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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2928-2935
Hematopoietic Growth Factors Signal Through the Formation of
Reactive Oxygen Species
By
Martin Sattler,
Thomas Winkler,
Shalini Verma,
Christopher H. Byrne,
Gautam Shrikhande,
Ravi Salgia, and
James D. Griffin
From the Department of Adult Oncology, Dana-Farber Cancer Institute,
Harvard Medical School, Boston, MA.
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ABSTRACT |
Hematopoietic growth factors (HGFs) stimulate growth,
differentiation, and prevent apoptosis of progenitor cells. Each growth factor has a specific cell surface receptor, which activates both unique and shared signal transduction pathways. We found that several
HGFs, including granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin-3 (IL-3), steel factor (SF), and thrombopoietin
(TPO) induce a rapid increase in reactive oxygen species (ROS) in
quiescent cells. In an effort to understand the potential biochemical
and biological consequences of increased ROS in these cells, we exposed
growth factor-deprived cells to hydrogen peroxide
(H2O2) at concentrations that increased
intracellular ROS. H2O2 induced a
dose-dependent increase in tyrosine phosphorylation, including
increased tyrosine phosphorylation of the GM-CSF receptor beta chain
( c), STAT5, and other signaling proteins.
H2O2 also induced expression of the early
response gene c-FOS, and G1- to S-phase transition, but not S- to
G2/M-phase transition of MO7e cells. The cell permeable antioxidant
pyrrolidine dithiocarbamate (PDTC) decreased the intracellular levels
of ROS and inhibited tyrosine phosphorylation induced by GM-CSF in MO7e
cells, suggesting that ROS generation plays an important role in GM-CSF
signaling. Consistent with this notion, PDTC and two other
antioxidants, N-acetyl cysteine and 2-mercaptoethanol, reduced growth
and viability of MO7e cells. These results suggest that generation of
ROS in response to HGFs may contribute to downstream signaling events, especially those involving tyrosine phosphorylation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC GROWTH factors (HGFs) bind
to specific cell surface receptors and rapidly activate cellular
tyrosine kinases or intrinsic receptor tyrosine kinase activity. In
many cases, the receptor itself becomes tyrosine phosphorylated, and phosphorylation sites in the receptor lead to recruitment of SH2 containing proteins that can activate downstream signaling
pathways.1-3 For example, the SH2 domain of SHC can be
recruited to a phosphotyrosine containing sequence in the
erythropoietin receptor followed by binding of GRB2 and SOS to
SHC.4,5 SOS is a nucleotide exchange factor and this
pathway leads to activation of p21RAS.6,7 In addition, many
growth factor receptors share common downstream signaling proteins such
as SHP-2, CBL, STATs, and PI3K.8 Such common pathways are
likely to lead to common biological events such as regulation of
proliferation, viability, or adhesion.
Recently, activation of the platelet-derived growth factor (PDGF)
receptor9 or ultraviolet
(UV)-irradiation10 have been shown to activate
intracellular regulation of redox processes through generation of
reactive oxygen species (ROS) such as H2O2 and
superoxide. It has been suggested in these cases that ROS may act as
second messengers to regulate activities of redox-sensitive enzymes,
including protein kinases and protein phosphatases. Of particular
interest is the fact that several protein tyrosine phosphatases are
highly sensitive to oxidation because of a critical thiol group in the
active site of the enzyme.11
In this study, we have investigated the potential role of ROS in signal
transduction of several hematopoietic growth factors, including
granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), steel factor (SF), and thrombopoietin (TPO), using the growth factor-dependent cell lines MO7e, TF1, and 32Dcl3. In
each cell line, growth factor stimulation increased the intracellular level of ROS as measured by 2', 7'-dichloro-fluorescein fluorescence. ROS levels were rapidly increased and sustained, suggesting that this
increase of ROS could, in part, be a signal due to multiple mechanisms,
including a direct response to signal transduction, as well as a
consequence of cell metabolism. To determine the potential significance
of increased ROS, more detailed signaling studies were performed in
MO7e cells to compare GM-CSF signaling with that induced by the ROS
H2O2. We found that
H2O2, like GM-CSF, induces tyrosine
phosphorylation of cellular proteins, c-FOS gene expression, G1 to S
phase transition, and cell migration.
