Martine Raes

IMPORTANCE OF SE-GLUTATHIONE PEROXIDASE, CATALASE, AND CU/ZN-SOD FOR CELL SURVIVAL AGAINST OXIDATIVE STRESS

Eukaryotic cells have to constantly cope with highly reactive oxygen-derived free radicals. Their defense against these free radicals is achieved by natural antioxidant molecules but also by antioxidant enzymes. In this paper, we review some of the data comparing the efficiency of three different antioxidant enzymes: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), catalase, and selenium-glutathione peroxidase. We perform our comparison on one experimental model (human fibroblasts) where the activities of these three antioxidant enzymes have been modulated inside the cells, and the repercussion of these changes was investigated in different conditions. We also focus our attention on the protecting role of selenium-glutathione peroxidase, because this enzyme is very rarely studied due to the difficulties linked to its biochemical properties. These studies evidenced that all three antioxidant enzymes give protection for the cells. They show a high efficiency for selenium-glutathione peroxidase and emphasize the fact that each enzyme has a specific as well as an irreplaceable function. They are all necessary for the survival of the cell even in normal conditions. In addition, these three enzymes act in a cooperative or synergistic way to ensure a global cell protection. However, optimal protection is achieved only when an appropriate balance between the activities of these enzymes is maintained. Interpretation of the deleterious effects of free radicals has to be analyzed not only as a function of the amount of free radicals produced but also relative to the efficiency and to the activities of these enzymatic and chemical antioxidant systems. The threshold of protection can indeed vary dramatically as a function of the level of activity of these enzymes.

Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (30 min, 37 degrees C) for hydrogen peroxide, 3 x 10(-4) M (15 min, 37 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, 37 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, 37 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia‐inducible factor‐1 (HIF‐1) is a transcription factor activated by hypoxia. The HIF‐1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF‐1 activation. We demonstrated that in human microvascular endothelial cells‐1 (HMEC‐1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia‐induced HIF‐1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF‐1α is phosphorylated in hypoxia by an ERK‐dependent pathway. These results evidence the role of mitogen‐activated protein kinase in the transcriptional response to hypoxia.

Hypoxia-induced activation of HIF-1: role of HIF-1α-Hsp90 interaction

The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors such as MyoD, a basic helix loop helix (bHLH) protein, and the bHLH‐Per‐aryl hydrocarbon nuclear translocator (ARNT)‐Sim (PAS) factors Sim and aryl hydrocarbon receptor (Ahr). The transcription factor hypoxia‐inducible factor‐1α (HIF‐1α), involved in the response to hypoxia, also belongs to the bHLH‐PAS family. This work was aimed to investigate the putative role of Hsp90 in HIF‐1 activation by hypoxia. Using a EGFP‐HIF‐1α fusion protein, co‐immunoprecipitation experiments evidenced that the chimeric protein expressed in COS‐7 cells interacts with Hsp90 in normoxia but not in hypoxia. We also demonstrated that Hsp90 interacts with the bHLH‐PAS domain of HIF‐1α. Moreover, Hsp90 is not co‐translocated with HIF‐1α into the nucleus. At last, we showed that Hsp90 activity is essential for HIF‐1 activation in hypoxia since it is inhibited in the presence of geldanamycin. These results indicate that Hsp90 is a major regulator in HIF‐1α activation.

Low levels of reactive oxygen species as modulators of cell function.

In this paper, we present various arguments supporting the hypothesis that reactive oxygen species (ROS) could be responsible for the modulation of various cellular functions, besides their well known toxic effects. We first review the recent evidence indicating that ROS are able to modulate genome expression through specific and precise mechanisms during cell activation. The role of the nitrogen reactive radicals such as nitric oxide is separately analyzed because of its specific role in the nervous and vascular systems. The action of the other ROS on gene activation will then be reviewed by first looking at their possible involvement in the activation of transcription factors like NF-kappa B. Arguments will then be developed in favor of the implication of the ROS in the cellular effects of PMA, TNF-alpha and other cytokines on the modulation of the genetic expression. Possible mechanisms will be presented for linking the production of the ROS with cell activation. In a general way we postulate that ROS can play a role of secondary messengers in several cell responses to external stimuli. In the second part of the paper, we will examine the long term influence of ROS and their possible roles in cellular aging. Different links exist between ROS and aging and the relationship between them is probably indirect. We propose to consider the effect of ROS as one of the multiple challenges that cells have to face, the cell being considered as a global system which must optimize its energy expenditure for carrying out its basic functions such as turnover, differentiated phenotype functions, multiplication, defense and repair processes. This thermodynamic point of view will help to understand the effect of low ROS stresses, among others, on accelerated aging.

Adenovirus-Mediated Gene Transfer of Human Platelet-Activating Factor–Acetylhydrolase Prevents Injury-Induced Neointima Formation and Reduces Spontaneous Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E–deficient mice. Methods and Results—Intravenous administration of an adenovirus (5×108 plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P <0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P <0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P <0.001), macrophages by 69% (P =0.006), and smooth muscle cells by 84% (P =0.002) in the arterial wall. This resulted in a 77% reduction (P <0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P =0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P =0.01 and P =0.04, respectively). Conclusions—Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E–deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

Is HIF-1α a pro- or an anti-apoptotic protein? ☆

Abstract Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor specifically activated by hypoxia. It induces the expression of different genes whose products play an adaptive role for hypoxic cells and tissues. Besides these protective responses, HIF-1 and/or hypoxia have also been shown to be either anti-apoptotic or pro-apoptotic, according to the cell type and experimental conditions. More severe or prolonged hypoxia rather induces apoptosis that is, at least in part, initiated by the direct association of HIF-1α and p53 and p53-induced gene expression. On the other hand, HIF-1α dimerized with ARNT, as an active transcription factor, can protect cells from apoptosis induced by several conditions. This review is aimed to describe the different mechanisms that account for these opposite effects of HIF-1α.

Regulation of gene expression by oxygen: NF-kappaB and HIF-1, two extremes.

Aerobic life is dependent on molecular oxygen for ATP regeneration, but only possible in a narrow range of oxygen concentrations. Increased oxygen tension is toxic through the generation of reactive oxygen species (ROS), while a decrease in oxygen concentration impairs energy availability and, hence, cell viability. Cells have developed strategies to respond to changes in oxygen tension: specific systems detect excessive ROS and hypoxia, leading to the activation of specific transcription factors and expression of appropriate target genes. The aim of this review is to describe how hypoxia-inducible factor-1 (HIF-1) and nuclear factor-kappaB (NF-kappaB) are regulated and what could be the sensors to the changes in oxygen levels. Some of the physiological responses initiated by these transcription factors are also mentioned.

A Haptoglobin-Hemoglobin Receptor Conveys Innate Immunity to Trypanosoma brucei in Humans

The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host immunity against the parasite.

Autotaxin Is Released from Adipocytes, Catalyzes Lysophosphatidic Acid Synthesis, and Activates Preadipocyte Proliferation UP-REGULATED EXPRESSION WITH ADIPOCYTE DIFFERENTIATION AND OBESITY

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904–910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipased-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabeticdb/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.