(Received for publication, November 6, 1995; and in revised form, December 13, 1995)
From the
The mitogen-activated protein kinase (MAPK) family is comprised
of key regulatory proteins that control the cellular response to both
proliferation and stress signals. In this study we investigated the
factors controlling MAPK activation by HO
and
explored the impact of altering the pathways to kinase activation on
cell survival following H
O
exposure. Potent
activation (10-20-fold) of extracellular signal-regulated protein
kinase (ERK2) occurred within 10 min of H
O
treatment, whereupon rapid inactivation ensued.
H
O
activated ERK2 in several cell types and
also moderately activated (3-5-fold) both c-Jun N-terminal kinase
and p38/RK/CSBP. Additionally, H
O
increased the
mRNA expression of MAPK-dependent genes c-jun, c-fos,
and MAPK phosphatase-1. Suramin pretreatment completely inhibited
H
O
stimulation of ERK2, highlighting a role for
growth factor receptors in this activation. Further, ERK2 activation by
H
O
was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol,
indicating that metal-catalyzed free radical formation mediates the
initiation of signal transduction by H
O
.
H
O
-stimulated activation of ERK2 was abolished
in PC12 cells by inducible or constitutive expression of the dominant
negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more
sensitive than wild-type PC12 cells to H
O
toxicity. Moreover, NIH 3T3 cells expressing constitutively
active MAPK kinase (MEK, the immediate upstream regulator of ERK) were
more resistant to H
O
toxicity, while those
expressing kinase-defective MEK were more sensitive, than cells
expressing wild-type MEK. Taken together, these studies provide insight
into mechanisms of MAPK regulation by H
O
and
suggest that ERK plays a critical role in cell survival following
oxidant injury.
The cellular response to diverse external stimuli is controlled
via a complex array of phosphorylation cascades. The extracellular
signal-regulated protein kinase (ERK) ()cascade is a
prominent component of the mitogen-activated protein kinase (MAPK)
family that in particular plays an integral role in both growth factor
and stress signaling (reviewed in (1) ). The majority of ERK
activity in most cell types arises from ERK1 (p42) and ERK2 (p44)
isoforms(1) , which are believed to have functional redundancy.
Interestingly, at least some stress signals (e.g. UVC irradiation(2) ) utilize the same signaling pathways
for ERK activation as do mitogens. This well characterized cascade
(reviewed in (3) ) is initiated by growth factor binding, which
stimulates receptor tyrosine kinases. The sequential activation of the
GTP-binding protein Ras and the serine kinase Raf then
ensues(3, 4, 5) . Raf then activates MAPK
kinase (MEK), a threonine/tyrosine dual specificity kinase that
directly activates ERK(6) . ERK activation culminates in the
phosphorylation of downstream cytosolic and nuclear factors that
control a variety of cellular processes(7) .
Oxidant injury
is thought to play a critical role in the degenerative alterations that
occur with aging and in the etiology of many disease processes
including cancer and
atherosclerosis(8, 9, 10) . Many of the basic
molecular aspects regulating the cellular response to oxidative stress
in bacteria are well established(11) . However, the pathways
mediating the control of gene expression by oxidants and sensitivity to
oxidant injury in mammalian systems are less well defined. Herein we
examine the activation of MAPK pathways by the oxidative agent
HO
, with particular focus on the cellular
consequences of modulating the ERK signaling cascade. Our findings
support a pivotal role for the ERK pathway in determining cell survival
following oxidant injury.
Figure 1:
Protein tyrosine phosphorylation and
kinase activation by HO
. A, Western
blot analysis using the antiphosphotyrosine antibody RC20H. NIH 3T3
cell lysates were prepared following H
O
treatment for the indicated times. Tyrosine-phosphorylated
proteins of 33, 35, 38, 41, and 44 kDa are indicated. B,
dose-response analysis of ERK2, JNK1/SAPK, and p38/RK/CSBP kinase
activation by H
O
. NIH 3T3 cells were treated
with the indicated doses of H
O
at the time of
maximal activation (10 min for ERK2; 15 min for JNK1/SAPK and
p38/RK/CSBP), and polyclonal anti-ERK2, anti-JNK1/SAPK, or
anti-p38/RK/CSBP antibodies were used for kinase immunoprecipitation
from the soluble fraction of cell lysates. Kinase activity was then
assessed by immune complex kinase assay using bovine brain MBP (for
ERK2 and p38/RK/CSBP) or GST-c-Jun (for JNK1/SAPK) as a
substrate.