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MATERIALS AND METHODS |
Cells.
The human megakaryocytic cell line, MO7e, was grown in Dulbecco's
modified Eagle's medium (DMEM) with 20% (vol/vol) fetal calf serum
(FCS) and 10 ng/mL GM-CSF (Immunex, Seattle, WA). The human
erythrocytic cell line, TF1, was grown in RPMI 1640 with 10% (vol/vol)
FCS and 10 ng/mL GM-CSF. The murine myeloid cell line, 32Dcl3, was
grown in RPMI 1640 with 10% (vol/vol) FCS and 10% (vol/vol) WEHI
conditioned medium (as a source of murine IL-3). MO7e cells were
deprived of growth factors for 18 hours in DMEM medium containing 1%
(wt/vol) bovine serum albumin (BSA) or TF1, and 32Dcl3 cells were
starved for the same period in RPMI 1640 medium containing 0.5%
(wt/vol) BSA. Cells were stimulated with recombinant human GM-CSF,
recombinant human IL-3 (Genetics Institute, Cambridge, MA), recombinant
human SF (Amgen, Thousand Oaks, CA), or recombinant murine IL-3
(Upstate Biotechnology Inc, Lake Placid, NY). Viability of cells was
determined by trypan blue exclusion or annexin V (Boehringer Mannheim,
Mannheim, Germany) staining.
Analysis of ROS in starved and growth factor-stimulated cells.
A total of 106 cells was incubated with 5 µmol/L DCF-DA
(2', 7'-dichloro-fluorescin-diacetate; Acros Organics, Pittsburgh, PA) for 5 minutes at 37°C and subsequently washed twice in cold Dulbecco's phosphate-buffered saline (PBS) before analysis using a
Coulter Epics XL flow cytometer (Coulter Corp, Miami, FL). DCF-DA is a
cell permeable dye commonly used to monitor intracellular changes in
ROS. This compound becomes fluorescent when oxidized by
either H2O2 or superoxide. The fluorescence of
oxidized DCF was measured with an excitation wavelength of 480 nm
and an emission wavelength of 525 nm.12,13
Stimulation of cells and preparation of cellular lysates.
For immunoprecipitation studies, growth factor-starved MO7e cells were
stimulated at 37°C for 7.5 minutes with GM-CSF (20 ng/mL) and SF
(40 ng/mL) or 20 minutes with H2O2 (5 mmol/L).
In some experiments, cells were pretreated for 3 hours with the
antioxidant pyrrolidine dithiocarbamate (PDTC; Sigma Co, St Louis,
MO) before stimulation with growth factors. Cells were
subsequently washed once in cold Dulbecco's PBS, and cell lysates were
prepared as described.14
Immunoprecipitation and immunoblotting.
Immunoprecipitation and immunoblotting using a chemiluminescence
technique was performed as described.14
Tyrosine-phosphorylated proteins were detected using the monoclonal
antibody 4G10 (kindly provided by Dr B. Druker, Oregon Health Sciences
University, Portland). A mouse monoclonal antibody against the GM-CSF
receptor chain (c; Santa Cruz Biotech, Santa Cruz, CA) and
polyclonal rabbit antisera against STAT5 (Santa Cruz Biotech) were used
for immunoprecipitation.
Northern blotting.
