Etude fonctionnelle de la signalisation intracellulaire médiée par l'intégrine alphaIIbbeta3 au cours de l'interaction avec le fibrinogène immobilisé

The platelet fibrinogen (fg) receptor integrin alphaIIbbeta3 plays a major role in platelet adhesion and platelet aggregation. Fibrinogen binding to integrin alphaIIbbeta3 is complicated since 2 recognition sites have been described: the universal tripeptide RGD site and a HHLGGAKQAGDV dodecapeptide sequence. However, it is still unclear whether these 2 recognition sites function independently, synergistically, or competitively. Here we have investigated the respective role of the dodecapeptide sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent CHO cell or human platelet spreading, by using intact fg, and well-characterized plasmin-generated fg fragments containing either the RGD motif (fragment C) or the dodecapeptide site (fragment D), and CHO cells expressing resting wt, constitutively active or non-functional receptors.Our data provide evidence that alphaIIbbeta3-dependent cell adhesion to immobilized fg is a two-step process: the dodecapeptide site by itself is first able to promote cell attachment by initiating alphaIIbbeta3 clustering, FAK phosphorylation and Rac1 activation while the RGD motif subsequently acts as a molecular switch on the beta3 subunit leading to mature focal adhesion formation, maximal RhoA activation, actin cytoskeleton organization and full cell spreading.L'intégrine alphaIIbbeta3 des plaquettes, principal récepteur du fibrinogène (fg), joue un rôle prépondérant dans l'adhésion et l'agrégation plaquettaires, en établissant un lien physique et dynamique entre le fg sanguin et les protéines plaquettaires intracellulaires de la signalisation et du cytosquelette. Le processus de reconnaissance du fg par l'intégrine alphaIIbbeta3 est complexe puisqu'au moins 2 sites distincts d'interaction sont connus: le site universel RGD et une séquence dodécapeptide HHLGGAKQAGDV. Cependant, il n'est pas encore clair si ces 2 sites fonctionnent indépendamment, de façon synergique ou par compétition. Nous avons donc examiné le rôle respectif des motifs dodécapeptide et RGD du fg au cours de l'étalement cellulaire médié par alphaIIbbeta3, au niveau de plaquettes sanguines humaines et de cellules CHO exprimant soit le recepteur sauvage, soit le récepteur constitutivement actif ou non-fonctionnel, et en utilisant le fg entier ou des fragments de fg, le fragment D ne contenant que le dodécapeptide, et le fragment C ne présentant que le motif RGD. Nos résultats mettent pour la première fois en évidence 2 voies de signalisation indépendantes mais synergiques, la première voie initiée par le dodécapeptide reconnu par alphaIIb, et conduisant à un attachement cellulaire, à la formation de complexes focaux, à la phosphorylation de FAK et à l'activation de Rac1, et la seconde voie impliquant le motif RGD qui joue un rôle de commutateur moléculaire au niveau de beta3, entraînant l'activation maximale de RhoA, la polymérisation et l'organisation de l'actine en fibres de stress, la formation d'adhérences focales matures et l'étalement cellulaire complet

Etude fonctionnelle de la signalisation intracellulaire médiée par l'intégrine alphaIIbbeta3 au cours de l'interaction avec le fibrinogène immobilisé

