The recruitment of p47(phox) and Rac2G12V at the phagosome is transient and phosphatidylserine dependent.

BACKGROUND INFORMATION: During phagocytosis, neutrophils internalise pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40(phox), p47(phox), p67(phox) and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood. Anionic phospholipids, phosphatidylserine (PS) and phosphoinositides (PPI) provide an important attachment site for numerous proteins, including several oxidase subunits. RESULTS: We investigated the kinetics of p47(phox) and Rac2 phagosomal membrane recruitment. Both subunits are known to interact with anionic phospholipids; we therefore addressed the role of PS in this recruitment. Phagosomal accumulation of p47(phox) and Rac2 tagged with fluorescent proteins was analysed by videomicroscopy. We used the C2 domain of lactadherin (lactC2) that interacts strongly and specifically with PS to monitor intracellular PS localisation and to decrease PS accessibility. During phagocytosis of opsonised zymosan, p47(phox) and constitutively active Rac2G12V briefly translocated to the phagosomal membrane, whereas ROS production continued for a longer period. However, in the presence of lactC2, Rac2G12V recruitment was inhibited and the kinetics of p47(phox) recruitment and detachment were delayed. A reduced phagosomal ROS production was also observed during the first 7 min following the phagosome closure. CONCLUSIONS: These results suggest that p47(phox) and Rac2 accumulate only transiently at the phagosome at the onset of NADPH activity and detach from the phagosome before the end of ROS production. Furthermore, lactC2, by masking PS, interfered with the phagosomal recruitment of p47(phox) and Rac2 and disturbed NADPH oxidase activity. Thus, PS appears as a modulator of NADPH oxidase activation.

Sphingosine kinases regulate NOX2 activity via p38 MAPK-dependent translocation of S100A8/A9.

Neutrophils play a fundamental role in host defense by neutralizing pathogens through the generation of ROS by NOX2. In nonexcitable cells, Ca(2+) influx is essentially mediated via SOCE, a complex mechanism in which depletion of intracellular Ca(2+) stores from the ER results in Ca(2+) entry through Ca(2+) SOCs at the plasma membrane. In this regard, it is well established that extracellular Ca(2+) entry participates to NOX2 activation. S1P, produced by SphKs, has been involved in Ca(2+) homeostasis and thus, could intervene in NOX2 regulation. The aim of this study was to characterize the importance of SphKs in NOX2 activation and the signaling cascade involved in this mechanism. Treatment of neutrophil-like dHL-60 cells by DHS, a SphK inhibitor, and SphK siRNA inhibited fMLF-induced NOX2 activity. Sequential activation of cells by thapsigargin and the phorbol ester PMA revealed that SphK-regulated NOX2 activity relies on intracellular Ca(2+) store depletion. Confocal microscopy and immunoblot analysis showed that stimulation by thapsigargin and PMA mediated S100A8/A9 recruitment to the plasma membrane and p38 MAPK activation. S100A8/A9 translocation decreased when SphK activity was blocked. This result was confirmed in purified human neutrophils, which were physiologically stimulated by fMLF. In addition, p38 MAPK was found to be regulated by SphKs. These results define a pathway leading to NOX2 activation, in which p38 MAPK-mediated S100A8/A9 translocation is regulated by Ca(2+) store depletion-dependent SphK activation.

An essential role of STIM1, Orai1, and S100A8-A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity.

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor.

iPLA2, a novel determinant in Ca2+- and phosphorylation-dependent S100A8/A9 regulated NOX2 activity.

The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to inflammatory processes, which could lead to the development of diseases such as atherosclerosis. In this paper, we characterize a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. Studies in cell-free or recombinant systems involved two Ca2+-binding proteins of the S100 family, namely S100A8 and S100A9, in NOX2 activation dependent on intracellular Ca2+ concentration ([Ca2+](i)) elevation. Using differentiated HL-60 cells as a model of neutrophils, we provide evidence that [Ca2+](i)-regulated S100A8/A9 translocation is mediated by an increase in [Ca2+](i) through intracellular Ca2+ store depletion. Moreover, we confirm that p38 MAPK induces S100A9 phosphorylation, a mandatory precondition for S100 translocation. Based on a pharmacological approach and an siRNA strategy, we identify iPLA(2) as a new molecular player aiding S100 translocation and NOX2 activity. Inhibition of p38 MAPK activity and S100A9 phosphorylation by bromoenol lactone, a selective inhibitor of iPLA(2), indicated that p38 MAPK-mediated S100A9 phosphorylation is dependent on iPLA(2). In conclusion, we have characterized a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation.

