Cellular response to oxidative stress: signaling for suicide and survival

Reactive oxygen species (ROS), whether produced endogenously as a consequence of normal cell functions or derived from external sources, pose a constant threat to cells living in an aerobic environment as they can result in severe damage to DNA, protein, and lipids. The importance of oxidative damage to the pathogenesis of many diseases as well as to degenerative processes of aging has becoming increasingly apparent over the past few years. Cells contain a number of antioxidant defenses to minimize fluctuations in ROS, but ROS generation often exceeds the cell's antioxidant capacity, resulting in a condition termed oxidative stress. Host survival depends upon the ability of cells and tissues to adapt to or resist the stress, and repair or remove damaged molecules or cells. Numerous stress response mechanisms have evolved for these purposes, and they are rapidly activated in response to oxidative insults. Some of the pathways are preferentially linked to enhanced survival, while others are more frequently associated with cell death. Still others have been implicated in both extremes depending on the particular circumstances. In this review, we discuss the various signaling pathways known to be activated in response to oxidative stress in mammalian cells, the mechanisms leading to their activation, and their roles in influencing cell survival. These pathways constitute important avenues for therapeutic interventions aimed at limiting oxidative damage or attenuating its sequelae.     

The stressed synovium

This review focuses on the mechanisms of stress response in the synovial tissue of rheumatoid arthritis. The major stress factors, such as heat stress, shear stress, proinflammatory cytokines and oxidative stress, are discussed and reviewed, focusing on their potential to induce a stress response in the synovial tissue. Several pathways of stress signalling molecules are found to be activated in the synovial membrane of rheumatoid arthritis; of these the most important examples are heat shock proteins, mitogen-activated protein kinases, stress-activated protein kinases and molecules involved in the oxidative stress pathways. The expression of these pathways in vitro and in vivo as well as the consequences of stress signalling in the rheumatoid synovium are discussed. Stress signalling is part of a cellular response to potentially harmful stimuli and thus is essentially involved in the process of synovitis. Stress signalling pathways are therefore new and promising targets of future anti-rheumatic therapies.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

This review focuses on the mechanisms of stress response in the synovial tissue of rheumatoid arthritis. The major stress factors, such as heat stress, shear stress, proinflammatory cytokines and oxidative stress, are discussed and reviewed, focusing on their potential to induce a stress response in the synovial tissue. Several pathways of stress signalling molecules are found to be activated in the synovial membrane of rheumatoid arthritis; of these the most important examples are heat shock proteins, mitogen-activated protein kinases, stress-activated protein kinases and molecules involved in the oxidative stress pathways. The expression of these pathways in vitro and in vivo as well as the consequences of stress signalling in the rheumatoid synovium are discussed. Stress signalling is part of a cellular response to potentially harmful stimuli and thus is essentially involved in the process of synovitis. Stress signalling pathways are therefore new and promising targets of future anti-rheumatic therapies.     

Oxidative stress in microorganisms—I

Oxidative stress in microbial cells shares many similarities with other cell types but it has its specific features which may differe in prokaryotic and eukaryotic cells. We survey here the properties and actions of primary sources of oxidative stress, the role of transition metals in oxidative stress and cell protective machinery of microbial cells, and compare them with analogous features of other cell types. Other features to be compared are the action of reactive oxygen species (ROS) on cell constituents, secondary lipid-or protein-based radicals and other stress products. Repair of oxidative injury by microorganisms and proteolytic removal of irreparable cell constituents are briefly described. Oxidative damage of aerobically growing microbial cells by endogenously formed ROS mostly does not induce changes similar to the aging of multiplying mammalian cells. Rapid growth of bacteria and yeast prevents accumulation of impaired macromolecules which are repaired, diluted or eliminated. During growth some simple fungi, such as yeast orPodospora spp., exhibit aging whose primary cause seems to be fragmentation of the nucleolus or impairment of mitochondrial DNA integrity. Yeast cell aging seems to be accelerated by endogenous oxidative stress. Unlike most growing microbial cells, stationaryphase cells gradually lose their viability because of a continuous oxidative stress, in spite of an increased synthesis of antioxidant enzymes. Unlike in most microorganisms, in plant and animal cells a severe oxidative stress induces a specific programmed death pathway-apoptosis. The scant data on the microbial death mechanisms induced by oxidative stress indicate that in bacteria cell death can result from activation of autolytic enzymes (similarly to the programmed mother-cell death at the end of bacillar sporulation). Yeast and other simple eukaryotes contain components of a proapoptotic pathway which are silent under normal conditions but can be activated by oxidative stress or by manifestation of mammalian death genes, such asbak orbax. Other aspects, such as regulation of oxidative-stress response, role of defense enzymes and their control, acquisition of stress tolerance, stress signaling and its role in stress response, as well as cross-talk between different stress factors, will be the subject of a subsequent review.     

