Sensitization of T cells to apoptosis—A role for ROS?

T cell homeostasis is achieved by balancing the production and proliferation of T cells with their apoptotic cell death. Activation of naïve T cells by antigen in the context of MHC results in the massive expansion of antigen-specific T cells and the production of reactive oxygen species (ROS). Following expansion, the majority of the T cells die via apoptosis, while a small number of them survive and differentiate into memory T cells. This cell fate decision is crucial to our understanding of how autoimmunity is avoided and how immunity is maintained. It has become increasingly clear that ROS can affect this cell fate decision by sensitizing T cells to apoptosis. Interestingly, ROS have effects on both intrinsic and extrinsic apoptosis pathways through modulation of expression of the major molecules in these pathways, Bcl-2 and FasL. In this review, we will focus on the pro-apoptotic effects of ROS and mechanisms by which they regulate the death of T cells.     

IEX-1 deficiency protects against colonic cancer

The immediate early response gene X-1 (IEX-1) is involved in regulation of various cellular processes including proliferation, apoptosis in part by controlling homeostasis of reactive oxygen species (ROS) at mitochondria. The present study shows reduced inflammatory responses and colorectal cancer in IEX-1 knockout (KO) mice treated with azoxymethane/dextran sulfate sodium (DSS). However, DSS induced worse colitis in RAG(-/-)IEX-1(-/-) double KO mice than in RAG and IEX-1 single KO mice, underscoring an importance of T cells in IEX-1 deficiency-induced protection against colon inflammation. Lack of IEX-1 promoted the differentiation of interleukin (IL)-17-producing T cells, concomitant with upregulation of Gαi2 expression, a gene that is well-documented for its role in the control of inflammation in the colon. In accordance with this, T-helper 17 (T(H)17) cell differentiation was compromised in the absence of Gαi2, and deletion of Gαi2 in T cells alone aggravated colon inflammation and colorectal cancer development after azoxymethane/DSS treatment. Null mutation of IEX-1 also enhanced both proliferation and apoptosis of intestinal epithelial cells (IEC) after injury. A potential impact of this altered IEC turnover on colon inflammation and cancer development is discussed. These observations provide a linkage of IEX-1 and Gαi2 expression in the regulation of T(H)17 cell differentiation and suggest a previously unappreciated role for IEX-1 in the control of colon epithelial homeostasis.     

Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis

Intracellular glutathione (GSH) depletion is an important hallmark of apoptosis. We have recently shown that GSH depletion by its extrusion regulates apoptosis independently of excessive reactive oxygen species accumulation. However, the mechanisms by which GSH depletion regulates apoptosis are still unclear. Because disruption of intracellular ionic homeostasis, associated with apoptotic volume decrease (AVD), is necessary for the progression of apoptotic cell death, we sought to evaluate the relationship between GSH transport and ionic homeostasis during Fas ligand (FasL)-induced apoptosis in Jurkat cells. GSH depletion in response to FasL was paralleled by distinct degrees of AVD identified by differences in cellular forward scatter and electronic impedance analysis. Inhibition of GSH efflux prevented AVD, K+ loss, and the activation of two distinct ionic conductances, mediated by Kv1.3 and outward rectifying Cl- channels. Reciprocally, stimulation of GSH loss accelerated the loss of K+, AVD, and consequently the progression of the execution phase of apoptosis. Although high extracellular K+ inhibited FasL-induced apoptosis, GSH depletion was largely independent of K+ loss. These results suggest that deregulation of GSH and ionic homeostasis converge in the regulation of apoptosis in lymphoid cells.     