The novel S527F mutation in the integrin beta3 chain induces a high affinity alphaIIbbeta3 receptor by hindering adoption of the bent conformation.

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor alphaIIbbeta3 in a patient with an ill defined platelet disorder: one in the beta3 gene (S527F) and two in the alphaIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant alphaIIbbeta3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the beta3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated alphaIIbbeta3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of alphaIIbbeta3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the alphaIIbbeta3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe(527) in beta3 and the calf-1 domain in alphaIIb and by decreased flexibility between I-EGF2 and I-EGF3.

Co-cultures of human coronary smooth muscle cells and dimethyl sulfoxide-differentiated HL60 cells upregulate ProMMP9 activity and promote mobility-modulation by reactive oxygen species.

Vascular cells and leukocytes, involved in the development of atherosclerosis, produce cytokines and/or reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) implicated in cell mobility. We investigated by co-culture experiments the effects of human coronary smooth muscle cells (HCSMC) on MMPs characteristics and mobility of neutrophil-like dimethyl sulfoxide-differentiated HL60 cells (not equal HL60). The effects of superoxide dismutase (SOD) and catalase were also analyzed. All the studied MMP2 characteristics remained unchanged. HCSMC stimulated MMP9 protein level, activity and mobility of not equal HL60 cells and expressed and secreted a variety of cytokines implicated in atherosclerosis. SOD and catalase increased MMP9 expression, protein level and activity of not equal HL60, but migration of not equal HL60 cells was only decreased by catalase, demonstrating that ROS are more efficient in modulating MMP9 activity of not equal HL60 than their mobility. Finally, HCSMC being able to stimulate not equal HL60, their co-cultures may represent an in vitro approach to study cellular interactions occurring in vivo during atherosclerosis.

The intermediate filament protein vimentin binds specifically to a recombinant integrin alpha2/beta1 cytoplasmic tail complex and co-localizes with native alpha2/beta1 in endothelial cell focal adhesions.

Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short alpha and beta cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin alpha2beta1 is a major collagen receptor but to date, only few proteins have been shown to interact with the alpha2 cytoplasmic tail or with the alpha2beta1 complex. In order to identify novel binding partners of a alpha2beta1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-alpha2 and GST-Jun alpha2 bound His-tagged calreticulin while GST-beta1 and GST-Fos beta1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun alpha2/GST-Fos beta1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with alphavbeta3-positive focal contacts. Here, we provide evidence that this interaction also occurs with alpha2beta1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.

A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain.

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.

Promoter characterization and genomic organization of the gene encoding integrin-linked kinase 1.

Integrin-linked kinase (ILK)-1 is a 59-kDa serine-threonine protein kinase, which associates with the cytoplasmic domain of beta 1, beta 2 and beta 3 integrins and acts as a receptor proximal kinase regulating integrin-mediated signal transduction. We have recently identified an isoform of ILK (ILK-2), which is expressed, in a TGF-beta 1-dependent manner, in a highly invasive tumor cell line but not in normal adult tissues. In contrast, ILK-1 is ubiquitously expressed in normal tissues and is up-regulated in various tumors independent of TGF-beta 1. Here, we report the structural organization and the promoter activity of the human ILK-1 gene, contained within a 8.8-kb genomic fragment cloned from a human BAC library. The mature protein is encoded by 13 exons. The last coding exon contains the entire 3' UTR of the ILK-1 gene, which overlaps with the complementary 3' UTR sequence of the TAF2H gene, a TATA box binding protein-associated factor. A major transcriptional initiation start site was found 138 bp upstream of exon 1 in close proximity to a consensus initiator element (Inr). The ILK gene is transcribed by a TATA-less and CAAT-less promoter with typical features of housekeeping genes. The promoter activity was characterized by a luciferase reporter assay and the minimal sequence conferring promoter activity was 349 bp in size and located immediately upstream of exon 1.