Tumor necrosis factor-alpha-induced reactive oxygen species formation is mediated by JNK1-dependent ferritin degradation and elevation of labile iron pool

Tumor necrosis factor alpha induces increased reactive oxygen species (ROS) generation in different experimental models. However, the nature of this phenomenon is still unknown. We hypothesized that TNF-induced ROS formation is due to JNK-regulated ferritin degradation and an increase in labile iron pool (LIP). We used as a model human prostate cancer cells, DU145. TNF treatment induced ROS formation, which was reduced to the control level in cells pretreated with desferrioxamine, an iron chelator. TNF induced a drop in light chain of the ferritin level, as judged by immunoblotting and an increase in LIP, evaluated by calcein fluorescence. Moreover, we observed that the JNK inhibitor SP600125 abolished TNF-induced changes in LIP, which suggests that JNK kinases are involved in this process. To explore which one of the JNK kinases is responsible for these effects, DU145 cells were transiently transfected with plasmids encoding inactive mutants of JNK1 or JNK2. The cells expressing inactive JNK1 mutant, but not cells expressing JNK2 mutant or possessing an empty vector, were completely resistant to TNF-induced ROS generation, ferritin degradation, and an increase in LIP. These data suggest that TNF-induced ROS formation is mediated by JNK1, which regulates ferritin degradation and thus the level of highly reactive iron.     

Neuroprotection by tempol in a model of iron-induced oxidative stress in acute ischemic stroke

Studies suggest iron exacerbates the damage caused by ischemic stroke. Our aim was to elucidate the effect of iron overload on infarct size after middle cerebral artery occlusion (MCAO) and to evaluate the efficacy of tempol, a superoxide dismutase mimetic, as a neuroprotective agent. Rats were administered iron +/- tempol before MCAO; control rats received saline. The middle cerebral artery was occluded for 24 h, and the size of the resultant infarct was assessed and expressed as the percentage of the hemisphere infracted (%HI). Iron treatment increased infarct size compared with control (51.83 +/- 3.55 vs. 27.56 +/- 3.28%HI iron treated vs. control, P = 0.01); pretreatment with tempol reversed this (51.83 +/- 3.55 vs. 26.09 +/- 9.57%HI iron treated vs. iron + tempol treated, P = 0.02). We hypothesized that reactive oxygen species (ROS) were responsible for the iron-induced damage. We measured ROS generated by exogenous iron in brain and peripheral vasculature from rats that had not undergone MCAO. There was no increase in ROS production in the brain of iron-treated rats or in brain slices incubated with iron citrate. However, ROS generation in carotid arteries incubated with iron citrate was significantly increased. ROS generation from the brain was assessed after MCAO by dihydroethidine staining; there was a dramatic increase in the ROS generation by the brain in the iron-treated rats compared with control 30 min after MCAO. We propose that iron-induced ROS generation in the cerebral vasculature adds to oxidative stress during an ischemic episode after the disruption of the blood-brain barrier.     

Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis

In the past, investigators have successfully used iron chelators to mitigate the cardiotoxicity of doxorubicin (DOX), a widely used anticancer drug that induces reactive oxygen species (ROS), oxidative damage, and apoptosis. Although intracellular iron plays a critical role in initiating DOX-induced apoptosis, the molecular mechanism(s) that link iron, ROS, and apoptosis are still unknown. In this study, we demonstrate that apoptosis results from the exposure of bovine aortic endothelial cells to DOX and that the apoptotic cell death is accompanied by a significant increase in cellular iron ((55)Fe) uptake and activation of iron regulatory protein-1. Furthermore, DOX-induced iron uptake was shown to be mediated by the transferrin receptor (TfR)-dependent mechanism. Treatment with the anti-TfR antibody (IgA class) dramatically inhibited DOX-induced apoptosis, iron uptake, and intracellular oxidant formation as measured by fluorescence using dichlorodihydrofluorescein. Treatment with cell-permeable iron chelators and ROS scavengers inhibited DOX-induced cellular (55)Fe uptake, ROS formation, and apoptosis. Based on these findings, we conclude that DOX-induced iron signaling is regulated by the cell surface TfR expression, intracellular oxidant levels, and iron regulatory proteins. The implications of TfR-dependent iron transport in oxidant-induced apoptosis in endothelial cells are discussed.     