Dual cell protective mechanisms activated by differing levels of oxidative stress in HT22 murine hippocampal cells

Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H₂O₂ induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H₂O₂ in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells.     

Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.     

The Hog1 MAP kinase pathway and the Mec1 DNA damage checkpoint pathway independently control the cellular responses to hydrogen peroxide

The DNA damage checkpoint pathway and the MAP kinase pathway respond to various forms of environmental as well as endogenous stresses through signal transduction mechanisms involving protein kinases. Both pathways are intertwined in mammalian cells, but potential crosstalk between these two pathways in budding yeast has not been examined yet. We show that the Rad53 checkpoint kinase and the Hog1 MAP kinase of Saccharomyces cerevisiae become phosphorylated upon exposure to hydrogen peroxide, indicative of activation of the DNA damage checkpoint and MAP kinase pathways in response to oxidative stress. Rad53 kinase is equally activated in wild type and in hog1-Delta cells. Likewise, the activation of Hog1 MAP kinase is not dependent on Mec1 kinase, the central checkpoint kinase in budding yeast. Mutants in either pathway are sensitive to hydrogen peroxide and the double mutants exhibit a near perfectly additive phenotype. These data demonstrate that the DNA damage checkpoint pathway and the MAP kinase pathway respond to oxidative stress independently of each other and suggest that these two stress signaling pathways are activated by different types of insults induced by hydrogen peroxide.     

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H(2)O(2) for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca(2+)-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.     

Untangling the oxidative cost of reproduction: An analysis in wild banded mongooses

The cost of reproduction plays a central role in evolutionary theory, but the identity of the underlying mechanisms remains a puzzle. Oxidative stress has been hypothesized to be a proximate mechanism that may explain the cost of reproduction. We examine three pathways by which oxidative stress could shape reproduction. The “oxidative cost” hypothesis proposes that reproductive effort generates oxidative stress, while the “oxidative constraint” and “oxidative shielding” hypotheses suggest that mothers mitigate such costs through reducing reproductive effort or by pre‐emptively decreasing damage levels, respectively. We tested these three mechanisms using data from a long‐term food provisioning experiment on wild female banded mongooses (Mungos mungo). Our results show that maternal supplementation did not influence oxidative stress levels, or the production and survival of offspring. However, we found that two of the oxidative mechanisms co‐occur during reproduction. There was evidence of an oxidative challenge associated with reproduction that mothers attempted to mitigate by reducing damage levels during breeding. This mitigation is likely to be of crucial importance, as long‐term offspring survival was negatively impacted by maternal oxidative stress. This study demonstrates the value of longitudinal studies of wild animals in order to highlight the interconnected oxidative mechanisms that shape the cost of reproduction.     

Loss of adaptation to oxidative stress as a mechanism for aortic damage in aging rats

Cells are armed with a vast repertoire of antioxidant defence mechanisms to prevent the accumulation of oxidative damage. The cellular adaptive response is an important antioxidant mechanism against physiological and pathophysiological oxidative alterations in a cell's microenvironment. The aim of this paper was to study, in the rat aorta, whether this adaptive response and the inflammation associated with oxidative stress were expressed throughout the aging process. We examined the rat aorta, as it is a very sensitive tissue to oxidative stress. Male Wistar rats of 1.5, 3, 12, 18 and 24 months of age were used. Superoxide anion (O2(-)) generation; levels of two antioxidant enzymes, superoxide dismutase (SOD) and catalase; and the levels of prostaglandin E2 (PGE2), an inflammatory marker, were measured. The results for rats at different ages were compared with those for 3 months of age. A balance between production of O2(-) and SOD activity was found in the aorta of rats from 1.5 to 12 months old. Oxidative stress was present in the aorta of old animals (18-24 months), due to a failure in the mechanisms of adaptation to oxidative stress. The observed increase in PGE2 levels in these rats reflected an inflammatory response. All together suggest that vascular oxidative stress and the inflammatory process observed in the old groups of rats could be closely related to vascular aging. Our results also remark the importance of the adaptative response to oxidative stress.     

Intracellular localization of endothelial cell annexins is differentially regulated by oxidative stress

Annexins are calcium-dependent phospholipid binding proteins that are implicated in the regulation of both intracellular and extracellular thrombostatic mechanisms in the vascular endothelium. Tight control of annexin gene expression and targeting of annexin proteins is therefore of importance in maintaining the health of the endothelium. Because annexins are abundant in vascular endothelial cells and could be either dysregulated by or contribute to anomalies in Ca2+ signaling, we investigated annexin gene expression and subcellular localization in human umbilical vein endothelial cells (HUVEC) in a model of chronic oxidative stress. HUVEC were cultured under mild hyperoxic conditions in a custom-built chamber to induce oxidative stress over a period of 12 days. Although annexin expression levels did not change significantly in response to hyperoxic stress, immunofluorescence analysis revealed striking effects on the subcellular localization of certain annexins, including the redistribution of annexins 5 and 6 from the cytosol to the nucleus. In addition, oxidative stress modulated the responses of certain annexins to stimulation with a range of pharmacological and physiological Ca2+-mobilizing agonists, in a manner that suggested that annexin localization is regulated via the complex integration of both Ca2+ and intracellular signaling pathways. These results show that differential regulation of annexin localization by oxidative stress may have a causative role in the cellular pathophysiology of vascular endothelial cell disease.     

