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Angela Santoni - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of Rho Family Small G-Protein Activity Induced by Integrin Ligation on Human Leukocytes
Methods in molecular biology (Clifton N.J.), 2004Co-Authors: Angela Gismondi, Fabrizio Mainiero, Angela SantoniAbstract:Integrin engagement by natural ligands or specific antibodies (Abs) initiates a series of intracellular signaling events that are important for regulating different cell functions, including migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. A central role in the Integrin-triggered signaling cascades is played by activation of the small GTP-binding proteins belonging to the Ras and Rho familie
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RAC1/P38 MAPK Signaling Pathway Controls β1 Integrin–Induced Interleukin-8 Production in Human Natural Killer Cells
Immunity, 2000Co-Authors: Fabrizio Mainiero, Angela Gismondi, Mario Piccoli, Luigi Frati, Alessandra Soriani, Raffaele Strippoli, Jordan Jacobelli, Angela SantoniAbstract:The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 Integrin Ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for Integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 Integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 Integrins.
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Integrin-mediated Ras-extracellular regulated kinase (ERK) signaling regulates interferon γ production in human natural killer cells
The Journal of experimental medicine, 1998Co-Authors: Fabrizio Mainiero, Angela Gismondi, Gabriella Palmieri, Mario Piccoli, Luigi Frati, Alessandra Soriani, Jordan Jacobelli, Marco Cippitelli, Angela SantoniAbstract:Recent evidence indicates that Integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 Integrin Ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 Integrin–mediated Ras activation. In addition, we demonstrate that Ligation of β1 Integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 Integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors.
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Proline-rich tyrosine kinase-2 activation by beta 1 Integrin fibronectin receptor cross-linking and association with paxillin in human natural killer cells.
Journal of immunology (Baltimore Md. : 1950), 1997Co-Authors: Angela Gismondi, Fabrizio Mainiero, Lorenzo Bisogno, Gabriella Palmieri, Mario Piccoli, Luigi Frati, Angela SantoniAbstract:Recent evidence indicates that Integrin Ligation results in activation of focal adhesion kinase (pp125FAK), the prototype of a new subfamily of nonreceptor protein tyrosine kinase (PTK), including FAKB and the proline-rich tyrosine kinase 2 (PYK-2), also termed cell adhesion kinase-beta or related adhesion focal tyrosine kinase. We have previously shown that cross-linking of alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors on human NK cells stimulates tyrosine phosphorylation of two proteins migrating at 105 and 115 kDa. Here we report that cross-linking of beta 1 Integrins on human NK cells stimulates tyrosine phosphorylation and PTK activity of PYK-2. PYK-2 tyrosine phosphorylation was maximal at 1 min and started to decline 20 min after stimulation. Engagement of alpha 4 beta 1 and alpha 5 beta 1 either with specific mAbs or after cell adhesion to fibronectin or its 120- and 40-kDa fragments also triggered PYK-2 tyrosine phosphorylation. Stimulation of PYK-2 tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by EGTA, indicating that PYK-2 tyrosine phosphorylation is PTK, but not calcium, dependent. We also demonstrate that PYK-2 is constitutively associated with paxillin, which undergoes tyrosine phosphorylation with the same kinetics of PYK-2 upon beta 1 Integrin Ligation.
Jean Plouet - One of the best experts on this subject based on the ideXlab platform.
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extracellular matrix bound vascular endothelial growth factor promotes endothelial cell adhesion migration and survival through Integrin Ligation
The FASEB Journal, 2003Co-Authors: Helen Hutchings, Nathalie Ortega, Jean PlouetAbstract:Vascular endothelial growth factor (VEGF), a major factor mediating endothelial cell survival, migration, and proliferation during angiogenesis, is expressed as five splice variants (121, 145, 165, 189, and 206 aminoacids) encoded by a single gene. Although the three shorter isoforms are mainly diffusible, the two longer ones are sequestered in cell membranes after secretion. However, their potential role as true components of the extracellular matrix has not been investigated. We determined that endothelial cells could adhere and spread on VEGF189 and VEGF165, but not on VEGF121. Adhesion was mediated by the alpha3beta1 and alpha(v)beta3 Integrins and other alpha(v) Integrins but not by the cognate VEGF receptors. Cells migrated on VEGF165 and VEGF189 and displayed a stellate morphology with numerous lamellopodia and FAK staining but no actin stress fibers. Tumstatin, an antiangiogenic peptide that interacts with the alpha(v)beta3 Integrin, could inhibit adhesion on VEGF, and this effect was potentiated by anti-alpha(v)beta3 blocking antibody. Immobilized VEGF almost totally abolished endothelial cell apoptosis through interactions with Integrins. The inhibition of alpha(v)beta3 engagement with immobilized VEGF by tumstatin inhibited most of its survival activity. We have thus determined a new VEGF receptor-independent role for immobilized VEGF in supporting cell adhesion and survival through interactions with Integrins.
