Signal transduction by reactive oxygen species in
National Institutes of Health, Bethesda, Maryland
Abstract: A growing body of evidence suggests a derived growth factor (PDGF) stimulation of vascular smooth
potential role for oxygen-derived radicals such as muscle cells (SMCs). Under these conditions, ROS production
superoxide anions and hydrogen peroxide as intra- is a rapid and transient phenomena. For the case of PDGF and
cellular signaling molecules. Recently, progress has SMCs, ROS production peaks within the first few minutes after
been made regarding the regulation of oxidant growth factor addition and returns to baseline within approxi-
production in non-phagocytic cells. Significant gaps mately 30 min (Fig. 1C). Inhibiting the growth factor-stimulated
in understanding persist, however, especially in burst of ROS appears to inhibit downstream signaling and in
regard to the source(s) of oxidant production and particular ligand-stimulated tyrosine phosphorylation [6, 7].
the direct intracellular target(s) of oxygen radicals. Although the exact target of ROS remains unclear, one
Nonetheless, numerous recent studies have impli- attractive candidate is intracellular tyrosine phosphatases. This
cated a dynamic change in the intracellular redox class of molecules has a redox-sensitive cysteine residue in the
state as an important determinant in a host of active site. Previous studies have demonstrated that exog-
cellular decisions ranging from growth, to apopto- enously added hydrogen peroxide could transiently and revers-
sis, to cellular senescence. J. Leukoc. Biol. 65: ibly inactivate phosphate activity . Nonetheless, it is
337–340; 1999. difficult to extrapolate from such studies whether ligand-
stimulated peroxide production is sufficiently robust to inacti-
vate phosphatase activity. Some recent evidence, however,
Key Words: NADPH oxidase · ras · rac · p53 · apoptosis
suggests that stimulation of the A431 epidermoid carcinoma
cell line with epidermal growth factor produces a burst of H2O2
sufficiently large to transiently inactivate phosphatase 1B .
Stimulation of a variety of non-phagocytic cells with either This suggests that one function of growth factor-stimulated ROS
peptide growth factors or cytokines has been demonstrated to production may be to reversibly regulate tyrosine phosphatases.
result in a burst of intracellular oxidant production [1–7]. This In a more general sense, oxidants might be involved in the
mini-oxidative burst shares some common characteristics with regulation of a variety of intracellular enzymatic pathways. As
the more widely studied oxidative burst of neutrophils. Nonethe- diagrammed in Figure 2, alterations in the redox state could,
less, in contrast to phagocytic cells, the oxidant production in in turn, alter enzymatic function in a number of discrete ways.
non-phagocytic cells appears to intersect with a host of Perhaps the most characterized is the oxidation of a reactive
intracellular pathways involved in growth, cell division, trans- cysteine molecule leading to protein dimerization. A number of
formation, apoptosis, and senescence. In the following brief receptors and intracellular enzymes appear to require dimeriza-
review, I will provide a selective outline of some of the progress tion for activity and it seems reasonable that the local redox
made in this emerging area. More complete reviews, or ones that state might regulate this process to some degree. Similarly,
cover other aspects in this field, are readily available [8–10]. although antioxidant proteins are generally thought of as
Intracellular oxidant production in living cells can be free-floating scavengers, recent evidence suggests that in
achieved by a variety of different methods. We have primarily certain cases they form protein complexes with intracellular
relied on the use of a fluorescent dye, 2’-7’-dihydrodichlorofluo- signaling molecules. In particular, studies suggest that Cu,Zn-
rescin diacetate (DCF). This compound rapidly diffuses into superoxide dismutase binds calcineurin  and thioredoxin
cells whereupon cellular esterases cleave the acetate moiety binds the signaling kinase ASK1 . In both cases, changes in
allowing for the accumulation of the now membrane-imper- the redox state regulate protein-protein interaction and enzy-
meant form. Interaction with intracellular hydrogen peroxide
produces a fluorescent moiety that can be conveniently imaged
by confocal microscopy. Such methods allow for a qualitative
Abbreviations: ROS, reactive oxygen species; DCF, dihydrodichlorofluores-
measurement of intracellular reactive oxygen species (ROS). cin diacetate; PDGF, platelet-derived growth factor; SMC, smooth muscle cells;
Nonetheless, it is currently impossible to convert levels of DCF EGF, epidermal growth factor; TNF- , tumor necrosis factor ; IL-1 ,
fluorescence to actual intracellular concentrations of ROS. interleukin-1 ; NAC, N-acetylcysteine.
Using such methodologies, it can be demonstrated that Correspondence: Toren Finkel, National Institutes of Health, Building 10,
Room 7B15, 10 Center Drive, Bethesda, MD 20892-1650. E-mail:
peptide growth factors stimulate the rapid production of email@example.com
hydrogen peroxide. As an example, Figure 1 demonstrates the Received September 8, 1998; revised November 12, 1998; accepted
level of DCF fluorescence before and 5 min after platelet- November 13, 1998.
Journal of Leukocyte Biology Volume 65, March 1999 337
Fig. 1. Ligands stimulate ROS production in non-phagocytic cells. Vascular smooth muscle cells were incubated with the peroxide-sensitive fluorophore DCF and
imaged by confocal microscopy. Levels of DCF fluorescence observed in (A) basal conditions or (B) 5 min after PDGF stimulation. (C) Time course of DCF
fluorescence after PDGF stimulation (Reprinted with permission from Sundaresan et al., Science (1995), 270:296-299.)
matic activity. Finally, a less characterized covalent modifica- Similarly, expression of an activated ras gene also increases
tion is glutathiolation, in which a glutathione residue is added to a the level of intracellular ROS [17, 18], although this effect
reactive cysteine. Such modifications have recently been described appears in NIH 3T3 cells to be predominantly through the
to cause reversible enzymatic regulation . In all three cases, ROS subsequent downstream activation of rac1. Spin trapping
could function as modulators of specific signaling pathways. experiments have demonstrated that the small GTPases pro-
The activity of the neutrophil NADPH-oxidase is regulated duce an increase in superoxide anions (O2 ), which is then
in part by the rac family of GTPases . A variety of emerging dismutated spontaneously or enzymatically to the more stable
evidence suggests that a similar role for rac proteins exists in H2O2 . Expression in NIH 3T3 cells of a dominant negative
non-phagocytic cells. Indeed, expression of constitutively ac- form of rac1 has been shown to block ROS production
tive mutants of rac1 in cells results in an increase in ROS levels as stimulated by a variety of ligands including PDGF, epidermal
measured by a variety of techniques including DCF fluorescence, growth factor (EGF), tumor necrosis factor (TNF- ), and
electron spin resonance, and enzymatic methods [17–20]. interleukin-1 (IL-1 ) . Taken together these results
suggest a critical role for the small GTPase in the regulation of
the intracellular redox state of non-phagocytic cells.
Although it has been appreciated for some time that the small
GTPases play an essential role in the regulation of cell growth
and transformation, emerging evidence suggests that growth
control by the small GTPases may be linked to their superoxide-
generating ability. NIH 3T3 cells transformed by ras but not by
c-Raf were demonstrated to produce an increase in superoxide
anions . This rise in ROS in turn appeared to proceed
through a rac1-dependent pathway. It is interesting to note that
S-phase progression of ras-transformed NIH 3T3 cells but not
Raf-transformed cells appeared to be sensitive to the cell-
permeant antioxidant N-acetylcysteine (NAC). Using effector
mutants of rac1, it has recently been demonstrated that the
ability of the small GTPase to generate superoxide co-
segregates with growth stimulation but is distinct from activa-
tion of the c-Jun amino-terminal kinase (JNK) or reorganization
of cytoskeleton . Furthermore, mutations in the unique
carboxyl-terminal insert region of rac1 also abrogate O2
production and growth stimulation. It is interesting to note that
this same region of rac has been shown to be required for the
activation of the neutrophil NADPH oxidase by rac proteins
[21, 22]. This suggests that the rac1-regulated oxidase in
non-phagocytic cells may be similar to the neutrophil superox-
ide generating system. It is also interesting that other compo-
nents of the NADPH oxidase are found in a variety of cell types
Fig. 2. Potential mechanism by which changes in ROS may alter known and appear to play a role in oxidative signaling .
