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Reactive Oxygen Species Signaling from the Perspective of the Stem Cell

Saghi Ghaffari1,2,3,4 and Raymond Liang1

1Department of Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

2Division of Hematology, Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

3Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

4Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

CHAPTER MENU

1. 1.1 Introduction
2. 1.2 ROS Regulation
3. 1.3 ROS Signaling
4. 1.4 ROS and Stem Cells
5. 1.4.1 Adult Stem Cells
6. 1.4.2 Embryonic Stem Cells
7. 1.5 ROS, Metabolism, and Epigenetic Influence
8. 1.6 Stem Cells and Mitochondria
9. 1.7 ROS and Stem Cell Aging
10. 1.8 Concluding Remarks
11. References

1.1 Introduction

Stem cells maintain tissue integrity and homeostasis by regenerating damaged or lost cells throughout life. Impaired stem cell function may promote defective response to stress, aging, and cancer. Work in the past decade has uncovered the critical role that redox signaling plays in the biology of stem cells. A major part of this work has taken place in blood-forming (hematopoietic) stem cells (HSCs) that are broadly used as a model system for adult stem cells. This chapter overviews the investigations of redox regulation of stem cells in the past decade.

1.2 ROS Regulation

ROS are generated from the reduction of molecular oxygen by one electron. ROS species are composed mainly of superoxide anions (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-). The superoxide anion is highly reactive and is rapidly reduced to H2O2 by the antioxidant enzyme superoxide dismutase (SOD) [1]. H2O2 can be further reduced to H2O and O2 by cellular antioxidants. ROS react adversely with and damage DNA, lipids, and proteins, the cumulative effects of which may cause cellular alterations or death. Overall ROS-mediated damage to macromolecules is thought to contribute to the physiological effects of aging [2]. ROS are also considered to be essential components in multiple biological processes as second messengers intimately implicated in the physiological regulation of signaling pathways [3]. Alterations of ROS generation versus scavenging, that is creating the redox milieu, may lead to disease as a result of either too much direct ROS damage (e.g., DNA mutations) or perhaps by impaired function of physiologically relevant ROS-dependent signaling pathways (e.g., myeloproliferative disorder; see succeeding text).

The main source of ROS in the cell is mitochondrial respiration. The generation of proton motive force by the electron transport chain-which leads to ATP production through ATP synthase in a process known as oxidative phosphorylation-is responsible for mitochondrial respiration. However, a small fraction, approximately 0.1-0.2% of O2, consumed by mitochondria form ROS through the premature electron flow to O2 mainly through complexes I and III [4]. The cell type, the environment, and ultimately the activity of mitochondria can influence greatly the precise proportion of ROS generated from mitochondrial respiration [5]. Thus, modulations of mitochondrial activity as well as metabolism in general regulate ROS levels; for instance, reduced ROS levels are achieved by decreasing the rate of mitochondrial respiration via minimizing oxidative phosphorylation. Furthermore, processes that regenerate oxidized glutathione, such as the pentose phosphate pathway, repress ROS levels. Another major source of ROS, in addition to mitochondria, is the membrane-bound protein NADPH oxidase (NOX), which consumes NADPH to generate O2 and subsequently H2O2. NOX generation of ROS has antimicrobial effects in host defense. In addition, NOX are also important for producing ROS in non-phagocytic cells to influence cellular signaling including growth factor (GF) signaling [6]. This includes increased NOX4-mediated ROS production in stem cells [7]. Notably differentiation of mesenchymal stem cells (MSCs) toward adipocytes or neuron-like cells has also been shown to employ NOX4-mediated H2O2 signaling as well as mitochondrial ROS [8, 9]. Elevated ROS in MSCs on the other hand reduces their engraftment potential and induces apoptosis after transplantation [7, 10].