Figure 2:
Activation of ERK2 by HO
in HeLa, Rat1, NIH 3T3, PC12, and SMC. The indicated cell types
were treated with 50 or 200 µM H
O
for 10 min, and ERK2 was analyzed in the soluble fraction of
lysates by immune complex kinase assay.
The
kinetics of ERK2 activation in NIH 3T3 cells following
HO
exposure are shown in Fig. 3.
H
O
stimulation of ERK2 activity occurred within
5 min of treatment and was maximal by 10 min after H
O
exposure (Fig. 3A). A rapid inactivation of ERK2
then ensued, with a return to basal ERK2 levels occurring within 30 min
of H
O
exposure. The increase in ERK2 kinase
activity following H
O
treatment was paralleled
by a shift in the electrophoretic mobility of ERK2 protein seen on
Western blots, indicating phosphorylation of ERK2 protein (Fig. 3B); however, the abundance of ERK2 protein
expressed remained unchanged.
Figure 3:
Time course for activation of ERK2 by
HO
. NIH 3T3 cells were treated with 200
µM H
O
for the indicated times,
after which cells were harvested and the soluble fraction was analyzed
for ERK2. A, kinetics of ERK2 activation in
H
O
-treated cells. ERK2 was immunoprecipitated
using a polyclonal anti-ERK2 antibody, and kinase activity was assayed
using bovine brain MBP as a substrate. B, Western blot
analysis of the expression and phosphorylation of
ERK2.
Figure 4:
HO
stimulates
MAPK-dependent gene expression. A, Northern blot analysis for
the induction of c-jun, c-fos, and MKP-1 mRNA
expression by H
O
. Following treatment with 200
µM H
O
for the indicated times, RNA
was isolated and Northern blots were probed with the indicated cDNAs.
The 18 S signal is shown as a control for variations in loading and
transfer. B, effect of cotransfection with rMKP-1 or rMKP-1as
on fos-luciferase expression stimulated by
H
O
(200 µM). C, effect of
cycloheximide pretreatment on ERK2 activation by
H
O
. Cycloheximide (40 µg/ml) was added 45
min prior to treatment with 200 µM H
O
for the indicated times, and ERK2 activity was assessed in a
soluble fraction of cell extracts by immune complex kinase
assay.
While MKP-1 has been implicated in
regulating ERK2 activity in response to growth factor
stimulation(18) , the physiological role of MKP-1 in mitigating
the rapid activation of ERK2 activity by HO
is
less well defined. The reversion of ERK2 protein to the
unphosphorylated state that accompanies the rapid loss of kinase
activity following ERK2 stimulation by H
O
in Fig. 3A suggests that protein phosphatases function in
ERK2 regulation following H
O
. However,
inhibition of protein synthesis by cycloheximide pretreatment did not
affect the kinetics of ERK2 activation by H
O
(Fig. 4C), indicating that newly synthesized
phosphatases such as MKP-1 do not participate in inactivating ERK2.
These results are consistent with the rapid kinetics of ERK2
inactivation and suggest that ERK2 activity is instead regulated by
preexisting phosphatases.
Figure 5:
Suramin inhibits ERK2 activation by
HO
. Suramin (0.3 mM) was added 45 min
before the direct addition of H
O
, and cells
were harvested 10 min later. ERK2 activity was analyzed in the soluble
fraction of cell lysates by immune complex kinase
assay.
The nature of
the chemical signal generated from HO
that
initiates the ERK2 cascade was also investigated. Enhancing the
cellular antioxidant potential by pretreatment with the glutathione
precursor N-acetyl-cysteine abolished the ability of
H
O
to stimulate ERK2 (Fig. 6). These
results confirm that oxidant stress initiates ERK2 activation by
H
O
. The iron chelator o-phenanthroline
also effectively inhibited ERK2 activation by H
O
(Fig. 6), suggesting that metal-dependent reactions are
required for kinase activation by H
O
. In the
presence of metal ions, H
O
can undergo
conversion via dismutation reactions to other oxygen-derived free
radical species including hydroxyl radical(21) . Indeed,
mannitol, a free radical scavenger with specificity for hydroxyl
radical, also blocked H
O
-mediated ERK2
activation. Taken together, these results suggest that
H
O
undergoes metal-catalyzed conversion to a
hydroxyl radical-like species and that oxidation by this free radical
initiates signal transduction leading to ERK2 activation by
H
O
.