Biological Membranes Are Nanostructures that Require Internal Heat and Imaginary Temperature as New,Unique Physiological Parameters Related to Biological Catalysts

In 1961, Peter Mitchell advanced a new idea for solving the problem of coupling between oxidation and phosphorylation, but some aspects of the relationship between the redox-chain as a potential energy donor and different energy acceptors remain largely unknown. The main structure–function relationships behind catalytic rate optimization in membrane enzymes are highly important, and comparative analyses of the energetics of catalytic reactions from membrane proteins of different destination are needed to advance our understanding. Moreover, the mode of control of primary radicals, such as reactive oxygen species (ROS), should be considered. For example, iron is essential for most organisms because it serves as an electron donor and acceptor in various metabolic processes. However, these chemical properties also allow iron to participate in the formation of ROS that cause substantial damage to lipids; iron can contribute to excess production of damaging ROS through Fenton chemistry. The evidence that iron contributes to various diseases of ageing is to be examined along with the need for low or moderate levels of iron, depending on homeostasis level. If this level in the organs and tissues is close to the optimal amount needed for an initiation of lipid-radical cycles, which may be responsible for the effectiveness of some membrane enzymes, this might minimize the ROS production and retard the processes related to ageing. To my mind, biological membranes possess an internal heat and imaginary temperature that are new, unique physiological parameters related to a role as factors of biological catalysis. This is speculation and additional studies will be needed to determine whether the imaginary temperature has an equal importance with the real temperature in cellular metabolism, membrane energetics (microsomal monooxygenase and ATP synthesis) and ageing.     

Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Neuroprotective activity of p-terphenyl leucomentins from the mushroom Paxillus panuoides

The neuroprotective mechanism of p-terphenyl leucomentins from the mushroom Paxillus panuoides was studied. Leucomentins showed potent inhibition of lipid peroxidation and H2O2 neurotoxicity, but free from any role as reactive oxygen species (ROS) scavengers. Iron-mediated oxidative damage has been implicated in these processes, as a provider of ROS via iron. Leucomentins can chelate iron when DNA is present with iron and H2O2, and so inhibiting DNA single strand breakage. These results suggest that the neuroprotective action of leucomentins is dependent on their ability to chelate iron.     

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g?=?4.3. This form of iron is referred to as "free" iron. EPR signals at g?=?4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S?=?5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g?=?4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.     

Iron-induced generation of mitochondrial ROS depends on AMPK activity

Deregulated iron homeostasis is generally believed to be implicated in neurodegenerative diseases, including Parkinson’s disease. Nevertheless, it is not fully understood how iron overload can elicit neuronal cell damage. Here we examined mitochondrial reactive oxygen species (ROS) levels in human dopaminergic neuroblastoma SH-SY5Y cells upon iron exposure. A relatively high concentration of iron could significantly increase mitochondrial ROS levels in SH-SY5Y cells. Pharmacological activation of AMP-activated protein kinase (AMPK) almost completely inhibited the effect of iron on mitochondrial ROS. By contrast, AMPK inhibition aggravated the neurotoxicity of iron and enhanced the production of mitochondrial ROS. Collectively, these findings suggested that excess iron may be able to perturb mitochondrial function, and AMPK activity is important for the association of iron and mitochondria.     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress

We have recently reported that red blood cells (RBC) promote T cell growth and survival by inhibiting activation-induced T cell death. In the present study, we have examined parameters of oxidative stress and intracellular iron in activated T cells and correlated these data with the expression of ferritin, heme oxygenase-1 (HO-1), and the transferrin receptor CD71. T cells growing in the presence of RBC had reduced levels of reactive oxygen species (ROS) and oxidatively modified proteins, suggesting that RBC efficiently counteracted ROS production on the activated T cells. Flow cytometry and immunodetection demonstrated that T cells dividing in the presence of RBC had increased levels of intracellular ferritin rich in L-subunits and HO-1 along with a downmodulation in CD71 expression. Finally, using the fluorescent iron indicator calcein and flow cytometry analysis, we were able to show that a relative amount of the labile iron pool (LIP) was upregulated in T cells growing in the presence of RBC. These findings are consistent with a typical response to iron overload. However, neither heme compounds nor ferric iron reproduced the levels of expansion and survival of T cells induced by intact RBC. Altogether, these data suggest that RBC inhibit apoptosis of activated T cells by a combination of ROS scavenging and upregulation of cytoprotective proteins such as ferritin and HO-1, which may counteract a possible toxic effect of the increased intracellular free iron.     