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extracellular matrix bound vascular endothelial growth factor promotes endothelial cell adhesion migration and survival through Integrin Ligation
The FASEB Journal, 2003Co-Authors: Helen Hutchings, Nathalie Ortega, Jean PlouetAbstract:SPECIFIC AIMSAlthough alternative splicing of vascular endothelial growth factor (VEGF) pre-mRNA regulates the binding of the isoforms to the extracellular matrix (ECM) and cell membranes, little is known about their specific biological responses as diffusible growth factors and nothing about their potential role as matrix-bound factors. In this report we examined the role of various immobilized VEGF isoforms on cell adhesion and survival of four endothelial strains.PRINCIPAL FINDINGS1. Endothelial cells adhere to VEGF independent of VEGF receptorsHuman umbilical artery endothelial cells (HUAEC) were plated in plastic wells that had been coated with BSA, fibronectin, VEGF121, VEGF165, or the full-length or cleaved forms of VEGF189. At various intervals, the wells were rinsed and cell adhesion was assessed by crystal violet staining. HUAEC adhered well to the VEGF189 isoforms (cleaved or full-length), moderately well to VEGF165, but not at all to VEGF121. The amount of cells adhered to VEGF189 was similar ...
John R Couchman - One of the best experts on this subject based on the ideXlab platform.
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syndecan 4 binding to the high affinity heparin binding domain of fibronectin drives focal adhesion formation in fibroblasts
Archives of Biochemistry and Biophysics, 2000Co-Authors: Anne Woods, Robert L Longley, Sarka Tumova, John R CouchmanAbstract:Abstract Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following Integrin Ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with Integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Cα, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.
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syndecan 4 proteoglycan regulates the distribution and activity of protein kinase c
Journal of Biological Chemistry, 1997Co-Authors: Eoksoo Oh, Anne Woods, John R CouchmanAbstract:Abstract During cell-matrix adhesion, both tyrosine and serine/threonine kinases are activated. Integrin Ligation correlates with tyrosine phosphorylation, whereas the later stages of spreading and focal adhesion and stress fiber formation in primary fibroblasts requires interactions of cell surface proteoglycan with heparin-binding moieties. This correlates with protein kinase C (PKC) activation, and PKCα can become localized to focal adhesions in normal, but not transformed, cells. PKC activation has been thought to be downstream of initial receptor-ligand interactions. We now show, however, that syndecan-4 transmembrane heparan sulfate proteoglycan and PKC co-immunoprecipitate and co-patch in vivo. The core protein of syndecan-4 can directly bind the catalytic domain of PKCα and potentiate its activation by phospholipid mediators. It can also directly activate PKCα in the absence of other mediators. This activity resides in the sequence LGKKPIYKK in the center of the short cytoplasmic domain, and other syndecans lack this sequence and PKC regulatory properties. Syndecan-4 is a focal adhesion component, and this interaction may both localize PKC and amplify its activity at sites of forming adhesions. This represents the first report of direct transmembrane signaling through cell surface proteoglycans.
Fabrizio Mainiero - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of Rho Family Small G-Protein Activity Induced by Integrin Ligation on Human Leukocytes
Methods in molecular biology (Clifton N.J.), 2004Co-Authors: Angela Gismondi, Fabrizio Mainiero, Angela SantoniAbstract:Integrin engagement by natural ligands or specific antibodies (Abs) initiates a series of intracellular signaling events that are important for regulating different cell functions, including migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. A central role in the Integrin-triggered signaling cascades is played by activation of the small GTP-binding proteins belonging to the Ras and Rho familie
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RAC1/P38 MAPK Signaling Pathway Controls β1 Integrin–Induced Interleukin-8 Production in Human Natural Killer Cells
Immunity, 2000Co-Authors: Fabrizio Mainiero, Angela Gismondi, Mario Piccoli, Luigi Frati, Alessandra Soriani, Raffaele Strippoli, Jordan Jacobelli, Angela SantoniAbstract:The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 Integrin Ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for Integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 Integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 Integrins.
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Integrin-mediated Ras-extracellular regulated kinase (ERK) signaling regulates interferon γ production in human natural killer cells
The Journal of experimental medicine, 1998Co-Authors: Fabrizio Mainiero, Angela Gismondi, Gabriella Palmieri, Mario Piccoli, Luigi Frati, Alessandra Soriani, Jordan Jacobelli, Marco Cippitelli, Angela SantoniAbstract:Recent evidence indicates that Integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 Integrin Ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 Integrin–mediated Ras activation. In addition, we demonstrate that Ligation of β1 Integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 Integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors.
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Proline-rich tyrosine kinase-2 activation by beta 1 Integrin fibronectin receptor cross-linking and association with paxillin in human natural killer cells.
Journal of immunology (Baltimore Md. : 1950), 1997Co-Authors: Angela Gismondi, Fabrizio Mainiero, Lorenzo Bisogno, Gabriella Palmieri, Mario Piccoli, Luigi Frati, Angela SantoniAbstract:Recent evidence indicates that Integrin Ligation results in activation of focal adhesion kinase (pp125FAK), the prototype of a new subfamily of nonreceptor protein tyrosine kinase (PTK), including FAKB and the proline-rich tyrosine kinase 2 (PYK-2), also termed cell adhesion kinase-beta or related adhesion focal tyrosine kinase. We have previously shown that cross-linking of alpha 4 beta 1 and alpha 5 beta 1 fibronectin receptors on human NK cells stimulates tyrosine phosphorylation of two proteins migrating at 105 and 115 kDa. Here we report that cross-linking of beta 1 Integrins on human NK cells stimulates tyrosine phosphorylation and PTK activity of PYK-2. PYK-2 tyrosine phosphorylation was maximal at 1 min and started to decline 20 min after stimulation. Engagement of alpha 4 beta 1 and alpha 5 beta 1 either with specific mAbs or after cell adhesion to fibronectin or its 120- and 40-kDa fragments also triggered PYK-2 tyrosine phosphorylation. Stimulation of PYK-2 tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by EGTA, indicating that PYK-2 tyrosine phosphorylation is PTK, but not calcium, dependent. We also demonstrate that PYK-2 is constitutively associated with paxillin, which undergoes tyrosine phosphorylation with the same kinetics of PYK-2 upon beta 1 Integrin Ligation.
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The adaptor protein Shc couples a class of Integrins to the control of cell cycle progression
Cell, 1996Co-Authors: Kishore K. Wary, Fabrizio Mainiero, Eugene E. Marcantonio, Steven J Isakoff, Filippo G GiancottiAbstract:We provide evidence that a class of Integrins combines with the adaptor Shc and thereby with Grb2. Coimmunoprecipitation and mutagenesis experiments indicate that the recruitment of Shc is specified by the extracellular or transmembrane domain of Integrin α subunit and suggest that this process is mediated by caveolin. Mutagenesis and dominant-negative inhibition studies reveal that Shc is necessary and sufficient for activation of the MAP kinase pathway in response to Integrin Ligation. Mitogens and Shc-activating Integrins cooperate to promote transcription from the Fos serum response element and transit through G1. In contrast, adhesion mediated by Integrins not linked to Shc results in cell cycle arrest and apoptosis even in presence of mitogens. These findings indicate that the association of specific Integrins with Shc regulates cell survival and cell cycle progression.
Clifford A. Lowell - One of the best experts on this subject based on the ideXlab platform.
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l selectin shedding affects bacterial clearance in the lung a new regulatory pathway for Integrin outside in signaling
Blood, 2019Co-Authors: Anika Cappenberg, Clifford A. Lowell, Andreas Margraf, Katharina Thomas, Bernadette Bardel, Dylan A Mccreedy, Veerle Van Marck, Alexander Mellmann, Alexander ZarbockAbstract:Pneumonia induced by Gram-negative bacteria is a common and serious disease associated with high morbidity and mortality. Elimination of bacterial pathogens relies on the recruitment and functions of neutrophils. The adhesion molecule L-selectin has recently been implicated in Integrin activation in neutrophils (inside-out signaling). However, the molecular mechanism by which L-selectin participates in host defense against Klebsiella pneumoniae-induced pulmonary inflammation is unknown. We demonstrate that L-selectin-deficient mice are prone to pulmonary infection compared with wild-type controls. Mechanistically, L-selectin cleavage from the neutrophil surface triggered by Integrin engagement is involved in neutrophil recruitment into the lung and bacterial clearance. Downstream of Integrin Ligation, the metalloproteinase A disIntegrin and metalloproteinase 17 (ADAM17) sheds L-selectin from the neutrophil surface in an IRhom2-dependent manner. L-selectin cleavage enhances Integrin-mediated outside-in signaling, resulting in increased neutrophil effector functions. Thus, we identify a novel regulatory mechanism in neutrophils required for an adequate immune response triggered by Integrin engagement during K pneumoniae-induced pulmonary inflammation.