Figure 6:
Role
of free radicals in HO
-mediated ERK2
activation. N-acetyl-cysteine (20 mM), o-phenanthroline (100 µM), or mannitol (100
mM) was added 45 min before the direct addition of
H
O
, and cells were harvested 10 min later for
analysis of ERK2 activity by immune complex kinase assay. Data are
expressed as the -fold induction in ERK2 activity over inhibitor alone
controls. The inhibitors alone did not activate
ERK2.
Figure 7:
Effect
of inducible and constitutive dominant-negative Ras-N-17 on
HO
-mediated ERK2 activation. PC12 cells were
treated with H
O
for 10 min, whereupon cells
were harvested and ERK2 activity in the soluble fraction of cell
lysates was assessed by immune complex kinase assay. Left,
PC12 cells expressing murine mammary tumor virus-Ras-N-17 were cultured
overnight in the presence or absence of dexamethasone (1
µM) prior to treatment with H
O
. Right, comparison of the -fold activation of ERK2 by
H
O
in parental PC12 cells and those
constitutively expressing Ras-N-17.
Figure 8:
Comparative effect of HO
on cell viability in parental and constitutive
Ras-N-17-expressing PC12 cells. PC12 cells were cultured overnight in
96-well plates, treated with H
O
in complete
medium, and stained 48 h later with crystal violet for assessment of
cell viability. Values represent mean ± S.E. for seven
wells.
In addition to mediating signal transduction
to ERK, Ras is known to participate in the activation of other MAPK
family members, including JNK1/SAPK(13, 25) . Thus,
the dramatic effect of Ras-N-17 on cell survival following
HO
may not solely arise as a consequence to
modulation of the ERK signaling pathway. In order to investigate the
possible contribution of the JNK1/SAPK pathway to the enhanced
sensitivity of PC12/Ras-N-17 cells to H
O
, we
compared H
O
-stimulated JNK1/SAPK activity in
Ras-N-17 and wild-type PC12 cells. However, we found that the modest
activation of JNK1/SAPK (4-fold with 200 µM H
O
) by H
O
was not
affected by cellular Ras status (data not shown). By contrast, the
reduced potential for cell survival following oxidant injury of
PC12/Ras-N-17 cells correlates with, and may indeed arise as a
consequence of, the decreased capacity for ERK activation in these
cells (Fig. 7).
In order to further address the function of
the ERK pathway in the cellular response to HO
,
we compared cell survival in NIH 3T3 cell lines in which the activity
of MEK, the immediate upstream regulator of ERK, had been
altered(26) . With increasing dosage of
H
O
, cell survival diminished accordingly in
cell lines expressing wild-type MEK (MEK
), constitutively
active MEK (MEK
), or MEK lacking a kinase domain
(MEK
) (Fig. 9). However, the sensitivity to
H
O
was correlated with MEK activity; MEK
cells exhibited enhanced resistance, while MEK
cells
showed diminished resistance, as compared with MEK
cells.
This effect of MEK was evidenced by a separation in the dose-response
curves for cell survival following H
O
exposure
in the three cell lines and in marked differences in the LD
for H
O
. While 120 µM resulted in a 50% decrease in survival in MEK
cells,
200 µM and 320 µM were required for the same
effect in MEK
and MEK
cells, respectively.
Likewise, the same dose of H
O
could
differentially affect cell survival in the three cell lines: 180
µM H
O
mediated a 10, 45, or 65%
loss of survival in MEK
, MEK
, and MEK
cells, respectively. Comparable findings resulted from colony
formation assays (data not shown). These results are consistent with,
and extend our findings in, PC12 cells expressing wild-type Ras or
Ras-N-17 to suggest that cell survival following exposure to
H
O
can be accordingly modulated by either
enhancing or suppressing the pathway to ERK activation.