The antioxidant effect of fermented papaya preparation involves iron chelation

Iron-overload is a major clinical problem in various diseases. Under this condition, serum iron which surpasses the binding capacity of transferrin is present as non-transferrin bound iron and cellular unbound Labile Iron Pool (LIP) is increased. LIP participates in the generation of free radicals, including reactive oxygen species (ROS). Increased ROS, with concomitant decrease in anti-oxidants, results in oxidative stress and toxicity to the liver, heart and other tissues, causing serious morbidity and eventually mortality. Therapeutic iron chelation reduces the LIP and thereby ameliorates oxidative stress-mediated toxicity. Many food-derived antioxidants have the capacities to scavenge ROS and chelate iron. We have reported that fermented papaya preparation (FPP) has ROS scavenging effect on blood cells in vitro or in vivo (in thalassemic patients and experimental animals). We now investigated FPP's iron chelating effect - its ability to prevent (and revert) LIP accumulation. Liver- and heart-derived cells, and RBCs were exposed to non-transferrin bound iron in the form of ferrous ammonium sulfate and the effect of FPP on their LIP content and ROS generation was measured by flow-cytometry. The results indicate that FPP reduces LIP and ROS, and suggests that its antioxidant mechanism is related, at least in part, to iron chelation.     

The many faces of the octahedral ferritin protein

Iron is an essential trace nutrient required for the active sites of many enzymes, electron transfer and oxygen transport proteins. In contrast, to its important biological roles, iron is a catalyst for reactive oxygen species (ROS). Organisms must acquire iron but must protect against oxidative damage. Biology has evolved siderophores, hormones, membrane transporters, and iron transport and storage proteins to acquire sufficient iron but maintain iron levels at safe concentrations that prevent iron from catalyzing the formation of ROS. Ferritin is an important hub for iron metabolism because it sequesters iron during times of iron excess and releases iron during iron paucity. Ferritin is expressed in response to oxidative stress and is secreted into the extracellular matrix and into the serum. The iron sequestering ability of ferritin is believed to be the source of the anti-oxidant properties of ferritin. In fact, ferritin has been used as a biomarker for disease because it is synthesized in response to oxidative damage and inflammation. The function of serum ferritin is poorly understood, however serum ferritin concentrations seem to correlate with total iron stores. Under certain conditions, ferritin is also associated with pro-oxidant activity. The source of this switch from anti-oxidant to pro-oxidant has not been established but may be associated with unregulated iron release from ferritin. Recent reports demonstrate that ferritin is involved in other aspects of biology such as cell activation, development, immunity and angiogenesis. This review examines ferritin expression and secretion in correlation with anti-oxidant activity and with respect to these new functions. In addition, conditions that lead to pro-oxidant conditions are considered.     

铁自噬与铁死亡及其相关疾病

铁是血红素、线粒体呼吸链复合体和各种生物酶的重要辅助因子,参与氧气运输、氧化还原反应和代谢物合成等生物过程。铁蛋白(ferritin)是一种铁存储蛋白质,通过储存和释放铁来维持机体内铁平衡。铁自噬(ferritinophagy)作为一种选择性自噬方式,介导铁蛋白降解释放游离铁,参与细胞内铁含量的调控。适度铁自噬维持细胞内铁含量稳定,但铁自噬过度会释放出大量游离铁。通过芬顿 (Fenton)反应催化产生大量的活性氧(reactive oxygen species, ROS),发生脂质过氧化造成细胞受损。因此,铁自噬在维持细胞生理性铁稳态中发挥至关重要的作用。核受体共激活因子4 (nuclear receptor co-activator 4, NCOA4)被认为是铁自噬的关键调节因子,与铁蛋白靶向结合,并传递至溶酶体中降解释放游离铁,其介导的铁自噬构成了铁代谢的重要组成部分。最新研究表明,NCOA4受体内铁含量、自噬、溶酶体和低氧等因素的调控。NCOA4介导的铁蛋白降解与铁死亡(ferroptosis)有关。铁死亡是自噬性细胞死亡过程。铁自噬通过调节细胞铁稳态和细胞ROS生成,成为诱导铁死亡的上游机制,与贫血、神经退行性疾病、癌症、缺血/再灌注损伤与疾病的发生发展密切相关。本文针对NCOA4介导的铁自噬通路在铁死亡中的功能特征,探讨NCOA4在这些疾病中的作用,可能为相关疾病的治疗提供启示。     

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

Background

High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.

