of B cells to LPS.

Animals↗
PubMed2005

Phosphatidylinositol 3-kinase is required for the transcriptional activation of cyclin D2 in BCR activated primary mouse B lymphocytes.

Induction of cyclin D2 is essential for mediating cell cycle entry in B cells activated by BCR cross-linking. In the present study we show that, like B lymphocytes lacking cyclin D2, the p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) or other components of the B cell signalosome, p110delta-null B cells fail to induce cyclin D2 and enter early G1 but not S phase of the cell cycle. The inhibitors of PI3K activity, LY294002 and Wortmannin, also abrogate cyclin D2 induction by BCR cross-linking, confirming that the class IA PI3K is necessary for cyclin D2 induction in response to BCR stimulation. Furthermore, using both p85alpha-null and p110delta-null B cells and inhibitors of PI3K, this study demonstrates for the first time, that BCR cross-linking induces cyclin D2 mRNA expression via transcriptional activation of the cyclin D2 promoter and that this transcriptional activation of cyclin D2 requires PI3K activity. Moreover, we identify a region between nucleotides -1624 and -1303 of the cyclin D2 promoter containing elements responsive to anti-IgM, which are PI3K dependent. Further characterisation of signalling intermediates downstream of the BCR revealed a perturbation of MAPK signalling pathways in p85alpha-null and p110delta-null B cells, and our data suggests that cross-talk exists between the PI3K and JNK pathways.

Androstadienes↗
PubMed2004

Vav1 and vav3 have critical but redundant roles in mediating platelet activation by collagen.

Vav family proteins are guanine nucleotide exchange factors for the Rho/Rac family of small GTP-binding proteins. In addition, they have domains that mediate protein-protein interactions, including one Src homology 2 (SH2) and two Src homology 3 (SH3) domains. Vav1, Vav2, and Vav3 play a crucial role in the regulation of phospholipase C gamma (PLC gamma) isoforms by immuno-tyrosine-based activation motif (ITAM)-coupled receptors, including the T- and B-cell antigen receptors. We have reported in platelets, however, that Vav1 and Vav2 are not required for activation of PLC gamma 2 in response to stimulation of the ITAM-coupled collagen receptor glycoprotein VI (GPVI). Here we report that Vav3 is tyrosinephosphorylated upon activation of GPVI but that Vav3-deficient platelets also exhibit a normal response upon activation of the ITAM receptor. In sharp contrast, platelets deficient in both Vav1 and Vav3 show a marked inhibition of aggregation and spreading upon activation of GPVI, which is associated with a reduction in tyrosine phosphorylation of PLC gamma 2. The phenotype of Vav1/2/3 triple-deficient platelets is similar to that of Vav1/3 double-deficient cells. These results demonstrate that Vav3 and Vav1 play crucial but redundant roles in the activation of PLC gamma 2 by GPVI. This is the first time that absolute redundancy between two protein isoforms has been observed with respect to the regulation of PLC gamma 2 in platelets.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2004

Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus.

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca(2+) flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca(2+) flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca(2+) flux has a PI3K-independent component, while Ca(2+) flux following mIg/CD19 coligation is totally PI3K dependent. The p85alpha and p110delta subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca(2+) flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110alpha becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.

Animals↗
PubMed2004

PLCgamma2 regulates Bcl-2 levels and is required for survival rather than differentiation of marginal zone and follicular B cells.

B cells from phospholipase C (PLC)gamma2-deficient mice express reduced levels of the pro-survival protein Bcl-2 and show a defect in the development of transitional T3 and marginal zone (MZ) B cells that reflects reduced B cell survival. Introduction of a bcl-2 transgene restored the numbers of MZ, T3 and follicular B cells in PLCgamma2(-/-) mice. Restricting the B cell repertoire in PLCgamma2-deficient mice by the introduction of a BCR transgene resulted in a striking reduction in the number of IgM-positive B cells and a paucity of IgD-expressing cells in the spleen which was also rescued by the bcl-2 transgene. BCR-stimulated ERK and IkappaBalpha phosphorylation were PLCgamma2 dependent, while calcium flux was reduced, but not abrogated, in the absence of PLCgamma2, suggesting an ancillary role for PLCgamma1. The bcl-2 transgene rescued development of PLCgamma2(-/-) B cells and serum IgM levels but did not restore BCR-mediated signaling, proliferation or serum IgG3 levels. These data suggest that PLCgamma2 performs a critical role in B cell development through regulation of survival rather than differentiation.

Animals↗
PubMed2004

Immunological function in mice lacking the Rac-related GTPase RhoG.

