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(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 H
The cellular response to diverse external stimuli is controlled
via a complex array of phosphorylation cascades. The extracellular
signal-regulated protein kinase (ERK) ( 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
H
Figure 1:
Protein tyrosine phosphorylation and
kinase activation by H
Figure 2:
Activation of ERK2 by H
The
kinetics of ERK2 activation in NIH 3T3 cells following
H
Figure 3:
Time course for activation of ERK2 by
H
Figure 4:
H
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 H
Figure 5:
Suramin inhibits ERK2 activation by
H
The nature of
the chemical signal generated from H
Figure 6:
Role
of free radicals in H
Figure 7:
Effect
of inducible and constitutive dominant-negative Ras-N-17 on
H
Figure 8:
Comparative effect of H
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
H In order to further address the function of
the ERK pathway in the cellular response to H
Figure 9:
Effect of cellular MEK status on
H
In this report, we demonstrate that multiple members of the
MAPK family are stimulated by H 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 H 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 H
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4138-4142
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
O
ROLE IN CELL SURVIVAL FOLLOWING OXIDANT INJURY (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O and
explored the impact of altering the pathways to kinase activation on
cell survival following HO exposure. Potent
activation (10-20-fold) of extracellular signal-regulated protein
kinase (ERK2) occurred within 10 min of HO treatment, whereupon rapid inactivation ensued.
HO activated ERK2 in several cell types and
also moderately activated (3-5-fold) both c-Jun N-terminal kinase
and p38/RK/CSBP. Additionally, HO increased the
mRNA expression of MAPK-dependent genes c-jun, c-fos,
and MAPK phosphatase-1. Suramin pretreatment completely inhibited
HO stimulation of ERK2, highlighting a role for
growth factor receptors in this activation. Further, ERK2 activation by
HO 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 HO.
HO-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 HO toxicity. Moreover, NIH 3T3 cells expressing constitutively
active MAPK kinase (MEK, the immediate upstream regulator of ERK) were
more resistant to HO 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 HO and
suggest that ERK plays a critical role in cell survival following
oxidant injury.
)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) .O, 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.
Cell Culture and Treatment
Primary cultures and
cell lines were cultured in a 37 °C humidified environment
containing 10% CO in air. Cell lines were cultured in
Dulbecco's modified Eagle's medium with gentamicin (50
ng/ml), supplemented with 10% calf serum (NIH 3T3), 10% fetal bovine
serum (HeLa and Rat1), or 10% fetal bovine serum and 5% horse serum
(PC12). Primary smooth muscle cells were isolated by enzymatic
digestion of rat aorta as described (12) and cultured in medium
199 supplemented with 20% calf serum, penicillin (100 units/ml), and
streptomycin (100 µg/ml). Serum starvation was achieved by
incubation in Dulbecco's modified Eagle's medium containing
0.5% fetal bovine serum for at least 16 h prior to the direct addition
of HO into this culture medium (with 200
µM HO equivalent to 1 µmol of
HO/10
cells). Suramin (0.3
mM), N-acetyl-cysteine (20 mM), o-phenanthroline (0.1 mM), mannitol (100
mM), or cycloheximide (40 µg/ml) were added to the culture
medium 45 min before the direct addition of HO. Immunoprecipitation and Kinase Activity
Assays
Cell cultures (60-80% confluent) were treated,
washed twice with ice-cold phosphate-buffered saline, and lysed in
buffer containing 20 mM HEPES (pH 7.4), 50 mM 
-glycerophosphate, 1% Triton X-100, 10% glycerol, 2 mM EGTA, 1 mM dithiothreitol, 10 mM sodium
fluoride, 1 mM sodium orthovanadate, 2 µM leupeptin, 2 µM aprotinin, 2 µM pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 0.5
µM okadaic acid. Soluble extracts were prepared by
centrifugation at 10,000 
g for 10 min at 4 °C.
Following normalization of protein content, endogenous ERK2, c-Jun
N-terminal kinase-1 (JNK1/SAPK), or p38/RK/CSBP were immunoprecipitated
from the cell extracts using rabbit polyclonal antibodies against
p42 or p46
(Santa Cruz Biotechnology,
Santa Cruz, CA), or p38
(provided by J.C. Lee,
Smithkline Beacham Pharmaceuticals, King of Prussia, PA), respectively.
Kinase activity was assayed for 20 min at 37 °C in the presence of
6 µg of substrate, 30 µM ATP, and 20 µCi of
[
-
P]ATP in 55 µl of assay buffer (20
mM MOPS, pH 7.2, 2 mM EGTA, 20 mM MgCl
). GST-c-Jun-(1-135) (13) was used
as a substrate for JNK1/SAPK, and myelin basic protein (MBP) was used
for assaying ERK2 and p38/RK/CSBP activity. After completion of kinase
assays, the proteins were resolved by SDS-polyacrylamide gel
electrophoresis, and the gels were dried and subjected to
autoradiography. The incorporation of P was quantitated
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis
Protein extracts were
prepared and subjected to Western analysis as described previously (14) using a monoclonal antibody specific for ERK2
(Transduction Laboratories, Lexington, KY). Phosphotyrosine was
detected using the monoclonal anti-phosphotyrosine antibody RC20H
(Transduction Laboratories). Immune complexes were visualized using an
enhanced chemiluminescence detection kit (Amersham Corp.) following the
manufacturer's specifications.RNA Isolation and Northern Analysis
Total RNA was
extracted from treated cells using RNA Stat-60 (Tel-Test
``B'', Friendswood, TX). RNA (10-20 µg/lane) was
size-separated in agarose/formaldehyde gels and transferred onto
GeneScreen Plus nylon membranes (DuPont NEN). cDNA probes for c-Fos,
c-Jun, and MKP-1 were labeled with [
-P]dCTP
using a random primer labeling kit (Boehringer Mannheim). Hybridization
and washes were performed according to the method of Church and
Gilbert(15) , and the hybridization signal was quantified using
a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Hybridization to
a 24-base pair oligonucleotide complementary to 18 S RNA
(5`-ACGGTATCTGATCGTCTCGAACC-3`) that had been 3`-end-labeled with
[
-P]ATP by terminal
deoxynucleotidyltransferase (Life Technologies, Inc.) was used to
control for variation in loading and transfer among samples.
Transfections and Luciferase Assay
HeLa cells were
transiently transfected by CaPO
precipitation with 1 µg fos-luciferase (16) together with either 10 µg of
carrier DNA (pSG5), 6 µg of carrier DNA and 4 µg of
pSG5-rMKP-1, or 10 µg of pSG5-MKP-1as(14) . Cells were
treated with 200 µM HO the
following day, and luciferase activity was measured in cell extracts
prepared 24 h after treatment using a luciferase assay system kit
(Promega, Madison, WI).Cell Viability Assays
Cells were plated at a
density of 50 10
cells/well (PC12) or 5
10 cells/well (NIH 3T3) into 96-well microtiter plates. The
plates were treated with HO the following day
and stained 48 h after treatment with crystal violet for assessment of
cell viability using a microplate spectrophotometer system (Molecular
Devices, Sunnyvale, CA)
H
As shown in Fig. 1A, HO Stimulates Protein Tyrosine
Phosphorylation and Potently Activates ERK2O
stimulated the rapid and
transient phosphorylation of several proteins of 33-44 kDa in NIH
3T3 cells. Kinases of the MAPK family have mobilities consistent with
the proteins that became recognized by the anti-phosphotyrosine
antibody within 5-10 min of H
O
treatment.
Indeed, direct measurement of kinase activity by immune complex kinase
assay confirmed that H
O
activated multiple
members of the MAPK family (Fig. 1B). While JNK1/SAPK
and p38/RK/CSBP were only activated 3-5-fold over control levels,
H
O
stimulated a striking increase in ERK2
activity of 25-fold. Enhancement of ERK2 activity was evident with as
little as 10 µM H
O
, with
dose-dependent increases up to 200 µM H
O
. To determine if ERK2 activation
constitutes a widespread cellular response to oxidants such as
H
O
, we examined the effect of
H
O
on ERK2 in several cell types including
HeLa, Rat1, NIH 3T3, and PC12 cell lines as well as in primary aortic
smooth muscle cell cultures (SMC). While minimal ERK2 activation by
H
O
has been reported in SMC(17) , we
found significant ERK2 stimulation (10-20-fold) in all cell types
tested, including these primary cultures (Fig. 2).
O. A, Western
blot analysis using the antiphosphotyrosine antibody RC20H. NIH 3T3
cell lysates were prepared following HO 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 HO. NIH 3T3 cells were treated
with the indicated doses of HO 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.
O in HeLa, Rat1, NIH 3T3, PC12, and SMC. The indicated cell types
were treated with 50 or 200 µM HO for 10 min, and ERK2 was analyzed in the soluble fraction of
lysates by immune complex kinase assay.
O exposure are shown in Fig. 3.
HO stimulation of ERK2 activity occurred within
5 min of treatment and was maximal by 10 min after HO exposure (Fig. 3A). A rapid inactivation of ERK2
then ensued, with a return to basal ERK2 levels occurring within 30 min
of HO exposure. The increase in ERK2 kinase
activity following HO 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.