T-cell death following immune activation is mediated by mitochondria-localized SARM

Following acute-phase infection, activated T cells are terminated to achieve immune homeostasis, failure of which results in lymphoproliferative and autoimmune diseases. We report that sterile α- and heat armadillo-motif-containing protein (SARM), the most conserved Toll-like receptors adaptor, is proapoptotic during T-cell immune response. SARM expression is significantly reduced in natural killer (NK)/T lymphoma patients compared with healthy individuals, suggesting that decreased SARM supports NK/T-cell proliferation. T cells knocked down of SARM survived and proliferated more significantly compared with wild-type T cells following influenza infection in vivo. During activation of cytotoxic T cells, the SARM level fell before rising, correlating inversely with cell proliferation and subsequent T-cell clearance. SARM knockdown rescued T cells from both activation- and neglect-induced cell deaths. The mitochondria-localized SARM triggers intrinsic apoptosis by generating reactive oxygen species and depolarizing the mitochondrial potential. The proapoptotic function is attributable to the C-terminal sterile alpha motif and Toll/interleukin-1 receptor domains. Mechanistically, SARM mediates intrinsic apoptosis via B cell lymphoma-2 (Bcl-2) family members. SARM suppresses B cell lymphoma-extra large (Bcl-xL) and downregulates extracellular signal-regulated kinase phosphorylation, which are cell survival effectors. Overexpression of Bcl-xL and double knockout of Bcl-2 associated X protein and Bcl-2 homologous antagonist killer substantially reduced SARM-induced apoptosis. Collectively, we have shown how T-cell death following infection is mediated by SARM-induced intrinsic apoptosis, which is crucial for T-cell homeostasis.     

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.     

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

Mammalian cell injury induced by hypothermia- the emerging role for reactive oxygen species

Hypothermia is a well-known strategem to protect biological material against injurious or degradative processes and is widely used in experimental and especially in clinical applications. However, hypothermia has also proved to be strongly injurious to a variety of cell types. Hypothermic injury to mammalian cells has long been attributed predominantly to disturbances of cellular ion homeostasis, especially of sodium homeostasis. For many years, reactive oxygen species have hardly been considered in the pathogenesis of hypothermic injury to mammalian cells. In recent years, however, increasing evidence for a role of reactive oxygen species in hypothermic injury to these cells has accumulated. Today there seems to be little doubt that reactive oxygen species decisively contribute to hypothermic injury in diverse mammalian cells. In some cell types, such as liver and kidney cells, they even appear to play the central role in hypothermic injury, outruling by far a contribution of the cellular ion homeostasis. In these cells, the cellular chelatable, redox-active iron pool appears to be decisively involved in the pathogenesis of hypothermic injury and of cold-induced apoptosis that occurs upon rewarming of the cells after a (sublethal) period of cold incubation.     

铁蛋白介导细胞凋亡的作用机制

铁蛋白是生物体广泛存在且高度保守的可溶性蛋白质,在铁离子稳态维持、胚胎发育调控、细胞增殖以及细胞凋亡等过程中具有重要作用。过量的铁离子能通过芬顿反应产生活性氧,过量的活性氧会造成氧化应激并直接损害DNA、脂质和蛋白质,最终导致细胞凋亡。铁蛋白能够螯合铁离子,进而保护细胞免受氧化应激诱导的细胞凋亡。铁蛋白表达受阻时,细胞内不稳定铁水平升高并诱导氧化应激,最终造成细胞凋亡。同时,氧化应激可在转录和翻译水平调节铁蛋白表达,升高的铁蛋白则参与维持机体氧化还原水平的稳定。本文主要从线粒体途径和死亡受体途径阐明铁蛋白介导细胞凋亡的分子机制,为深入研究铁蛋白功能以及相关疾病治疗提供理论支持。     

Activation of mitochondrial-driven apoptosis in skeletal muscle cells is not mediated by reactive oxygen species production