The expression of c-FOS after H2O2 stimulation
in MO7e cells was analyzed by Northern blotting using standard methods.
cDNA probes against c-FOS (431 bp) and G3PDH (glycerinaldehyde
3-phosphate dehydrogenase; 331 bp) were generated by reverse
transcriptase-polymerase chain reaction (RT-PCR). The following
oligonucleotides were used for c-FOS:
5'-AGCTCCCTCCTCCGGTTGCGGCAT-3' (antisense primer) and 5'-CTACGAGGCGTCATCCTCCCG-3' (sense primer) and for G3PDH:
5'-TTCAAGGGGTSTACATGGCAACTG-3' (antisense primer) and
5'-GGGCATCCTGGGCTACACTG-3' (sense primer). The cDNA probes
were labeled using Klenow fragment (High Prime Kit; Boehringer
Mannheim) with 32P-deoxycytidine triphosphate
(dCTP) and purified with ProbeQuant G-50 micro columns
(Pharmacia Biotech, Piscataway, NJ). Total RNA was
isolated with Trizol reagent (Life Technologies, GIBCO-BRL, Gaithersburg, MD) and used to prepare mRNA (Message Maker; Life Technologies, GIBCO-BRL) to evaluate gene expression. Bound probe was
analyzed by phosphorimaging analysis (FLA-2000 Fluorescent Image
Analyzer; Fuji Photo Film Corp, Stamford, CT).
Cell cycle analysis.
Starved MO7e cells were treated at 37°C with GM-CSF, 0.05 mmol/L
H2O2 in water (Sigma, 30% [wt/wt] solution)
or an equal volume water and analyzed after propidium iodide staining
using standard methods. In brief, 0.5 × 106 cells per
sample were washed once in cold Dulbecco's PBS and resuspended in 500 µL staining solution containing 50 µg/mL propidium iodide, 0.1%
(vol/vol) NP-40, and 0.1% (wt/vol) sodium citrate. Cells were
incubated at 4°C in the dark for 15 minutes and then analyzed by
flow cytometry.
Transwell migration assay.
The membranes of transwell chambers (8-µm pore size polycarbonate
membrane, Corning Costar Corp, Cambridge, MA) were coated with 10 µg/mL fibronectin (Life Technologies, GIBCO-BRL) for 18 hours.
Starved cells (0.2 × 106) were transferred to the
upper chamber in DMEM medium containing different stimuli. After 5 hours, cells in the lower compartment were concentrated by
centrifugation and living cells counted by trypan blue exclusion.
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RESULTS |
GM-CSF, IL-3, SF, and TPO alter the intracellular redox status of
hematopoietic cell lines.
Activation of tyrosine kinases, in particular the PDGF receptor, has
been shown to upregulate ROS levels. The relative ROS levels in
hematopoietic cell lines, treated and untreated with GM-CSF, were
measured and compared with other growth factors using the fluorochrome
2', 7'-dichloro-fluorescin-diacetate. The human megakaryocytic cell
line, MO7e (Fig 1A); the human erythrocytic cell line, TF1 (Fig 1B); and the murine myeloid cell line, 32Dcl3 (Fig
1C) were studied. Figure 1A (lower panel) shows that the relative ROS
levels in MO7e cells are increased on GM-CSF, IL-3, TPO, or SF
stimulation compared with growth factor-deprived cells. Similarly, the
relative ROS levels were increased in TF1 cells after GM-CSF or IL-3
stimulation (Fig 1B, lower panel) and in 32Dcl3 cells after IL-3
stimulation (Fig 1C, lower panel). Growth factor-deprived
and stimulated cells had equal levels of autofluorescence as tested by
fluorescence-activated cell sorting (FACS) analysis using no
fluorochrome (Fig 1A through C, upper panel). The kinetics of changes
in relative levels of ROS were analyzed in MO7e cells treated for 0 to
18 hours with GM-CSF. The levels of ROS continously increased within
the first 2 hours until they reached a plateau and increased only
slightly over the next 16 hours (Fig 1D).
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| Fig 1.
GM-CSF, IL-3, SF, and TPO increase the intracellular
level of ROS in hematopoietic cell lines. (A through C) The relative
levels of ROS were measured in starved cells (dotted line) and in
growth factor-treated cells (straight line) as indicated using 2',
7'-dichloro-fluorescin-diacetate (bottom panel) or the
autofluorescence of these cells was measured without fluorochrome (top
panel). (A) MO7e cells were treated with either GM-CSF (10 ng/mL), IL-3
(10 ng/mL), SF (20 ng/mL), or TPO (100 ng/mL) for 18 hours. (B) TF1
cells were treated with either GM-CSF (10 ng/mL) or IL-3 (10 ng/mL) for
18 hours. (C) 32Dcl3 cells were treated with IL-3 (10 ng/mL) for 18 hours. (D) The increase in relative levels of ROS was measured in MO7e
cells stimulated for 0 to 18 hours with 20 ng/mL GM-CSF.
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The antioxidant PDTC decreases ROS levels and viability in
GM-CSF-treated cells.
To determine if the intracellular ROS levels can be manipulated by
adding reducing agents, MO7e cells grown in GM-CSF- or IL-3-containing medium without FCS were treated with the reducing agent PDTC (25 µmol/L) for 3 hours and then analyzed. PDTC was not
cytotoxic under these conditions, and the cells retained full viability
for the duration of the experiment as assessed by trypan blue exclusion
and annexin V staining. PDTC decreased the intracellular levels of ROS
in MO7e cells treated with either growth factor (Fig 2A, bottom panel), but
did not alter the autofluorescence of these cells (Fig 2A, top panel).
Finally, the effects of PDTC on proliferation of 3-day cultures of MO7e
cells in GM-CSF (10 ng/mL) were tested. In contrast to the relatively
high concentration of PDTC that was required for a 3-hour treatment to
suppress GM-CSF- and IL-3-induced increase in ROS, the dose that was
required to suppress cell growth in a 3-day culture was lower. As shown
in Fig 2B, 0.5 µmol/L PDTC completely suppressed growth and
viability of GM-CSF-stimulated MO7e cells. Equivalent results were
obtained with PDTC-treated BaF3 cells (data not shown). Similar to
PDTC, the antioxidants, N-acetyl cysteine and 2-mercaptoethanol,
suppressed growth and viability of GM-CSF-treated MO7e cells. These
effects were dose-dependent and the lowest concentrations that reduced cell growth in a 3-day culture were 10 mmol/L for N-acetyl cysteine and
50 µmol/L for 2-mercaptoethanol. These data suggest that antioxidants block a pathway in GM-CSF-stimulated MO7e cells that is important for
cell growth.
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| Fig 2.
The antioxidant PDTC reduces intracellular levels of ROS
and cell growth in MO7e cells. (A) MO7e cells were stimulated for 18 hours with 10 ng/mL GM-CSF or 10 ng/mL IL-3 before treatment for 3 hours with 25 µmol/L PDTC (dotted line) or left untreated (straight
line) as indicated. The autofluorescence of these cells (top panel) or
the relative levels of ROS using 2', 7'-dichloro-fluorescin-diacetate
(bottom panel) were measured. (B) MO7e cells were treated with the
indicated doses PDTC, NAC, and 2-mercaptoethanol (2-ME) for 72 hours,
and cell growth was measured by trypan blue exclusion. The error bars
indicate the standard error of the mean (SEM) (n = 4).
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Oxidative stress increases tyrosine phosphorylation of cellular
proteins in MO7e cells.
Signaling through HGF receptors correlates with the activation of
tyrosine kinases such as JAK2. Biological effects activated by growth
factors are therefore thought to be mediated through tyrosine
phosphorylation of cellular proteins. We sought to determine the
biochemical consequences of an exogenously given ROS to unstimulated cells and to compare it with GM-CSF-stimulated cells.