L'intégrine alphaIIbbeta3 des plaquettes, principal récepteur du fibrinogène (fg), joue un rôle prépondérant dans l'adhésion et l'agrégation plaquettaires, en établissant un lien physique et dynamique entre le fg sanguin et les protéines plaquettaires intracellulaires de la signalisation et du cytosquelette. Le processus de reconnaissance du fg par l'intégrine alphaIIbbeta3 est complexe puisqu'au moins 2 sites distincts d'interaction sont connus: le site universel RGD et une séquence dodécapeptide HHLGGAKQAGDV. Cependant, il n'est pas encore clair si ces 2 sites fonctionnent indépendamment, de façon synergique ou par compétition. Nous avons donc examiné le rôle respectif des motifs dodécapeptide et RGD du fg au cours de l'étalement cellulaire médié par alphaIIbbeta3, au niveau de plaquettes sanguines humaines et de cellules CHO exprimant soit le recepteur sauvage, soit le récepteur constitutivement actif ou non-fonctionnel, et en utilisant le fg entier ou des fragments de fg, le fragment D ne contenant que le dodécapeptide, et le fragment C ne présentant que le motif RGD. Nos résultats mettent pour la première fois en évidence 2 voies de signalisation indépendantes mais synergiques, la première voie initiée par le dodécapeptide reconnu par alphaIIb, et conduisant à un attachement cellulaire, à la formation de complexes focaux, à la phosphorylation de FAK et à l'activation de Rac1, et la seconde voie impliquant le motif RGD qui joue un rôle de commutateur moléculaire au niveau de beta3, entraînant l'activation maximale de RhoA, la polymérisation et l'organisation de l'actine en fibres de stress, la formation d'adhérences focales matures et l'étalement cellulaire complet.The platelet fibrinogen (fg) receptor integrin alphaIIbbeta3 plays a major role in platelet adhesion and platelet aggregation. Fibrinogen binding to integrin alphaIIbbeta3 is complicated since 2 recognition sites have been described: the universal tripeptide RGD site and a HHLGGAKQAGDV dodecapeptide sequence. However, it is still unclear whether these 2 recognition sites function independently, synergistically, or competitively. Here we have investigated the respective role of the dodecapeptide sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent CHO cell or human platelet spreading, by using intact fg, and well-characterized plasmin-generated fg fragments containing either the RGD motif (fragment C) or the dodecapeptide site (fragment D), and CHO cells expressing resting wt, constitutively active or non-functional receptors.Our data provide evidence that alphaIIbbeta3-dependent cell adhesion to immobilized fg is a two-step process: the dodecapeptide site by itself is first able to promote cell attachment by initiating alphaIIbbeta3 clustering, FAK phosphorylation and Rac1 activation while the RGD motif subsequently acts as a molecular switch on the beta3 subunit leading to mature focal adhesion formation, maximal RhoA activation, actin cytoskeleton organization and full cell spreading.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

OAG induces an additional PKC-, PI3K-, and Rac2-mediated signaling pathway up-regulating NOX2 activity, independently of Ca 2+ entry

The requirement of calcium ion (Ca(2)(+)) entry for neutrophil NADPH oxidase (NOX2) regulation is clearly established. However, its role in the signaling pathway leading to NOX2 activation is still elusive. 1-oleoyl-2-acetyl-sn-glycerol (OAG) causes an increase in NOX2 activity and has been shown to directly modulate Ca(2)(+) channels unrelated to the well-known store-operated Ca(2)(+) entry (SOCE) mechanism. In our study, we have investigated the potential role of OAG in Ca(2)(+) influx-mediated NOX2 activity in neutrophil-like-differentiated HL-60 cells to further characterize second signals involved in the regulation of NOX2. OAG inhibited fMLF- and thapsigargin-induced Ca(2)(+) entry, a phenomenon that was not restored by protein kinase C (PKC) or PI3K blockade. Addition of OAG resulted in a rapid decrease of maximal intracellular Ca(2)(+) concentration induced by thapsigargin. Both results suggest that OAG has an inhibitory effect, independent of PI3K and PKC, on the regulation of SOCE. In contrast to SOCE inhibition, OAG-induced NOX2 activation was mediated by PKC and PI3K. Our data establish that both kinases exert their effects through the regulation of Rac2 activity. In addition, OAG potentiated the effect of fMLF on the activation of NOX2 and led to a discernible activity of NOX2 upon thapsigargin stimulation. In conclusion, our results demonstrate that an additional PKC- and/or PI3K-dependent signal may act in synergy with Ca(2)(+) influx to trigger NOX2 activation