While the acquisition of apoptosis resistance is part of the differentiation program of skeletal muscle cells, differentiated muscle cells can undergo apoptosis in response to physiological or pathological stimuli. The generation of reactive oxygen species by mitochondria plays a major role in the control of apoptosis in many cell types. Indeed their involvement in controlling apoptosis in differentiated muscle cells, or in generating resistance to apoptosis remains unknown. Moreover, differentiated muscle cells specifically express the uncoupling protein-3, a mitochondrial protein potentially involved in controlling reactive oxygen species production. To study the role of mitochondrial reactive oxygen species in the control of apoptosis in skeletal muscle cells, L6E9 myoblasts and myotubes were exposed to staurosporine, an inducer of apoptosis via mitochondrial pathways. Staurosporine activated apoptotic pathways (i.e. caspase-3 and caspase-9) increasing reactive oxygen species in myoblasts and, to a minor extent, in myotubes. However, the increase in reactive oxygen species was not needed to induce apoptosis nor was it involved in the differential sensitization of myoblasts and myotubes to apoptosis. Moreover, expression of uncoupling protein-3 in myotubes did not affect reactive oxygen species production, although it produced a slight sensitization for staurosporine-induced apoptosis. Results indicate that apoptotic activation in skeletal muscle cells mainly involves reactive oxygen species-independent mechanisms and that mitochondrial uncoupling protein-3 is not protective either for reactive oxygen species production or for apoptotic activation in muscle cells.     

线粒体代谢与干细胞的命运决定

线粒体在真核细胞多种生物学过程中扮演重要角色,如能量产生、钙平衡、细胞内物质代谢、活性氧产生、细胞信号传导和凋亡等。线粒体的高度动态性,如生物发生、动态融合、分裂和退化等代谢特征与细胞种类、组织的需求密切相关。干细胞是一类具有自我更新和多向分化潜能的细胞。目前研究表明,线粒体的代谢与干细胞发育、命运决定紧密相关。本文综述干细胞干性维持及定向分化过程中,线粒体代谢改变与线粒体形态、结构和功能变化。     

Modelling the role of the Hsp70/Hsp90 system in the maintenance of protein homeostasis

Neurodegeneration is an age-related disorder which is characterised by the accumulation of aggregated protein and neuronal cell death. There are many different neurodegenerative diseases which are classified according to the specific proteins involved and the regions of the brain which are affected. Despite individual differences, there are common mechanisms at the sub-cellular level leading to loss of protein homeostasis. The two central systems in protein homeostasis are the chaperone system, which promotes correct protein folding, and the cellular proteolytic system, which degrades misfolded or damaged proteins. Since these systems and their interactions are very complex, we use mathematical modelling to aid understanding of the processes involved. The model developed in this study focuses on the role of Hsp70 (IPR00103) and Hsp90 (IPR001404) chaperones in preventing both protein aggregation and cell death. Simulations were performed under three different conditions: no stress; transient stress due to an increase in reactive oxygen species; and high stress due to sustained increases in reactive oxygen species. The model predicts that protein homeostasis can be maintained during short periods of stress. However, under long periods of stress, the chaperone system becomes overwhelmed and the probability of cell death pathways being activated increases. Simulations were also run in which cell death mediated by the JNK (P45983) and p38 (Q16539) pathways was inhibited. The model predicts that inhibiting either or both of these pathways may delay cell death but does not stop the aggregation process and that eventually cells die due to aggregated protein inhibiting proteasomal function. This problem can be overcome if the sequestration of aggregated protein into inclusion bodies is enhanced. This model predicts responses to reactive oxygen species-mediated stress that are consistent with currently available experimental data. The model can be used to assess specific interventions to reduce cell death due to impaired protein homeostasis.     

Underlying Mechanism of Quercetin-induced Cell Death in Human Glioma Cells

There has been considerable interest in recent years in the anti-tumor activities of flavonoids. Quercetin, a ubiquitous bioactive flavonoid, can inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the precise molecular mechanism by which quercetin induces apoptosis in cancer cells is poorly understood. The present study was undertaken to examine the effect of quercetin on cell viability and to determine its underlying mechanism in human glioma cells. Quercetin resulted in loss of cell viability in a dose- and time-dependent manner and the decrease in cell viability was mainly attributed to cell death. Quercetin did not increase reactive oxygen species (ROS) generation and the quercetin-induced cell death was also not affected by antioxidants, suggesting that ROS generation is not involved in loss of cell viability. Western blot analysis showed that quercetin treatment caused rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Transient transfection with constitutively active forms of MEK and Akt protected against the quercetin-induced loss of cell viability. Quercetin-induced depolarization of mitochondrial membrane potential. Caspase activity was stimulated by quercetin and caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin resulted in a decrease in expression of survivin, antiapoptotic proteins. Taken together, these findings suggest that quercetin results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of ERK, Akt, and survivin.     

Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) pump and consequent loss in the ER-Ca(2+) homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells. Downstream of the ER-Ca(2+) emptying, both caspase-dependent and -independent pathways are activated to ensure cell demise. The induction of apoptosis as a cell death modality is dependent on the availability of proapopototic Bax and Bak proteins, which are essential effectors of the mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase activation. In Bax(-/-)/Bak(-/-) cells a nonapoptotic pathway dependent on sustained autophagy commits the oxidatively damaged cells to death. These results argue that the decision to die in this paradigm of oxidative stress is taken upstream of Bax-dependent MOMP and that the irreversible photodamage to the ER induced by hypericin-PDT acts as a trigger for an autophagic cell death pathway in apoptosis-deficient cells.     

High-Dose Nicotinamide Suppresses ROS Generation and Augments Population Expansion during CD8+ T Cell Activation

During T cell activation, mitochondrial content increases to meet the high energy demand of rapid cell proliferation. With this increase, the level of reactive oxygen species (ROS) also increases and causes the rapid apoptotic death of activated cells, thereby facilitating T cell homeostasis. Nicotinamide (NAM) has previously been shown to enhance mitochondria quality and extend the replicative life span of human fibroblasts. In this study, we examined the effect of NAM on CD8⁺ T cell activation. NAM treatment attenuated the increase of mitochondrial content and ROS in T cells activated by CD3/CD28 antibodies. This was accompanied by an accelerated and higher-level clonal expansion resulting from attenuated apoptotic death but not increased division of the activated cells. Attenuation of ROS-triggered pro-apoptotic events and upregulation of Bcl-2 expression appeared to be involved. Although cells activated in the presence of NAM exhibited compromised cytokine gene expression, our results suggest a means to augment the size of T cell expansion during activation without consuming their limited replicative potential.     

ZIP1 and Zinc Inhibits Fluoride-Induced Apoptosis in MC3T3-E1 Cells

Excess fluoride intake could induce apoptosis in the cells. As an essential micronutrient and cytoprotectant, zinc is involved in many types of apoptosis. Here, we studied the effects of zinc and ZIP1 on fluoride-induced apoptosis in mouse MC3T3-E1 cells and examined the underlying molecular mechanisms. Our study found that fluoride not only inhibited cell proliferation and increased the intracellular reactive oxygen species (ROS) but also induced cell apoptosis. Whereas pretreatment with zinc significantly attenuated fluoride-induced ROS production and partly protected cells against fluoride-induced apoptosis through MAPK/ERK signaling pathway. Our study also found that fluoride upregulated the expression of ZIP1 in a time-dependent manner. Moreover, overexpression of ZIP1 also inhibited fluoride-induced apoptosis by activation of PI3K/Akt pathway. This cytoprotective effect of zinc and ZIP1 may be new factors that affect the physiological activity of fluoride and need study further.     

Rac1 drives intestinal stem cell proliferation and regeneration

Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology.     

Differentiation and cell death: lessons from the immune system

The immune system provides a unique vantage point from which to view the interrelationship between differentiation and cell death, as apoptosis is a prominent feature of B- and T-lymphocyte development. Two common themes emerge from recent experimental observations. First, survival signals are crucial during the differentiation process. The upregulation of Bcl-2 during positive selection suggests that this molecule serves as a survival signal to maintain lymphoid homeostasis. Second, if cell death is repressed, cellular differentiation can occur in the absence of inductive signals.     

Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state

gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.