Figure 3 (top panel) shows that
H2O2, like GM-CSF, increases the tyrosine phosphorylation of the common c and STAT5 compared with growth factor-deprived MO7e cells. We also found tyrosine phosphorylation of
other signaling proteins after H2O2 stimulation
including c-KIT, the receptor for SF, the adapter protein SHC, and the
protein tyrosine phosphatase SHP-1 (data not shown). These data suggest that a change in the intracellular redox status on growth factor stimulation contributes to the increased tyrosine phosphorylation of
cellular proteins.
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| Fig 3.
The redox status in MO7e cells regulates the tyrosine
phosphorylation of growth factor receptors and STAT5. MO7e cells were
left untreated (CTRL) or treated for 7.5 minutes with either GM-CSF (20 ng/mL), 20 minutes H2O2 (5 mmol/L) (PEROX), or
pretreated with 1 mmol/L PDTC for 3 hours and then stimulated with
GM-CSF (GM-CSF/PDTC) for 7.5 minutes. Tyrosine-phosphorylated proteins
were detected in GM-CSF receptor c and STAT5 immunoprecipitates by
immunoblotting with an antiphosphotyrosine antibody (p-Tyr).
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We excluded the possibility that the increase in tyrosine
phosphorylation induced by H2O2 occurred during
the subsequent lysis of cells. The buffer that was used to prepare
lysates of peroxide-stimulated cells contains vanadate that could
hypothetically react with peroxide to generate pervanadate, a potent
phosphatase inhibitor and known stimulator of cellular tyrosine
phosphorylation. However, omission of vanadate from this buffer did not
reduce H2O2-induced tyrosine phosphorylation
(data not shown).
Because HGF-stimulated cells have increased levels of ROS and increased
levels of ROS are associated with tyrosine phosphorylation, we asked if
reducing agents such as PDTC might decrease tyrosine phosphorylation of
cellular proteins in MO7e cells. Figure 3 (bottom panel) shows that
PDTC reduced tyrosine phosphorylation of cellular proteins previously
shown to be phosphorylated by either H2O2 or
GM-CSF. Pretreatment of MO7e cells with PDTC resulted in reduced tyrosine phosphorylation of the c and STAT 5 after GM-CSF
stimulation. These data suggest that PDTC is effective in reducing
tyrosine phosphorylation of cellular proteins in growth
factor-stimulated cells. It can also be appreciated that the reduction
in phosphotyrosine signal was significant, but incomplete.
H2O2 induces c-FOS expression and cell cycle
progression.
The previous results suggested that treatment of MO7e cells with
H2O2 mimics at least some of the signaling
pathways normally activated by growth factor receptors, such as
receptor phosphorylation. Therefore, we sought to determine if
H2O2 can also activate some of the fundamental
processes that are required for cell growth and viability, such as gene
expression. We have previously shown that c-FOS is upregulated in MO7e
cells after GM-CSF stimulation. MO7e cells were treated for 30 minutes,
60 minutes, or 3 hours with H2O2, GM-CSF, or
left untreated and then expression of c-FOS was analyzed by Northern
blotting (Fig 4A). c-FOS was induced by both GM-CSF and
H2O2 after 30 minutes of stimulation and
thereafter decreased and became undetectable after 3 hours of
stimulation. The membrane was stripped and reprobed with a G3PDH probe,
demonstrating equal loading of mRNA.
Because H2O2 induced early gene expression, we
sought to determine whether this oxidant could also induce cell cycle
progression. Starved MO7e cells were either treated with GM-CSF,
H2O2, or control medium, and the DNA content
was analyzed for 0 to 33 hours by propidium iodide staining
(Fig 4B). Due to the short half-life of
dilute H2O2, the stimulation was repeated by
resuspending the cells in fresh medium every 3 hours for the first 12 hours of stimulation. Growth factor-deprived cells remained in
G1-phase for the 33 hours of this study. Both GM-CSF and
H2O2 induced a fraction of cells to enter
S-phase after 24 hours (44% and 15%, respectively), but further cell
cycle progression to G2/M-phase was only observed in GM-CSF-stimulated
cells. This suggests that H2O2 can induce
G1-arrested cells to enter S-phase, but stimulation with the oxidant
alone is insufficient to bring cells into G2/M-phase and, thus, induce
proliferation.
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| Fig 4.
H2O2 mimics GM-CSF-induced G1-S
phase transition, early gene expresion, and random transwell migration
in MO7e cells. (A) Gene expression was analyzed using mRNA from MO7e
cells left untreated (CTRL) or stimulated for the indicated times with
GM-CSF (20 ng/mL) or H2O2 (PEROX, 250 µmol/L). The expression of c-FOS and G3PDH was detected by Northern
blotting using specific probes. (B) MO7e cells were either left
untreated or treated with GM-CSF (20 ng/mL) or
H2O2 (PEROX, 50 µmol/L). Cell cycle analysis
was performed using propidium iodide at the indicated time points and
the samples were analyzed by flow cytometry. (C) MO7e cells were either
left untreated or treated with GM-CSF, SF, or
H2O2 (PEROX) as indicated and used for a random
transwell migration assay. The number of viable cells in the lower
chamber was determined after 5 hours of migration by trypan blue
exclusion. The error bars indicate the SEM (n = 4).
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H2O2 increases random transwell migration of
MO7e cells.
Another prominent effect of growth factors such as GM-CSF is induction
of migration. Therefore, the potential ability of
H2O2 to increase the number of MO7e cells
migrating through a fibronectin-coated membrane in a transwell
migration assay was measured. MO7e cells were placed in the upper
transwell chambers, while equal concentrations of the stimulant were in
both chambers. Therefore, the number of cells migrating to the lower
chamber is random and depends on the ability of the stimulant to
increase cell motility, as well as to activate the ability to migrate
through the transwell membrane. In this assay, growth factor-deprived
MO7e cells were stimulated with either 0.1 mmol/L, 0.25 mmol/L, or 0.5 mmol/L H2O2, or 20 ng/mL GM-CSF or 40 ng/mL SF.
As shown in Fig 4C, both cytokines increased transwell migration
compared with starved cells in this assay, fourfold after GM-CSF
stimulation, and eightfold after SF stimulation. The random transwell
migration of MO7e cells was also increased with
H2O2. This effect was dose-dependent and at the
highest concentration (0.5 mmol/L H2O2) was
increased threefold over the level in starved MO7e cells. These data
suggest that oxidants such as H2O2, like
GM-CSF, might contribute to the complex biologic processes that
regulate cytoskeletal function and result in cell migration.
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DISCUSSION |
We have shown that GM-CSF and other growth factors including IL-3, SF,
and TPO are associated with increased levels of ROS in different
hematopoietic cell lines compared with unstimulated cells. Selectively
increasing intracellular ROS by adding H2O2 induced tyrosine phosphorylation and other signaling events, while pretreatment of cells with the reducing agent, PDTC, suppressed formation of ROS, as well as GM-CSF-activated signal transduction, suggesting that ROS contribute to growth factor signal transduction. Reducing agents such as PDTC, 2-mercaptoethanol, and N-acetyl cysteine
suppressed growth of several hematopoietic cell lines, including MO7e
cells, suggesting that growth factor-induced generation of ROS may be
biologically significant.