RGD, the Rho'd to cell spreading

Some RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding. However, the precise involvement of each of these recognition sites during cell adhesion is still unclear. Here we review recent investigations on integrin alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen providing evidence that the fibrinogen synergy gamma(400-411) sequence by itself promotes cell attachment by initiating alphaIIbbeta3 clustering and recruitment of intracellular proteins to focal complexes, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to induce a conformational change necessary for RhoA activation and full cell spreading

A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2

Ca2+-dependent regulation of NOX2 activity via MRP proteins in HL-60 granulocytes

Recently, two proteins of the S100 protein family, the myeloid-related calcium-binding proteins MRP-8 and MRP-14 have been implicated in the Ca2+-induced activation of the neutrophil NADPH oxidase (NOX2) but the mechanism underlying this process remains unclear. In this study, the role of MRP-8/14 in the Ca2+-dependent regulation of NOX2 activity was characterized in neutrophil-like HL-60 cells using small interfering RNAs (siRNAs) to knock-down endogenous MRP-8 and/or MRP-14 expression. Real-time PCR and Western blot revealed that MRP-8 and MRP-14 expression was 20 times higher in dimethylsulfoxide-differentiated neutrophil-like HL-60 cells compared to quiescent HL-60 cells. Knock-down of MRP-8 and MRP-14 in differentiated HL-60 cells decreased protein levels by 30 and 45% respectively. The impact of the reduced MRP-8/14 protein expression on NOX2 activity was investigated by measuring fMLF-induced H2O2 production. In cells simultaneously transfected with MRP-8 and MRP14 siRNAs, H2O2 production was reduced by 50%, suggesting that both MRP-8 and MRP-14 are required for NOX2 activity; single knock-downs were inefficient. To elucidate the role of Ca2+ in MRP8/14, and consequently in NOX2 activation, siRNA-transfected cells were treated with the Ca2+ ionophore ionomycin prior to stimulation with PMA, a Ca2+-independent protein kinase C activator. PMA-induced H2O2 production was enhanced by ionomycin. This amplification of NOX2 activity was abolished by MRP8/14 knock-down, indicating that both MRP-8 and MRP-14 are necessary to regulate Ca2+-induced NOX2 activation. Taken together, our results suggest that the mechanism of MRPs activation is highly dependent on the increase of intracellular Ca2+ level for a full activation of NOX2

Role of Calprotectin as a Modulator of the IL27-Mediated Proinflammatory Effect on Endothelial Cells

An underlying endothelial dysfunction plays a fundamental role in the pathogenesis of cardiovascular events and is the central feature of atherosclerosis. The protein-based communication between leukocytes and inflamed endothelial cells leading to diapedesis has been largely investigated and several key players such as IL6, TNFα, or the damage associated molecular pattern molecule (DAMP) calprotectin are now well identified. However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification. Therefore, we examined the inflammatory impact of IL27 on primary endothelial cells and the potentially modulatory effect of calprotectin on both transcriptome and proteome levels. A qPCR-based screening demonstrated high IL27-mediated gene expression of IL7, IL15, CXCL10, and CXCL11. Calprotectin time-dependent downregulatory effects were observed on IL27-induced IL15 and CXCL10 gene expression. A mass spectrometry-based approach of IL27 ± calprotectin cell stimulation enlightened a calprotectin modulatory role in the expression of 28 proteins, mostly involved in the mechanism of leukocyte transmigration. Furthermore, we showed evidence for STAT1 involvement in this process. Our findings provide new evidence about the IL27-dependent proinflammatory signaling which may be under the control of calprotectin and highlight the need for further investigations on molecules which might have antiatherosclerotic functions

Overexpression of the partially activated alpha(IIb)beta3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet-like extensions in Chinese hamster ovary cells

BACKGROUND: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. RESULTS: Here we demonstrate that the partially activated alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. CONCLUSIONS: CHO alpha(IIb)beta3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree