Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells.

Angiotensin II, a hypertrophic/anti-apoptotic hormone, utilizes reactive oxygen species (ROS) as growth-related signaling molecules in vascular smooth muscle cells (VSMCs). Recently, the cell survival protein kinase Akt/protein kinase B (PKB) was proposed to be involved in protein synthesis. Here we show that angiotensin II causes rapid phosphorylation of Akt/PKB (6- +/- 0.4-fold increase). Exogenous H(2)O(2) (50-200 microM) also stimulates Akt/PKB phosphorylation (maximal 8- +/- 0.2-fold increase), suggesting that Akt/PKB activation is redox-sensitive. Both angiotensin II and H(2)O(2) stimulation of Akt/PKB are abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002 (2(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), suggesting that PI3-K is an upstream mediator of Akt/PKB activation in VSMCs. Furthermore, diphenylene iodonium, an inhibitor of flavin-containing oxidases, or overexpression of catalase to block angiotensin II-induced intracellular H(2)O(2) production significantly inhibits angiotensin II-induced Akt/PKB phosphorylation, indicating a role for ROS in agonist-induced Akt/PKB activation. In VSMCs infected with dominant-negative Akt/PKB, angiotensin II-stimulated [(3)H]leucine incorporation is attenuated. Thus, our studies indicate that Akt/PKB is part of the remarkable spectrum of angiotensin II signaling pathways and provide insight into the highly organized signaling mechanisms coordinated by ROS, which mediate the hypertrophic response to angiotensin II in VSMCs.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

Differential thiol oxidation of the signaling proteins Akt,PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle

Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation; whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.     

Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance

Salt imposes immediate problems for plant cells, such as osmotic stress, impaired ion homeostasis and sodium toxicity, followed by a secondary oxidative stress caused by generation of reactive oxygen species (ROS). Here, we analyzed the production of ROS during salt stress. We show that salt stress triggered plasma membrane internalization, resulting in the production of ROS within endosomes. The intracellular ROS were produced by NADPH oxidase in response to the ionic but not the osmotic stress. Both endocytosis and ROS production were suppressed in phosphatidylinositol (PtdIns) 3-kinase (PI3K) mutants, PI3K being a key regulator of vesicle trafficking in animals and plants, and by wortmannin, which is a specific inhibitor of PI3K and PI4K. Endocytosis and the production of ROS were rescued by supplementation of seedlings with exogenous PtdIns 3-phosphate (PtdIns3P), less with PtdIns4P, but not with PtdIns(4,5)P(2). Surprisingly, despite reduced oxidative stress, the mutants and the wortmannin-treated plants exhibited a phenotype overly sensitive to salt, as also resulted from treatment with diphenyleneiodonium, a suicide inhibitor of NADPH oxidase, suggesting a positive role for ROS in salt tolerance. In summary, our results show that salt stress responses, such as increased plasma membrane endocytosis and the intracellular production of ROS, are coordinated by phospholipid-regulated signaling pathways, and suggest that ROS act in the signal transduction of the salt tolerance response.     

The role of melatonin as an antioxidant in human lens epithelial cells

Abstract

Oxidative stress plays a significant role in pathophysiology of cataracts and also known to affect the phosphatidylinositol-3-kinase/ protein kinase B (PI3K/Akt) signaling pathway. This well-documented pathway is involved in protecting against apoptosis-inducing insults, including oxidative stress. Melatonin (N-acetyl-5-methoxy-tryptamine), the major secretory product of the pineal gland, was identified as a powerful free radical scavenger and a broad-spectrum antioxidant that defends against various oxidative stress-associated diseases. This study was conducted to determine whether melatonin could prevent hydrogen peroxide (H₂O₂)-induced oxidative stress in human lens epithelial cells (HLECs) and to elucidate the molecular pathways involved in this protection. HLECs were subjected to various concentrations of H₂O₂ in the presence or absence of melatonin at different concentrations. Cell viability was monitored by a 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl-tetrazoliumbromide (MTT) assay, and the apoptosis rate and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry using annexin V-FITC and propidium iodide (PI) staining. The expression levels of HO-1, Nrf-2, CAT, and MDA were measured using Western blot analysis. Akt activation was also evaluated by Western blot analysis. The data from our study showed that cells pretreated with melatonin can reduce H₂O₂-induced intracellular ROS generation and thus protect HLECs from cell apoptosis. Furthermore, we found that melatonin is a potent activator of Akt in HLECs. Our findings suggest that in addition to functioning as a direct free radical scavenger, melatonin can elicit cellular signaling pathways that are protective against oxidative stress-induced cataracts.     

Oxidative and nitrosative signaling in plants: Two branches in the same tree?

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) constitute key features underpinning the dynamic nature of cell signaling systems in plants. Despite their importance in many aspects of cell biology, our understanding of oxidative and especially of nitrosative signaling and their regulation remains poorly understood. Early reports have established that ROS and RNS coordinately regulate plant defense responses to biotic stress. In addition, evidence has accumulated demonstrating that there is a strong cross-talk between oxidative and nitrosative signaling upon abiotic stress conditions. The goal of this mini-review is to provide latest findings showing how both ROS and RNS comprise a coordinated oxidative and nitrosative signaling network that modulates cellular responses in response to environmental stimuli.Key words: abiotic stress, nitrosative stress, oxidative stress, reactive nitrogen species, reactive oxygen species, signaling     

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.     