Role of sugarcane straw allelochemicals in the growth suppression of arrowleaf sida

Previous studies suggested that allelochemicals from sugarcane straw may suppress the growth of arrowleaf sida (Sida rhombifolia L.). A study was conducted to establish: (1) the direct or indirect role of the organic molecules from sugarcane straw leachate on the growth suppression of arrowleaf sida and (2) if leachate phytotoxins induce proline accumulation in arrowleaf sida tissues as an adaptative response to a water or an oxidative stress. Inhibition of root elongation was the primary effect of sugarcane straw leachate on arrowleaf sida grown in unsterile soil. Addition of activated charcoal to unsterile soil before incorporation of straw leachate reduced the inhibition in root growth suggesting a direct participation of organic molecules in leachate phytotoxicity on arrowleaf sida. Inorganic straw constituents did not inhibit root growth while microbial activity increased leachate phytotoxicity. Soil chemical analysis suggested a direct action of organic molecules in leachate phytotoxicity rather than variations in macro and micronutrients or nutrient microbial immobilization. Straw leachate induced proline accumulation in roots and cotyledons of arrowleaf sida. Proline increase was related with oxidative stress in the roots but not in the cotyledons. Our results indicate a direct action of organic compounds from sugarcane straw and/or their microbial transformation products on root growth of arrowleaf sida. These substances induced proline accumulation in roots mainly as consequence of an oxidative stress while water stress may be the main cause of high proline content in the cotyledons. Although the observed responses could be due to phenolic compounds, the involvement of organic molecules with other chemical nature could not be excluded.     

Activated c-Abl is degraded by the ubiquitin-dependent proteasome pathway.

C-Abl is a nonreceptor tyrosine kinase that is tightly regulated in the cell. Genetic data derived from studies in flies and mice strongly support a role for Abl kinases in the regulation of the cytoskeleton (reviewed in [1,2]). C-Abl can be activated by several stimuli, including oxidative stress [3], DNA damage [4], integrin engagement [5], growth factors, and Src family kinases [6]. Structural alterations elicit constitutive activation of the c-Abl tyrosine kinase, leading to oncogenic transformation. While the mechanisms that activate c-Abl are beginning to be elucidated, little is known regarding the mechanisms that downregulate activated c-Abl. Here, we show for the first time that activated c-Abl is downregulated by the ubiquitin-dependent degradation pathway. Activated forms of c-Abl are more unstable than wild-type and kinase-inactive forms. Moreover, inhibition of the 26S proteasome leads to increased c-Abl levels in vitro and in cells, and activated c-Abl proteins are ubiquitinated in vivo. Significantly, inhibition of the 26S proteasome in fibroblasts increases the levels of tyrosine-phosphorylated, endogenous c-Abl. Our data suggest a novel mechanism for irreversible downregulation of activated c-Abl, which is critical to prevent the deleterious consequences of c-Abl hyperactivation in mitogenic and cytoskeletal pathways.     

Biological response of human diploid keratinocytes to quinone-producing compounds: role of NAD(P)H:quinone oxidoreductase 1

Reactive oxygen species (ROS) and quinones are known to determine redox balance alteration, oxidative stress and carcinogenicity. Keratinocytes of the human epidermis, a tissue particularly exposed to oxidant stimuli, possess a wide range of antioxidant and detoxifying mechanisms aimed to avoid oxidative damage of the tissue. In the present study, we evaluate the response of diploid and transformed human keratinocytes to exposure to L-dopa and tetrahydropapaveroline (THP), catechol compounds susceptible to undergo oxidation to form quinones with concomitant production of reactive oxygen species. We demonstrated that these compounds elicit up-regulation of intracellular antioxidant enzymes, in a different degree in normal cells with respect to transformed ones. Normal diploid keratinocytes adequately scavenge toxic substances through the activation of several, concurrent pathways. Conversely, in transformed cells, the whole oxidative burden must be detoxified by the limited set of conserved pathways that, accordingly, have to be highly activated. The biological response to catechol toxicity appears to rely on the pathway of NAD(P)H:quinone oxidoreductase 1 (NQO1). In conclusion, NAD(P)H:quinone oxidoreductase 1 confirms its antioxidant and detoxifying role contributing to the capacity of keratinocytes to protect epidermis against oxidative stress. Being retained in almost any cell, it represents a mechanism of general relevance in cell physiology.