Under normal physiological conditions, the generation of ROS is tightly regulated by the ROS scavenging system. ROS scavengers are antioxidant enzymes that can neutralize ROS by directly reacting with and accepting electrons from ROS. When ROS production outpaces ROS scavenging, an excessive accumulation of ROS occurs, leading to oxidative stress and adverse effects on multiple cellular components including proteins, lipids, and nucleotides. To counteract this, the cell contains multiple types of antioxidants specific to different species of ROS, which helps to prevent pathological levels of ROS and to repair oxidative damage to cellular components. These include SOD, catalase, peroxiredoxins (PRX), thioredoxin (TRX), glutathione peroxidase (GPX), and glutathione reductase (GR). Glutathione, a tripeptide, is one of the most abundant antioxidants synthesized by the cell. Oxidized proteins and H2O2 are reduced by glutathione through the glutaredoxin and TRX system. Other key antioxidants include SOD and catalase, which reduce O2- and H2O2, respectively. The subcellular localization of antioxidants at areas of high ROS generation, such as within the mitochondria, may further enhance the efficiency of ROS scavenging.

1.3 ROS Signaling

Despite their deleterious properties, cumulating evidence in the past three decades has established ROS as pivotal signals in cell fate regulation [11, 12]. There is little doubt that oxygen radicals serve as signaling messengers that variably influence cellular behavior [13, 14]. ROS reaction with proteins such as transcription factors, kinases, and phosphatases alters processes that regulate cell cycle, apoptosis, quiescence, or differentiation [15-17]. GF and oncogenic signaling [18-23] are some examples of ROS signaling. ROS also influence transcriptional activity and likely epigenetics [24-26]. The main ROS species involved in intracellular signaling are Hydrogen peroxide (H2O2) mostly due to their relatively longer half-life and ability to easily diffuse through membranes relative to other types of ROS [27]. H2O2 is also among ROS species with substrate specificity that generates reversible oxidation that is likely to trigger signaling cascade in in vivo physiological settings [12].

ROS signal via direct modification of proteins by amino acid oxidation, the most common of which is oxidation of cysteine residues [28]. ROS signaling to amino acids can cause functional changes in a range of proteins. Proteins directly modified by ROS-known as redox sensors-undergo a conformational change as a result of oxidative modification that influences their function, stability, subcellular localization, interactions with other proteins, and other critical processes. A major example is provided by ROS modulation of protein tyrosine phosphatases (PTP) [1]. It has been shown recently that ROS-mediated inhibition of PTP1B (encoded by PTPN1) in oncogenic-induced senescent cells results in the upregulation of cell cycle inhibitor p21CIP, cell cycle arrest, and senescence by a mechanism involving miRNAs. These studies showed that argonaute that regulates miRNA loading is a target of PTP1B whose repression results in tyrosine phosphorylation of argonaute and reduced loading of miRNAs targeting p21CIP leading to cell cycle arrest and senescence [29]. These studies illustrate the extent of ROS signaling impact and further reiterate the function of ROS as rheostat in cell signaling [30]; in addition by establishing a link between ROS, inhibition of phosphatases, and regulation of miRNAs, these studies expand the scope of ROS-mediated modulations of signaling pathways.

ROS regulation of protein function is complicated by many feedback loops. While ROS can modify protein function, a growing network of proteins modulates ROS levels. These include PTEN and sirtuins (SIRTs) (specifically SIRT1 and SIRT3), ataxia telangiectasia mutated (ATM), p38 mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (mTOR), and protein kinase B (AKT) protein kinases as well as the multifunctional apurinic/apyrimidinic (AP) endonuclease1/redox factor-1 (APE/Ref-1) protein. Transcription factors such as nuclear factor kappa B (NF?B) mediate ROS transactivation of the hypoxia-inducible factor 1 alpha (HIF-1a) [31]; Forkhead box O (FOXO) family; nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2; PR domain containing 16 (PRDM16); and p53 tumor suppressor [32-37]. Among these, many proteins considered as redox sensors that also modulate ROS levels have key functions in the regulation of stem cell fate (reviewed in [13, 38]) (Figure 1.1). For instance, changes of ROS and p53 activity by thioredoxin-interacting protein (TXNIP) may be implicated in hematopoietic stem cell (HSC) function specifically with age [39]. The polycomb group member BMI1 also regulates stem cell function, modulates ROS levels, and is implicated in regulating...