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.     

ASK Family Proteins in Stress Response and Disease

Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.     

Oxidative stress in the haematopoietic niche regulates the cellular immune response in Drosophila

Oxidative stress induced by high levels of reactive oxygen species (ROS) is associated with the development of different pathological conditions, including cancers and autoimmune diseases. We analysed whether oxidatively challenged tissue can have systemic effects on the development of cellular immune responses using Drosophila as a model system. Indeed, the haematopoietic niche that normally maintains blood progenitors can sense oxidative stress and regulate the cellular immune response. Pathogen infection induces ROS in the niche cells, resulting in the secretion of an epidermal growth factor-like cytokine signal that leads to the differentiation of specialized cells involved in innate immune responses.     

Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a copper stress.     

Functional distinctions of protein kinase B/Akt isoforms defined by their influence on cell migration

The three mammalian members of the protein kinase B/Akt (PKB/Akt) family have been implicated in a plethora of cellular signaling processes with key functions in control of cellular metabolism, growth, proliferation and apoptosis. As a major target of phosphatidylinositol (PI) 3-kinase signaling, the PKB/Akt isoforms also have central roles in a variety of human cancers, with effects on tumor initiation, progression and metastasis. It has been shown that isoform-specific functions of PKB/Akt family members can contribute to tumorigenesis on multiple levels. A series of recent studies documents the isoform-specific functions of PKB/Akt family members in regulation of cellular motility and migration by influencing numerous cellular targets involved in organization of the actin cytoskeleton, cellular interaction with the extracellular matrix, expression of motility genes and establishment of cellular polarity. A thorough insight into the isoform-specific roles of PKB/Akt proteins is essential for a full understanding of the complex biological outcomes elicited by PI 3-kinase and PKB/Akt signaling.     

In vivo ROS production and use of oxidative stress-derived biomarkers to detect the onset of diseases such as Alzheimer’s disease,Parkinson’s disease,and diabetes

Breakthroughs in biochemistry have furthered our understanding of the onset and progression of various diseases, and have advanced the development of new therapeutics. Oxidative stress and reactive oxygen species (ROS) are ubiquitous in biological systems. ROS can be formed non-enzymatically by chemical, photochemical and electron transfer reactions, or as the byproducts of endogenous enzymatic reactions, phagocytosis, and inflammation. Imbalances in ROS homeostasis, caused by impairments in antioxidant enzymes or non-enzymatic antioxidant networks, increase oxidative stress, leading to the deleterious oxidation and chemical modification of biomacromolecules such as lipids, DNA, and proteins. While many ROS are intracellular signaling messengers and most products of oxidative metabolisms are beneficial for normal cellular function, the elevation of ROS levels by light, hyperglycemia, peroxisomes, and certain enzymes causes oxidative stress-sensitive signaling, toxicity, oncogenesis, neurodegenerative diseases, and diabetes. Although the underlying mechanisms of these diseases are manifold, oxidative stress caused by ROS is a major contributing factor in their onset. This review summarizes the relationship between ROS and oxidative stress, with special reference to recent advancements in the detection of biomarkers related to oxidative stress. Further, we will introduce biomarkers for the early detection of neurodegenerative diseases and diabetes, with a focus on our recent work.     

Activation of protein kinase B induced by H(2)O(2) and heat shock through distinct mechanisms dependent and independent of phosphatidylinositol 3-kinase

Protein kinase B (PKB) is a downstream target of phosphatidylinositol (PI) 3-kinase in the signaling pathway of growth factors, and is activated by cellular stress such as H(2)O(2) and heat shock. To study the mechanism of the stress-induced activation of PKB, PI 3-kinase products were measured in stress-stimulated cells. Both PI 3,4-bisphosphate and PI 3,4, 5-trisphosphate increased in H(2)O(2)-treated cells, and the elevation of these phospholipids and activation of PKB were concurrently blocked by wortmannin, a potent inhibitor of PI 3-kinase. In heat-shocked cells, the level of PI 3,4-bisphosphate did not change while that of PI 3,4,5-trisphosphate increased slightly, and an association between PKB molecules was observed. Two active PKB fractions, presumably monomeric and oligomeric forms, were resolved from heat-shocked cells by gel filtration column chromatography. Activation of the former was suppressed by pretreatment with wortmannin, whereas the generation and activation of the latter were not blocked by the PI 3-kinase inhibitor. Only the monomeric form, but not the oligomeric form, was recovered from H(2)O(2)-treated cells, and its activation was prevented by wortmannin. These results indicate that PKB is activated by two distinct mechanisms that are dependent and independent of PI 3-kinase in stress-stimulated cells.     

Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.     

Reactive oxygen species generation and signaling in plants

The introduction of molecular oxygen into the atmosphere was accompanied by the generation of reactive oxygen species (ROS) as side products of many biochemical reactions. ROS are permanently generated in plastids, peroxisomes, mitochiondria, the cytosol and the apoplast. Imbalance between ROS generation and safe detoxification generates oxidative stress and the accumulating ROS are harmful for the plants. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses. Here, we summarize the generation of ROS in the different cellular compartments and the signaling processes which are induced by ROS.Keyword: reactive oxygen species, signal transduction, plastids