In other cell systems, ROS have previously been shown to be involved in
various biologic functions. ROS are formed in cells after a variety of
stimuli, including UV-irradiation10 or cytokines, such as
transforming growth factor- (TGF-),15 epidermal
growth factor (EGF),16 or PDGF.9 Interestingly,
depending on the cell type or the stimulus, changes in the oxidative
state can result in apparently opposing pathways such as p53-mediated
apoptosis through ROS17,18 or induction of apoptosis by
antioxidants.19,20 The role of ROS has been best described
in PDGF-stimulated vascular smooth muscle cells. PDGF stimulated the
formation of H2O2 in these cells, which could
be suppressed by overexpression of catalase. PDGF-induced tyrosine
phosphorylation, mitogen-activated protein (MAP) kinase stimulation,
DNA synthesis, and chemotaxis were found to be dependent on the
increase of H2O2 in these cells.9
Here we show that GM-CSF, IL-3, SF, and TPO induce increased formation
of ROS, and that some of the biochemical and biologic effects of these
cytokines are mimicked by H2O2. These effects include tyrosine phosphorylation of cellular proteins, increased migration, c-FOS gene expression, and G1 to S-phase cell cycle progression. In MO7e cells, H2O2 was as
effective as GM-CSF in inducing cellular tyrosine phosphorylation, and
the antioxidant, PDTC, reduced GM-CSF-stimulated tyrosine
phosphorylation, suggesting that ROS might contribute to signaling by
stimulating or augmenting tyrosine phosphorylation. GM-CSF and ROS
induced tyrosine phosphorylation of some of the same substrates,
including the c chain21 and STAT5.22,23
The studies presented here expand the cell lineages and signaling
events already potentially linked to intracellular formation of ROS.
However, the exact mechanisms of ROS action are not well understood.
Recent work indicates that an important function of ROS may be to
modulate the function of protein tyrosine phosphatases. Protein
tyrosine phosphatases contain a critical cysteine residue in their
active site that is a potential target for redox regulation, and this
residue must be in the reduced state for full phosphatase activity.11 For example, H2O2 can
specifically inhibit the protein tyrosine phosphatase activity of LAR
(leukocyte antigen-related) and PTP1, but has no apparent effect on
serine/threonine protein phosphatases, including PP2C- and
calcineurin. The inhibitory effect is due to the selective oxidation of
the cysteine residue in the catalytic domain by
H2O2.24 This is of special interest because inhibition of tyrosine phosphatases through redox modulation would explain the broad spectrum of H2O2 on
biologic activities. In the current study, we have shown that
H2O2 increases tyrosine phosphorylation of
cellular proteins and the antioxidant, PDTC, inhibits cytokine
receptor-induced kinase activity and reduces the total cellular
tyrosine phosphorylation. Tyrosine phosphatases are crucial for the
regulation of many signaling pathways,25,26 and if ROS
inhibit one or more tyrosine phosphatases in hematopoietic cells, this
could explain many of the biologic activities associated with ROS that
are shown here.
The results presented here also show that ROS generated outside the
cell can activate intracellular signaling in hematopoietic cells. This
is consistent with previous studies in other cell types showing
induction of tyrosine phosphorylation or activation of a number of
signaling molecules by H2O2, including
SHC,27 LCK,28 SYK,29
FAK,30 protein kinase C (PKC),31
activation of mitogen-activated protein kinase (MAPK),27
and other signaling molecules. Redox-sensitive cysteine residues in key
proteins are likely to play a critical role in mediating these
signaling events. For example, oxidation of Cys118 in
p21RAS has been reported to activate its GTPase activity and increase
the activity of the downstream effector MAPK.32
Interestingly, in contrast to the GM-CSF-induced transient tyrosine
phosphorylation of cellular proteins, the fluorescence of DCF was
sustained in MO7e cells for at least 18 hours. We cannot exclude that
the production of ROS is a result of mitochondrial metabolism as a
consequence of cell growth. However, our results also suggest that ROS
can play a role in processes that promote cell growth. Cells stimulated
with H2O2 have increased tyrosine phosphorylation, increased cell cycle progression and gene expression, and increased transwell migration. These processes occur later than the
observed tyrosine phosphorylation. Our data also show that PDTC-induced
reduction of ROS is insufficient to completely block cellular tyrosine
phosphorylation. This suggests that ROS contribute only in part to the
regulation of cellular tyrosine kinases.