RhoG is a low-molecular-weight GTPase highly expressed in lymphocytes that activates gene transcription and promotes cytoskeletal reorganization in vitro. To study the in vivo function of RhoG, we generated mice homozygous for a targeted disruption of the RhoG gene. Despite the absence of RhoG, the development of B and T lymphocytes was unaffected. However, there was an increase in the level of serum immunoglobulin G1 (IgG1) and IgG2b as well as a mild increase of the humoral immune response to thymus-dependent antigens. In addition, B- and T-cell proliferation in response to antigen receptor cross-linking was slightly increased. Although RhoG deficiency produces a mild phenotype, our experiments suggest that RhoG may contribute to the negative regulation of immune responses. The lack of a strong phenotype could indicate a functional redundancy of RhoG with other Rac proteins in lymphocytes.

Animals↗
PubMed2003

RhoG regulates gene expression and the actin cytoskeleton in lymphocytes.

RhoG, a member of the Rho family of GTPases, has been implicated as a regulator of the actin cytoskeleton. In this study, we show a novel function for the small GTPase RhoG on the regulation of the interferon-gamma promoter and nuclear factor of activated T cells (NFAT) gene transcription in lymphocytes. Optimal function of RhoG for the expression of these genes requires a calcium signal, normally provided by the antigen receptor. In addition, RhoG potentiation of NFAT requires the indirect activity of Rac and Cdc42; however, pathways distinct from those activated by Rac and Cdc42 mediate RhoG activation of NFAT-dependent transcription. Using effector domain mutants of RhoG we found that its ability to potentiate NFAT-dependent transcription correlates with its capacity to increase actin polymerization, supporting the suggestion that NFAT-dependent transcription is an actin-dependent process. RhoG also promotes T-cell spreading on fibronectin, a property that is independent of its ability to enhance NFAT-dependent transcription. Hence, these results implicate RhoG in leukocyte trafficking and the control of gene expression induced in response to antigen encounter.

Actins↗
PubMed2003

Regulation of Vav localization in membrane rafts by adaptor molecules Grb2 and BLNK.

Despite the importance of the Vav family proteins for B cell receptor (BCR) signaling, their activation mechanisms remain poorly understood. We demonstrate here that adaptor molecules Grb2 and BLNK, in addition to Vav, are required for efficient Rac1 activation in response to BCR stimulation. Loss of either Grb2 or BLNK results in decreased translocation of Vav3 to membrane rafts. By expression of Vav3 as a raft-targeted construct, the defective Rac1 activation in Grb2- or BLNK-deficient B cells is restored. Hence, our findings suggest that Grb2 and BLNK cooperate to localize Vav into membrane rafts, thereby contributing to optimal activation of Vav in B cells.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C.

We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.

Animals↗
PubMed2002

A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation.

Mice lacking the p110delta catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110delta(-/-) B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110delta function is required for BCR-mediated calcium flux, activation of phosphlipaseCgamma2, and Bruton's tyrosine kinase. Thus, p110delta plays a critical role in B cell homeostasis and function.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2002

True anti-anionic phospholipid immunoglobulin M antibodies can exert lupus anticoagulant activity.

True (cofactor-independent) anticardiolipin antibodies (aCL) are thought to lack lupus anticoagulant (LA) activity and pathogenic potential. A serum monoclonal immunoglobulin Mlambda (mIgMlambda) with aCL and LA activities found in a man with a splenicIgMlambda+ B-cell lymphoplasmacytic lymphoma (LPL) without thrombotic events has been characterized. LPL-derived hybridoma clones (designated HY-FRO) producing the serum mIgMlambda were obtained. mIgMlambda secreted by HY-FRO grown in protein-free culture medium, like that purified from serum, (i) showed binding, in a cofactor-free system, to solid-phase CL and phosphatidylserine (PS) and to the membrane of PS-expressing cells (apoptotic cells and activated platelets); (ii) failed to bind neutral phospholipids (PL), beta2Glycoprotein, histone, ssDNA, dsDNA, human IgG and umbilical vein endothelial cells. Absorption with apoptotic cells abolished its binding to anionic plate-bound CL and PS. IgMlambda-FRO used poorly mutated VH and Vlambda region genes, with a pattern that was inconsistent with an antigen-driven selection. Basic amino acids were present in the IgH complementarity determining region 3 (CDR3), which can be important for binding to anionic PL. These findings demonstrate unequivocally that true anti-anionic PL IgM antibodies can exert LA and indicate this anti-PL type does not involve thrombophilia.

Aged↗