Original Contributions
Reactive oxygen species activate focal adhesion kinase, paxillin and P130CAS tyrosine phosphorylation in endothelial cells

https://doi.org/10.1016/S0891-5849(98)00134-8Get rights and content

Abstract

Reactive oxygen species (ROS), particularly hydroxyl radical (HOradical dot), 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 μM) and herbimycin A (0.9 μM), 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 HOradical dot. 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 μM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HOradical dot, 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.

Introduction

During different pathological events where ischaemia reperfusion occurs and that affect mainly the cardiovascular and pulmonary systems, endothelium is the principal target for reactive oxygen species (ROS). Indeed, the production of reactive oxygen species (ROS) such as superoxide anion (O2•−), hydrogen peroxide (H2O2) and hydroxyl radical (HOradical dot) increases in blood and tissues during reperfusion and have been shown to be the cause of injury in the heart and other organs [1], [2], [3]. They are released by tissue xanthine oxidase (XO) concentrated in endothelial cells and derived from xanthine dehydrogenase conversion during ischemia [4], [5] or by polymorphonuclear neutrophils (PMN), recruited during the inflammatory response [6] and accumulated in extravascular tissues after their migration between endothelial cells [7], [8], [9], [10]. Before their migration, neutrophils must adhere to the endothelium, this adhesion being mediated by various adhesion molecules such as selectins, integrins or intercellular adhesion molecules (ICAM) [11]. These molecules are expressed on endothelial cells and on neutrophils after activation by various stimuli such as cytokines, endotoxin, thrombin, and other inflammatory mediators. In previous studies [10], [12], [13], we and others have shown that ROS, in vitro, stimulated PMN adhesion, to human umbilical vein endothelial cells (HUVEC), suggesting an important role of ROS in PMN recruitment during inflammation; this adhesion seemed to be due to intracellular hydroxyl radical (HOradical dot) produced by a Fenton reaction inside HUVEC [10], [14] since we have shown that hydroxyl radical scavengers decreased it. However, the intracellular mechanism by which HOradical dot mediates the adhesiveness of HUVEC is unclear. A growing body of evidence suggests that in other cell types, ROS, produced directly or by γ-irradiation, may function as intracellular signaling molecules, by activating different various tyrosine kinases [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. Tyrosine-phosphorylated proteins play an important role in the regulation of cell-adhesion molecules [16], [25], [26], [27]. Indeed, both genistein and herbimycin A attenuated the adhesion of neutrophils and monocytes to endothelal cells in response to both interleukin 1 and tumor necrosis factor alpha [28].

P125FAK, is a member of a non-receptor protein-tyrosine kinases family which plays a role in regulating the changes in actin cytoskeleton organization. p125FAK in fibroblasts is associated to focal adhesions; its tyrosine phosphorylation and that of the cytoskeleton-associated protein paxillin (PAX) and p130cas [29], [30] can be activated by β1 and β3 integrins [31], [32], [33], by a variety of regulatory peptides and lipids mediating cell growth and differentiation and acting through G-protein-coupled receptors[34], [37], and by several other compounds [25], [26], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44]. Recently VEGF has been shown in endothelial cells to rapidly tyrosine phosphorylate p125FAK and paxillin, this event being potentially involved in the migratory cell response to this factor [45].

Our aim in this study was to investigate the effects of ROS on p125FAK, paxillin and p130cas phosphorylation, in endothelial cells, and their involvement in the modification of cytoskeleton which could mediate an increase in PMN-HUVEC adhesion.

We show that ROS and possibly hydroxyl radicals HOradical dot are able to stimulate the phosphorylation of p125FAK, paxillin and p130cas in endothelial cells and to increase the cell membrane tyrosine phosphorylation. As cytochalasin D was able to inhibit at the same time tyrosine phosphorylation and PMN adhesion, we postulated that a correlation may exist between phosphorylation of cytoskeleton proteins, modification of cell membrane structure and cytoskeleton organization which may play a role in the increase neutrophil adhesion mediated by ROS.

Section snippets

Chemicals

Hank’s balanced salt solution with or without Ca2+, Mg2+ (HBSS, HBSS wo) was from Gibco (Paisley, UK). Xanthine oxidase (XO), hypoxanthine (HX), Triton X-100, catalase, bovine serum albumin (BSA), FITC-conjugated-phalloidin, paraformaldehyde, 2-mercaptoethanol, acrylamide, bis-acrylamide, persulfate, Temed, bromophenol blue, cytochalasine D, glycerol, sodium pyrophosphate, phenylmethylsulfonylfluoride (PMSF), genistein, trizma base, trizma hydrochloride (Tris-HCl), RNase and propidium iodide

Adherence of PMN to ROS-stimulated huvec

We have previously shown that hydroxyl radical scavengers such as dimethylthiourea (DMTU), pentoxifylline (Ptx), and dimethylsulfoxide (DMSO) were able to inhibit the adhesion of PMN to HX-XO-treated endothelial cells [14]. In order to identify the oxidative species involved, HUVEC were incubated for 24 h with two iron-chelators able to inhibit the Fenton reaction producing HOradical dot, viz. DF (1 mM) or HBED (0.5 mM), and treated for 15 min with HX-XO as described in Materials and Methods. The decrease

Discussion

We report here that ROS and probably hydroxyl radicals increase PMN adherence to endothelial cells and trigger tyrosine phosphorylation of HUVEC proteins, as shown by western blotting and by confocal microscopy around the cell membrane of ROS-stimulated HUVEC. Moreover, tyrosine kinase inhibitors such as herbimycin A and genistein also inhibited the adherence of PMN to ROS-stimulated HUVEC [14], suggesting that the intracellular mechanism of adherence involves tyrosine kinase. Antioxidants

Acknowledgements

We thank Germain Trugnan (INSERM U410, Faculté X. Bichat, Paris) for confocal microscopy analysis. HBED was a generous gift from J.B. Galey (L’Oréal, Paris). We also thank C. Babin-Chevaye for HUVEC preparation, and the midwives of the Clinique Marie-Louise and clinique Sainte Thérèse for providing umbilical cords.

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