Valérie Andrieu

Reactive oxygen species activate focal adhesion kinase, paxillin and p130cas tyrosine phosphorylation in endothelial cells.

Reactive oxygen species (ROS), particularly hydroxyl radical (HO*), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 microM) and herbimycin A (0.9 microM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120-130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO*. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 microM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO*, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.

Phlebotomies or erythropoietin injections allow mobilization of iron stores in a mouse model mimicking intensive care anemia

Objective:Anemia in critically ill patients is frequent and consists of chronic disease associated with blood losses. These two mechanisms have opposite effects on iron homeostasis, especially on the expression of the iron regulatory hormone hepcidin. We developed a mouse model mimicking the intensive care anemia to explore iron homeostasis. Design:Experimental study. Setting:University-based research laboratory. Subjects:C57BL/6 mice. Interventions:Mice received either a single intraperitoneal injection of lipopolysaccharide followed 1 week later by zymosan, or were subjected to repeated phlebotomies by retro-orbital punctures, or both. Several subsets of mice were analyzed over a 14-day period to describe the mouse model of intensive care anemia. Additional mice received erythropoietin injections with or without the zymosan treatment and were killed at day 5, to perform a more detailed analysis. Measurements and Main Results:We observed anemia as soon as 5 days after zymosan injection, together with increased messenger RNA (mRNA) levels for interleukin-6 and hepcidin. Phlebotomies alone fully suppressed hepcidin mRNA expression. Interestingly, in mice treated with zymosan and phlebotomies, hepcidin expression was suppressed, despite the persistent increase in interleukin-6. Stimulation of erythropoiesis by erythropoietin injections also led to a decrease in hepcidin mRNA in zymosan-treated mice. In these situations combining inflammation and erythropoiesis stimulation, there was no change in ferroportin, the membrane iron exporter, at the mRNA level, whereas ferroportin protein increased. Macrophage iron stores (assessed by histology using diaminobenzidine staining, or by quantification of nonheme iron and ferritin concentrations) were depleted in the spleen. Conclusions:These results suggest that the erythroid factor dominates over inflammation for hepcidin regulation, and that iron could be mobilized in these situations combining inflammation and erythropoiesis stimulation.

Focal Adhesion Kinase Regulation by Oxidative Stress in Different Cell Types

Focal adhesion kinase (FAK) is a tyrosine kinase ubiquitously expressed in cells. It was initially shown to be the initiator of focal adhesion formation in adherent cells, after its binding to integrins which induce its autophosphorylation. However, it can be also activated by a great variety of other stimuli able to act on different intracellular signaling. Reactive oxygen species (ROS), which have been shown to act as external or internal cell stimuli, induce tyrosine phosphorylation of FAK. Its autophosphorylation is followed by a submembranous localization which is crucial for many of the biological roles of FAK, including cell spreading, cell migration, cell proliferation, and prevention of apoptosis. It plays an important role in development of tumor cells, its regulation could be thus a way of impairing cell proliferation in cancer. We describe in this review the structure, activity, and functions of FAK in different cells and how ROS are able, like other stimuli, to induce its phosphorylation and modification of cell morphology and structure. The link between ROS and FAK activation could explain the role of ROS in mediating cell proliferation, cell migration, or apoptosis.

Erythropoietin stimulates spleen BMP4-dependent stress erythropoiesis and partially corrects anemia in a mouse model of generalized inflammation

Mouse bone marrow erythropoiesis is homeostatic, whereas after acute anemia, bone morphogenetic protein 4 (BMP4)-dependent stress erythropoiesis develops in the spleen. The aim of this work was to compare spleen stress erythropoiesis and bone marrow erythropoiesis in a mouse model of zymosan-induced generalized inflammation, which induces long-lasting anemia and to evaluate the ability of erythropoietin (Epo) injections to correct anemia in this setting. The effects of zymosan and/or Epo injections on erythroid precursor maturation and apoptosis, serum interferon-γ levels, hematologic parameters, and spleen BMP4 expression were analyzed, as well as the effect of zymosan on red blood cell half-life. We found that bone marrow erythropoiesis is suppressed by inflammation and does not respond to Epo administration, despite repression of erythroblast apoptosis. On the contrary, a robust erythropoietic response takes place in the spleen after Epo injections in both control and zymosan-induced generalized inflammation mice. This specific response implies Epo-mediated induction of BMP4 expression by F4/80(+) spleen macrophages, proliferation of stress burst-forming units-erythroid, and increased number of spleen erythroblasts. It allows only partial recovery of anemia, probably because of peripheral destruction of mature red cells. It is not clear whether similar BMP4-dependent stress erythropoiesis can occur in human bone marrow after Epo injections.

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox.

NADPH oxidase is an O‐generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane‐bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G‐protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X‐100 or octyl glucoside buffer and separated into detergent‐soluble and detergent‐insoluble fractions, p40phox and p67phox were mainly associated with the detergent‐insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox‐deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X‐100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH‐oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes. J. Leukoc. Biol. 66: 1014–1020; 1999.

12 – Redox Regulation of Cell Adhesion Processes

This chapter discusses the role of oxidants and antioxidants in the regulation of cell adhesion processes as well as the molecular mechanisms of such regulation. Cell adhesion processes play a major role in the regulation of immune functions as well as of other vital biological processes, such as embryogenesis, cell growth, differentiation, and wound repair. Cell adhesion, which is a multistep process including rolling, firm attachment, and transmigration of leukocytes, is mediated by several classes of cell adhesion molecules. Cell adhesion molecule expression and adhesive properties of cells are modified greatly in several diseased conditions involving redox imbalances, such as cancer, atherosclerosis, diabetes, chronic inflammation, and ischemiareperfusion injury. Several stimuli such as cytokines, chemokines, and phorbol ester are known to activate the expression and function of cell adhesion molecules. Both these evidences indicate that the overall cell adhesion process is redox regulated—direct activation of cell adhesion processes by oxidants and inhibitory action of antioxidants on cell adhesion molecule expression and function.