Superoxide and Derived Reactive Oxygen Species in the Regulation of Hypoxia‐Inducible Factors
Introduction
An adequate supply of oxygen is mandatory for the function of diverse processes within all aerobic organisms. Thereby, O2 can often be transformed into highly reactive derivatives, ROS. In the eucaryotic cell, ROS can be generated through multiple sources, including the electron transport chain in mitochondria, ionizing radiation, and enzymes producing superoxide anion radicals. Superoxide anion radical formation is often the initial step in ROS generation. Given that superoxide anion radicals and ROS are cytotoxic, cells have developed antioxidant mechanisms, which include enzymes that dismutate O2– into H2O2 (superoxide dismutases) or degrade H2O2 (catalase, glutathione peroxidases, and peroxiredoxins). When cellular production of ROS overwhelms its antioxidant capacity, a state of oxidative stress is reached, leading to serious cellular injuries contributing to the pathogenesis of a number of diseases. Nevertheless, when generated in lower concentrations, ROS can act as second messengers in signal transduction and gene regulation in a variety of cell types and under several biological conditions.
Due to the importance of oxygen for cell metabolism, elaborate mechanisms have been evolved to allow adaptation when oxygen availability drops. These mechanisms include responses ensuring energy and oxygen supply to compensate the drop in O2 tension. Although the identity of a cellular oxygen sensor remains elusive, a family of transcription factors, HIFs, has been reported to primarily mediate the cellular response to hypoxia.
Interestingly, a number of factors, including growth, coagulation, hormones, and inflammatory cytokines, as well as cellular stress factors (e.g., physical and chemical stress), have been shown to activate HIF transcription factors by using ROS as signaling molecules. In the following sections, we will therefore summarize the role of ROS in the regulation of HIF.
Section snippets
Reactive Oxygen Species Act as Signaling Molecules
Transfer of one electron to O2 results in the production of superoxide anion radicals (O2−•), which often are the precursors for formation of other reactive species, such as hydrogen peroxide (H2O2), hydroxyl radicals (OH•), peroxynitrite (ONOO‐), hypochlorous acid (HOCl), and singlet oxygen (1O2). In mammalian cells, ROS are formed in response to toxic reagents or as (by‐) products of enzyme reactions. One interesting enzyme that actively generates O2−• is a multi‐protein complex known as
HIFs are Sensitive to Oxygen
The first identified HIF transcription factor was HIF‐1 (Beck 1993, Semenza 1992, Wang 1995, Wang 1995a, Wang 1995b, Wang 1995c); however, now this group represents a small family with the additional members HIF‐2 and HIF‐3. All of these factors are heterodimers consisting of an α‐subunit and a β‐subunit, which both are basic helix‐loop‐helix and Per‐ARNT‐Sim (bHLH‐PAS) domain–consisting proteins. While the α‐subunits were found to be new proteins named HIF‐1α, HIF‐2α and HIF‐3α, the β‐subunits
Reactive Oxygen Species Modulate HIF
In addition to hypoxia, HIF‐1α is also responsive to a variety of non‐hypoxic stimuli, such as insulin (Kietzmann 2003b, Treins 2002, Zelzer 1998), platelet‐derived growth factor (PDGF), transforming growth factor (TGF)‐β, insulin‐like growth factor (IGF)‐1 (Fukuda 2002, Görlach 2001, Richard 2000), epidermal growth factor (EGF) (Liu et al., 2006), thrombin (Görlach et al., 2001), angiotensin‐II (Richard et al., 2000), cytokines (Stiehl et al., 2002), carbachol (Hirota et al., 2004), chromium
How are HIFs Regulated by Reactive Oxygen Species?
Reactive oxygen species may influence HIFs at different levels: they may influence mechanisms regulating HIF‐α synthesis or HIF‐α stability; they may act directly on HIF‐α, thereby modifying stability or activity; or they may act via interference with a regulatory signaling pathway farther upstream.
Summary
Together, a vast body of results has shown that ROS derived from superoxide anion radicals has a profound effect on the HIF system, which appears to react sensitively toward changes in the cellular redox state. Thereby, a threshold concentration of ROS may drive the response in dependence of the cell type and/or stimulus. Thus, only slight changes in the redox state may be required to activate the HIF pathway.
Methods
Since changes in ROS levels appear to be critical for HIF induction, the determination of ROS levels under various conditions is essential for understanding the involvement of ROS in the regulation of HIF. ROS can interfere with a variety of substances, thus requiring controlled conditions when measuring ROS levels in cells. In the following sections, we summarize some techniques that appear to be suitable for ROS measurements alone or in combination.
The Cytochrome C Reduction Assay for Detection of Extracellular Reactive Oxygen Species
Cytochrome C is a heme protein and part of the mitochondrial electron transport chain. The ferricytochrome C can be directly reduced by superoxide to ferrocytochrome C:
This reduction leads to an increase in the absorbance spectrum of cytochrome C at 550 nm, which can be spectrophotometrically monitored. Since the reduction of cytochrome C is proportional to the amount of the reducing agent, it is possible to quantify the ROS production (Johnston 1978, Murrant 2001).
Chemiluminescence Assay for Detection of Extracellular Reactive Oxygen Species
Chemiluminescence methods for superoxide detection have been frequently employed because of the alleged specificity of the reaction of the chemiluminescent probe with superoxide, minimal cellular toxicity, and relatively high sensitivity. Frequently used probes are luminol, lucigenin, or the chemiluminescence Cypridina luciferin derivative, 2‐methyl‐6‐(4‐methoxyphenyl)‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one, hydrochloride (MCLA, Invitrogen, Carlsbad, CA).
MCLA is used to monitor superoxide
Measuring Intracellular Production of Reactive Oxygen Species using Fluorescent Dyes
Intracellularly formed ROS can be determined using fluorescent dyes. These dyes, including dihydroethidine (DHE), dihydroflorescein (H2DCF), and dihydrorhodomine 123 (DHR), are initially nonfluorescent; however, they become fluorescent upon reaction with ROS to give hydroethidium (HE), dichlorofluorescin (DCF), and rhodamine 123, respectively. Subsequently, fluorescence intensity can be easily determined using a fluorometer or fluorescence reader.
Detection of Reactive Oxygen Species by Electron Paramagnetic Resonance
Electron paramagnetic resonance (EPR) is a spectroscopic technique that detects paramagnetic species that have unpaired electrons, including free radicals, many transition metal ions, and defects in materials. It is also often called electron spin resonance (ESR).
Electron paramagnetic resonance is a magnetic resonance technique used to detect the transitions of unpaired electrons in an applied magnetic field. Like a proton, the electron has “spin,” which gives it a magnetic property known as a
Acknowledgments
This work was supported by DFG GO709/4–4, the 6th European framework program (EUROXY), and Fondation Leducq to A. G. and by DFG SFB 402 A1, GRK 335, Deutsche Krebshilfe (106429), Fonds der Chemischen Industrie, and Fondation Leducq to T. K.
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