O. NIH 3T3 cells were treated with 200
µM HO for the indicated times,
after which cells were harvested and the soluble fraction was analyzed
for ERK2. A, kinetics of ERK2 activation in
HO-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.
MAPK-dependent Gene Expression following
H
An important cellular
consequence of MAPK activation is the modulation of the expression of
transcription factors and other key regulatory proteins. Therefore, we
investigated the effect of HO ExposureO on the expression
of several such early response genes. As shown in Fig. 4A,
HO
treatment stimulated a transient but
dramatic increase in the mRNA expression of MKP-1, c-jun, and
c-fos. H
O
also activated a fos promoter-luciferase reporter gene construct in transiently
transfected HeLa cells (Fig. 4B). The dependence of fos promoter activation by H
O
on the
ERK pathway was further demonstrated by cotransfection experiments, in
which coexpression of MKP-1 (which is known to abrogate MAPK
activity(18, 19) ), attenuated fos-luciferase
activity by H
O
, while expression of antisense
MKP-1 was without effect.
O stimulates
MAPK-dependent gene expression. A, Northern blot analysis for
the induction of c-jun, c-fos, and MKP-1 mRNA
expression by HO. Following treatment with 200
µM HO 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
HO (200 µM). C, effect of
cycloheximide pretreatment on ERK2 activation by
HO. Cycloheximide (40 µg/ml) was added 45
min prior to treatment with 200 µM HO for the indicated times, and ERK2 activity was assessed in a
soluble fraction of cell extracts by immune complex kinase
assay.
O 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 HO in Fig. 3A suggests that protein phosphatases function in
ERK2 regulation following HO. However,
inhibition of protein synthesis by cycloheximide pretreatment did not
affect the kinetics of ERK2 activation by HO (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.Initiation of ERK2 Signaling by
H
Mitogen-stimulated signal transduction leading to ERK2
activation is initiated through the interaction of peptide growth
factors with their receptors. Receptor activation can also be mediated
by sulfhydryl oxidation, and through such mechanisms
HO: Role of Growth Factors and Free
RadicalsO has been postulated to mimic the actions of
insulin and other receptor-binding proteins(20) . We therefore
explored the role of growth factor receptors in the initiation of ERK2
activation by HO. Suramin is known to block
ligand-receptor interactions and can inhibit ERK2 activation by
epidermal growth factor as well as by UVC irradiation (2) . As
shown in Fig. 5, suramin pretreatment also blocked
HO-stimulated ERK2 activation.
O. Suramin (0.3 mM) was added 45 min
before the direct addition of HO, and cells
were harvested 10 min later. ERK2 activity was analyzed in the soluble
fraction of cell lysates by immune complex kinase
assay.
O 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
HO to stimulate ERK2 (Fig. 6). These
results confirm that oxidant stress initiates ERK2 activation by
HO. The iron chelator o-phenanthroline
also effectively inhibited ERK2 activation by HO (Fig. 6), suggesting that metal-dependent reactions are
required for kinase activation by HO. In the
presence of metal ions, HO 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 HO-mediated ERK2
activation. Taken together, these results suggest that
HO 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
HO.
O-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
HO, 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.
Role of Ras in ERK2 Activation by
H
Although Ras plays a prominent role
in the activation of multiple MAPK family members in response to both
mitogen stimulation and UVC irradiation(5) , MAPK activation
can also occur through Ras-independent pathways(22) . To
determine if Ras is a component of the signaling cascade leading to
ERK2 activation by HOO, we examined the
inhibitory effect of the dominant negative Ras-N-17 allele on ERK2
activation in PC12 cells(23, 24) . Dexamethasone
pretreatment of PC12 cells carrying a murine mammary tumor virus-driven
Ras-N-17 expression vector resulted in a near complete abolition of
ERK2 activation by HO (Fig. 7, left
panel), suggesting that Ras is required for ERK2 activation by
HO. Similar results were obtained in PC12 cells
constitutively expressing the dominant negative Ras-N-17 allele (Fig. 7, right panel). By comparison with that in
wild-type PC12 cells, ERK2 activation by HO in
these Ras-N-17-expressing cells was markedly reduced. Taken together,
the results from these two model systems of mutant Ras expression
indicate a requirement for Ras in mediating ERK2 activation by
HO.
O-mediated ERK2 activation. PC12 cells were
treated with HO 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 HO. Right, comparison of the -fold activation of ERK2 by
HO in parental PC12 cells and those
constitutively expressing Ras-N-17.