intracellular signaling pathways. Included among the possibilities is redox
regulation of protein dimerization, regulation of protein-protein interaction, and
One pathway that has been consistently observed to be under
glutathiolation in which glutathione (GSH) is covalently added to a reactive redox regulation is the activation of NF- B. Indeed, although
cysteine residue . See text for details. NF- B is activated by a number of diverse stimuli, many of
338 Journal of Leukocyte Biology Volume 65, March 1999 http://www.jleukbio.org
these inducers stimulate ROS production and indeed NF- B a variety of evidence has implicated ROS in determining the
activity is consistently blocked or attenuated in the presence of Haflick limit, i.e. the point beyond which cells in culture
antioxidants [24, 25]. The observation that the small GTPase remain viable but fail to divide. Previous studies have demon-
rac1 regulate ROS production led us to hypothesize that the strated that culturing primary cells in low oxygen, a condition in
redox-dependent activation of NF- B could be accounted for, which ROS levels are inhibited, appears to prolong in vitro
in part, by the stimulation of a rac-dependent pathway. Such a life-span . Similarly, treatment of cells with non-lethal
hypothesis has been subsequently verified by us  and others concentrations of exogenous H2O2 mimics many aspects of the
. Unfortunately, it remains unclear what are the direct molecular senescent state . It is interesting to note that expression of
targets of rac1-stimulated ROS production, which in turn lead to an activated ras gene in primary cells has been recently
NF- B activation. Further characterization of the effects of ROS on demonstrated to produce a senescent phenotype . Our
the activity of downstream kinases such as MEKK or the more preliminary evidence suggests that ras-induced senescence is
recently described I B kinases may be informative. secondary to a change in the intracellular level of reactive
Although ROS appear to play a role in growth factor oxygen species. Our initial results suggest that the source of
signaling, they may also serve as effector molecules in apopto- ras-induced ROS in primary cells in the mitochondria and as
sis. It should be noted that, although a number of studies have such may differ from the ras-regulated pathway involved in cell
observed a rise in ROS levels preceding apoptosis, it remains growth and transformation.
controversial whether ROS are required or are irrelevant to cell In summary, the regulation of the intracellular redox state
death . We have noted that p53 overexpression resulted in appears to be an active process that directly or indirectly
the apoptotic death of human SMCs . It is interesting that in influences a host of cellular pathways. A number of studies
these cells p53 expression resulted in a rise in intracellular have implicated the small GTPases ras and rac1 in the
DCF fluorescence that preceded cell death. Treatment of cells regulation of ROS production in non-phagocytic cells. Although
with antioxidants inhibited p53-dependent apoptosis. A similar such studies suggest that there may be some conservation
result was obtained by Vogelstein and colleagues using colonic between ROS generation in phagocytic and non-phagocytic
epithelial cells . Analysis of the genes strongly induced by cells, the large number of redox-dependent pathways suggest
p53 in these epithelial cells demonstrated that a number of that there may be multiple sources of oxidants and multiple
points of regulation. Further characterization of the oxidant
p53-dependent gene products were involved in redox regula-
source and the protein targets of intracellular ROS should
tion. This led the authors to conclude that p53 may induce
greatly aid in the understanding of the emerging area.
apoptosis through its ability to regulate transcription of a set of
gene products that function to alter the intracellular level of
ROS. Very recently, it has been demonstrated the expression of
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340 Journal of Leukocyte Biology Volume 65, March 1999 http://www.jleukbio.org