Figure 9:
Effect of cellular MEK status on
HO
-stimulated loss of cell viability. NIH 3T3
cells expressing MEK
, MEK
, or MEK
were cultured overnight in 96-well plates, treated with
H
O
in complete medium, and stained 48 h later
with crystal violet for assessment of cell viability. Values represent
mean ± S.E. for seven wells.
In this report, we demonstrate that multiple members of the
MAPK family are stimulated by HO
and that ERK2
in particular is highly activated in a variety of cell types ( Fig. 1and 2). The rapid and transient nature of ERK2 activation
by H
O
(Fig. 3) highlights the reversible
and direct nature of alterations stimulated by H
O
and underscores a role for phosphatases in regulating this
response. However, the involvement of newly synthesized proteins in
regulating ERK2 activation following H
O
exposure is precluded by both the rapidity of inactivation and
the cycloheximide insensitivity of ERK2 activation (Fig. 4C). Dephosphorylation of ERK2 protein may
instead be reliant on preexisting
phosphatases(19, 27, 28) . The early temporal
control of ERK2 activation by both kinase and phosphatase activity is
also reflected in the transient stimulation of MAPK-dependent gene
expression by H
O
(Fig. 4A).
The function of numerous cellular proteins, including transcription
factors, calcium-regulatory proteins, and other cell and organelle
surface molecules, is subject to redox
regulation(10, 29, 30, 31, 32, 33, 34) .
Oxidation-reduction mechanisms are in fact a likely physiological means
for reversible regulation of protein function and provide a likely
target through which exogenous oxidants can usurp normal signal
transduction pathways. For example, many growth factor and cytokine
receptors have cysteine-rich motifs, the oxidation of which can
simulate ligand binding(35, 36) . That suramin can
block HO
-stimulated ERK2 activation (Fig. 5) suggests that oxidation of such cell surface receptors
may mediate signal initiation by H
O
. Indeed,
the sulfhydryl reactivity of the oxidant signal generated from
H
O
was confirmed by the inhibitory actions of N-acetyl-cysteine (Fig. 6). Free radical species
generated from H
O
may directly oxidize and
thereby activate cell surface receptors, although the oxidative
modification of other molecules, including those involved in
phosphatase regulation, may also function in the regulation of ERK2 by
H
O
. These findings further suggest that free
radicals or other redox mechanisms may constitute a critical component
of the signaling pathways to ERK activation normally utilized by growth
factors and other stimuli. Our demonstration that ERK2 can be activated
by exposure to low doses of H
O
(10
µM), such as may typically occur in cells(37) ,
supports this assertion. That H
O
-stimulated
ERK2 is regulated through Ras (Fig. 7), as has been reported for
serum and growth factors(5) , further emphasizes the
significant overlap between the pathways for oxidative stress and
normal physiological signals.
While MAPK activation has been
reported in response to both proliferation and stress
stimuli(1) , an understanding of the function of the ERK
phosphorylation cascade in regulating the downstream cellular effects
that occur pursuant to stimulation is only beginning to emerge. Recent
reports have provided evidence that constitutive MAPK activation is
associated with the transformed phenotype (38) and that
likewise unregulated activation of MEK, the immediate upstream
activator of MAPK, can alone cause cellular
transformation(26, 39) . These findings are in keeping
with the known oncogenic potential of other molecules (i.e. Ras and Raf) that play a critical role in signal transduction
pathways (reviewed in (40) ). In the present study we
demonstrate in two model systems that modulation of the pathway to ERK
activation by HO
affects cellular survival
following H
O
. Expression of dominant negative
Ras in PC12 cells and kinase-defective MEK in NIH 3T3 cells results in
enhanced sensitivity to H
O
, while a
constitutively active MEK variant engendered greater resistance. Thus,
we provide evidence that altered responsiveness to extracellular stress
is an important consequence of these potentially oncogenic alterations.
This type of ``response modification'' has been proposed to
contribute to carcinogenic development by oxidant and other
stimuli(41, 42) . While the precise MAPK-dependent
cellular alterations engendering a modified response to oxidants remain
to be defined, the present study provides strong support of a crucial
role for the MAPK pathway in regulating cellular protection and
proliferation in response to oxidative stress.