Effect of Modulating ERK Activation on Survival following
H
To investigate the potential
physiological role of ERK in mediating the cellular response to
oxidative stress, we undertook a comparative analysis of cell survival
following HO ExposureO exposure in PC12/Ras-N-17 cells
and wild-type PC12 cells. HO stimulated a
dose-dependent decrease in cell survival in both cell lines as assessed
by crystal violet staining. However, PC12/Ras-N-17 cells exhibited
significantly greater sensitivity to HO than
their wild-type counterparts, as evidenced by reduced survival at
identical doses of HO (Fig. 8). Indeed,
the LD for H
O was 150 µM in PC12/Ras-N-17 cells, as compared with 380 µM in
wild-type PC12 cells. Similar results were found by colony formation
assay (data not shown).
O on cell viability in parental and constitutive
Ras-N-17-expressing PC12 cells. PC12 cells were cultured overnight in
96-well plates, treated with HO in complete
medium, and stained 48 h later with crystal violet for assessment of
cell viability. Values represent mean ± S.E. for seven
wells.
O 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 HO, we
compared HO-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 HO) by HO 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).O,
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
HO, 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 HO 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.
O-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.
O 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 HO (Fig. 3) highlights the reversible
and direct nature of alterations stimulated by HO and underscores a role for phosphatases in regulating this
response. However, the involvement of newly synthesized proteins in
regulating ERK2 activation following HO 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 HO (Fig. 4A). O-stimulated ERK2 activation (Fig. 5) suggests that oxidation of such cell surface receptors
may mediate signal initiation by HO. Indeed,
the sulfhydryl reactivity of the oxidant signal generated from
HO was confirmed by the inhibitory actions of N-acetyl-cysteine (Fig. 6). Free radical species
generated from HO 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
HO. 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 HO (10
µM), such as may typically occur in cells(37) ,
supports this assertion. That HO-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.O affects cellular survival
following HO. Expression of dominant negative
Ras in PC12 cells and kinase-defective MEK in NIH 3T3 cells results in
enhanced sensitivity to HO, 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.
), wild-type MEK;
MEK
, constitutively active MEK; MEK
,
kinase-defective MEK; MKP-1, MAPK phosphatase-1; rMKP-1, rat MKP-1;
SMC, aortic smooth muscle cell primary cultures; MOPS,
4-morpholinepropanesulfonic acid; p38/RK/CSBP, 38-kDa MAPK-related
protein/reactivating kinase/cytokine-suppressive anti-inflammatory drug
binding protein; UVC, short wavelength UV radiation.
We are grateful to Dr. S. Mansour and Dr. N. Ahn for
NIH 3T3/MEK cells, to Dr. G. Cooper for PC12/Ras-N-17 cells, to Dr. J.
Bos for fos-luciferase, and to Dr. J. Kyriakis, Dr. J.
Woodgett, and Dr. J. C. Lee for protocols and reagents for immune
complex kinase assays.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 T. P. Rygiel, A. E. Mertens, K. Strumane, R. van der Kammen, and J. G. Collard The Rac activator Tiam1 prevents keratinocyte apoptosis by controlling ROS-mediated ERK phosphorylation J. Cell Sci., April 15, 2008; 121(8): 1183 - 1192. [Abstract] [Full Text] [PDF]
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 L. Wang, P. Chanvorachote, D. Toledo, C. Stehlik, R. R. Mercer, V. Castranova, and Y. Rojanasakul Peroxide Is a Key Mediator of Bcl-2 Down-Regulation and Apoptosis Induction by Cisplatin in Human Lung Cancer Cells Mol. Pharmacol., January 1, 2008; 73(1): 119 - 127. [Abstract] [Full Text] [PDF]
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 L. A. Tephly and A. B. Carter Constitutive NADPH oxidase and increased mitochondrial respiratory chain activity regulate chemokine gene expression Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1143 - L1155. [Abstract] [Full Text] [PDF]
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 L. A. Tephly and A. B. Carter Differential Expression and Oxidation of MKP-1 Modulates TNF-{alpha} Gene Expression Am. J. Respir. Cell Mol. Biol., September 1, 2007; 37(3): 366 - 374. [Abstract] [Full Text] [PDF]
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 E. Babilonia, D. Lin, Y. Zhang, Y. Wei, P. Yue, and W.-H. Wang Role of gp91phox-Containing NADPH Oxidase in Mediating the Effect of K Restriction on ROMK Channels and Renal K Excretion J. Am. Soc. Nephrol., July 1, 2007; 18(7): 2037 - 2045. [Abstract] [Full Text] [PDF]
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 A. Zheng, A. Kallio, and P. Harkonen Tamoxifen-Induced Rapid Death of MCF-7 Breast Cancer Cells Is Mediated via Extracellularly Signal-Regulated Kinase Signaling and Can Be Abrogated by Estrogen Endocrinology, June 1, 2007; 148(6): 2764 - 2777. [Abstract] [Full Text] [PDF]
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