In addition to tyrosine phosphorylation, ROS can induce a specific
response by activation or induction of transcription factors. The
response of regulators of transcription to changes in the cellular
redox status has been well described. p53, NF- B, and AP-1, the
family of JUN/FOS transcription factors, can be regulated in response
to redox signaling. For example, on a posttranscriptional level,
oxidants and reductants can regulate the activity of p53, AP-1, and
NF-B, likely through critical redox-sensitive cysteine residues in
these proteins. Both, c-JUN and c-FOS levels are known to be induced by
oxidants, including H2O2.33 This is
consistent with our data showing upregulation of c-FOS, a component of
AP-1, in MO7e cells as a response to H2O2 and
GM-CSF. Interestingly, the UV-irradiation-induced increase in AP-1
activity can be decreased by antioxidants.34 However,
oxidants have also been shown to decrease the AP-1-mediated gene
induction.35 The regulation of AP-1 by redox processes
shows an important role of ROS for mediating the biologic function of
this transcription factor in cell growth and
proliferation.36
The origin of ROS associated with activated cytokine receptors is
unknown. There are several possible mechanisms, which could contribute
to modulating ROS levels. The overall activity or protein expression of
enzymes that generate ROS such as NADH (nicotinamide-adenine dinucleotide, reduced)/NADPH (nicotinamide-adenine dinucleotide phosphate, reduced) oxidases or xanthine oxidases could be
elevated. Also, enzymes such as catalase, superoxide dismutase,
glutathione peroxidase, or thiols such as thioredoxin or glutathione
reduce ROS, and it is possible that activated cytokine receptors affect the activity of one of these pathways. Superoxide dismutase generates H2O2 from superoxide anions, while catalase
reduces H2O2 to water. The glutathione
peroxidases, which also include the phospholipid hydroperoxide
glutathione peroxidase, reduce peroxides by using reduced glutathione
(GSH) as an electron donor and generate the dimeric form of glutathione
(GSSG).37 ROS levels can also be regulated by exogenous
antioxidants such as -tocopherols, -carotene, or ascorbic
acid.38 The two thiol group containing reducing drugs used
in this study, PDTC and NAC, have been shown to directly reduce
intracellular free radicals and modulate the intracellular redox
status.39
The mechanisms which generate ROS have been best studied in cells
involved in host defense mechanism. ROS are released by phagocytes
including neutrophils, monocytes, and eosinophils, as well as by B
cells through a mechanism called the respiratory burst. This
respiratory burst is part of a defense mechanism aimed at the
destruction of invading pathogens. The production of ROS starts with
the activation of the membrane-associated NADPH oxidase and formation
of superoxide, a key precursor of other ROS.40,41 However,
the formation of ROS during a respiratory burst is for a very different
biologic purpose than for intracellular signaling.
In any case, it is likely that further characterization of
redox-sensitive proteins regulated through ROS will be helpful in
further understanding the signaling of cytokine receptors. Of
particular interest would be to first identify the specific ROS that is
involved in HGF signaling. These specific ROS or set of ROS could be
used directly to dissect the signaling pathways that are stimulated by
these molecules. Similarly, overexpression of enzymes that are
metabolizing these oxidants, like catalase, which has the ability to
reduce H2O2, will be further helpful to
understand the significance of this pathway. Overall, our results suggest that several HGFs induce rapid and sustained accumulation of
ROS and further suggest that these ROS have the potential ability to
modulate signal transduction involving tyrosine phosphorylation events.
Finally, our data show that ROS, at least when added to cells in the
form of H2O2, can mediate important biologic
events, including cell cycle progression and migration. Further
definition of the role of ROS in signaling are called for.
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FOOTNOTES |
Submitted November 12, 1998; accepted January 4, 1999.
Supported by José Carreras International Leukemia Foundation
fellowship FIJC-95/INT (to M.S.) and Grants No. CA01730 (to R.S.) and
CA36167 (to J.D.G.) from the National Institutes of Health (NIH).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to James D. Griffin, MD,
Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard
Medical School, 44 Binney St, Boston, MA 02115; e-mail:
james_griffin{at}dfci.harvard.edu.
| |
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TOP
ABSTRACT
INTRODUCTION