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STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense.
Blood, 2014Co-Authors: Hong Zhang, Regina A. Clemens, Fengchun Liu, Yoshihiro Baba, Pierre Theodore, Tomohiro Kurosaki, Clifford A. LowellAbstract:The stromal-interacting molecule 1 (STIM1) is a potent sensor of intracellular calcium, which in turn regulates entry of external calcium through plasma membrane channels to affect immune cell activation. Although the contribution of STIM1 to calcium signaling in lymphocytes has been well studied, the role of this protein in neutrophil-mediated inflammation and host defense is unknown. We report that STIM1-deficient murine neutrophils show loss of store-operated calcium entry (SOCE) in response to both soluble ligands that activate G-proteins as well as Fcγ-receptor or Integrin Ligation that activates tyrosine kinase signaling. This results in modest defects in phagocytosis and degranulation responses but a profound block in superoxide production by the phagocyte oxidase. We trace the primary intracellular target of calcium to be protein kinase C isoforms α and β (PKCα and PKCβ), which in turn phosphorylate subunits of the oxidase leading to superoxide production. In vivo the loss of SOCE in stim1−/− chimeric mice results in marked susceptibility to bacterial infections but also protection from tissue injury in hepatic ischemia/reperfusion injury. These results demonstrate the critical role of STIM1-mediated SOCE and define major protein targets of calcium signaling in neutrophil activation during inflammatory disease.
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pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection
Journal of Immunology, 2011Co-Authors: Lynn A Kamen, Joseph Schlessinger, Clifford A. LowellAbstract:The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2−/− mice, we found that this kinase was required for Integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following Integrin Ligation, which likely accounts for the poor degranulation and cell migration. Pyk2−/− mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the Integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.
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Convergence of immunoreceptor and Integrin signaling.
Immunological reviews, 2007Co-Authors: Clare L. Abram, Clifford A. LowellAbstract:Summary: A common signaling pathway is known to operate downstream of immunoreceptors, such as the T-cell, B-cell, or Fc receptors, following engagement by their respective ligands. This pathway involves Src family kinase-mediated tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) that recruit and activate spleen tyrosine kinase (Syk) or Zap70 (ζ-associated protein of 70 kDa) kinases, which in turn activate a variety of downstream signals. Evidence has been building from a variety of sources, particularly mouse models, that molecules involved in the immunoreceptor signaling pathway are also required for signals initiated by Integrins. Integrins are the major cell surface receptors that mediate adhesion of leukocytes to a variety of extracellular matrix proteins and counter-receptors expressed on endothelial cells. Integrin Ligation is a critical step in the activation of leukocyte effector functions (such as neutrophil degranulation or lymphocyte proliferation). Integrin signaling through pathways common to those utilized by immunoreceptors provides a mechanism by which leukocyte adhesion can regulate activation of cellular responses. In animal models, Integrin-mediated signal transduction plays a critical role in inflammatory disease. In this review, we discuss the convergence of immunoreceptor and Integrin signaling, focusing on how these pathways modulate leukocyte activation.
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Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs
Nature immunology, 2006Co-Authors: Attila Mócsai, Clare L. Abram, Zoltán Jakus, Lewis L. Lanier, Clifford A. LowellAbstract:At sites of inflammation, Ligation of leukocyte Integrins is critical for the activation of cellular effector functions required for host defense. However, the signaling pathways linking Integrin Ligation to cellular responses are poorly understood. Here we show that Integrin signaling in neutrophils and macrophages requires adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs). Neutrophils and macrophages lacking two ITAM-containing adaptor proteins, DAP12 and FcRγ, were defective in Integrin-mediated responses. Activation of the tyrosine kinase Syk by Integrins required that DAP12 and FcRγ were first phosphorylated by Src family kinases. Retroviral transduction of neutrophils and macrophages with wild-type and mutant Syk or DAP12 demonstrated that the Src homology 2 domains of Syk and the ITAM of DAP12 were required for Integrin signaling. Our data show that Integrin signaling for the activation of cellular responses in neutrophils and macrophages proceeds by an immunoreceptor-like mechanism.