Results

One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.

Conclusion

Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.     

Iron homeostasis and iron-regulated ROS in cell death,senescence and human diseases

BackgroundIron is essential for many types of biological processes. However, excessive iron can be cytotoxic and can lead to many diseases. Since ferroptosis, which is an iron-dependent regulated form of necrosis, was recently discovered, iron and iron-catalysed oxidative stress have attracted much interest because of their sophisticated mechanism of cellular signalling leading to cell death and associated with various diseases.Scope of reviewIn this review, we first focus on how iron catalyses reactive oxygen species (ROS). Next, we discuss the roles of iron in cell death and senescence and, in particular, the downstream signalling pathways of ROS. Finally, we discuss the potential regulation mechanism of iron as a therapeutic target for various iron-related diseases.Major conclusionsBoth labile iron released from organelles upon various stresses and iron incorporated in enzymes produce ROS, including lipid ROS. ROS produced by iron activates various signalling pathways, including mitogen-activated protein kinase (MAPK) signalling pathways such as the apoptosis signal-regulating kinase 1 (ASK1)-p38/JNK pathway. These ROS-activated signalling pathways regulate senescence or cell death and are linked to cancer, ischaemia-reperfusion injury during transplantation and ageing-related neurodegenerative diseases.General significanceIron overload damages cells and causes harmful effects on the body through oxidative stress. Thus, understanding the spatiotemporal availability of iron and the role of iron in generating ROS will provide clues for the suppression of ROS and cytotoxic redox-active iron. Moreover, elucidating the molecular mechanisms and signalling pathways of iron-dependent cytotoxicity will enable us to find novel therapeutic targets for various diseases.     

Iron Overload Inhibits Osteoblast Biological Activity Through Oxidative Stress

Iron overload has recently been connected with bone mineral density in osteoporosis. However, to date, the effect of iron overload on osteoblasts remains poorly understood. The purpose of this study is to examine osteoblast biological activity under iron overload. The osteoblast cells (hFOB1.19) were cultured in a medium supplemented with different concentrations (50, 100, and 200 μM) of ferric ammonium citrate as a donor of ferric ion. Intracellular iron was measured with a confocal laser scanning microscope. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescin diacetate fluorophotometry. Osteoblast biological activities were evaluated by measuring the activity of alkaline phosphatase (ALP) and mineralization function. Results indicated that iron overload could consequently increase intracellular iron concentration and intracellular ROS levels in a concentration-dependent manner. Additionally, ALP activity was suppressed, and a decline in the number of mineralized nodules was observed in in vitro cultured osteoblast cells. According to these results, it seems that iron overload probably inhibits osteoblast function through higher oxidative stress following increased intracellular iron concentrations.     

Inhibition of Iron Uptake Is Responsible for Differential Sensitivity to V-ATPase Inhibitors in Several Cancer Cell Lines

Many cell lines derived from tumors as well as transformed cell lines are far more sensitive to V-ATPase inhibitors than normal counterparts. The molecular mechanisms underlying these differences in sensitivity are not known. Using global gene expression data, we show that the most sensitive responses to HeLa cells to low doses of V-ATPase inhibitors involve genes responsive to decreasing intracellular iron or decreasing cholesterol and that sensitivity to iron uptake is an important determinant of V-ATPase sensitivity in several cancer cell lines. One of the most sensitive cell lines, melanoma derived SK-Mel-5, over-expresses the iron efflux transporter ferroportin and has decreased expression of proteins involved in iron uptake, suggesting that it actively suppresses cytoplasmic iron. SK-Mel-5 cells have increased production of reactive oxygen species and may be seeking to limit additional production of ROS by iron.