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Experimental Cell Research 297 (2004) 461–471
K+ channel activity and redox status are differentially required for JNK
activation by UV and reactive oxygen species
Jie Gao,a,1 Dan Wu,a,1 Taylor B. Guo,a,b Qin Ruan,c Tie Li,d Zhenyu Lu,a Ming Xu,a,b,e
Wei Dai,a,b,c,* and Luo Lua,b,d,*
aDepartment of Medical Genetics, Shanghai Second Medical University, PR ChinabHealth Science Center, Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China
cDivision of Molecular Carcinogenesis, Department of Medicine, New York Medical College, Valhalla, NY 10595, USAdDivision of Molecular Medicine, Harbor-UCLA Medical Center, School of Medicine, University of California Los Angeles, Torrance, CA 90502, USA
eDepartment of Cell Biology Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
Received 2 January 2004, revised version received 19 March 2004
Available online 6 May 2004
Abstract
Upon exposure to ultraviolet (UV) radiation, osmotic changes or the presence of reactive oxygen species (ROS) c-Jun N-terminal kinases
(JNKs) are rapidly activated. Extensive studies have elucidated molecular components that mediate the activation of JNKs. However, it
remains unclear whether activation of JNKs by various stress signals involves different pathways. Here we show that K+ channel activity is
involved in mediating apoptosis induced by UV but not by H2O2 in myelocytic leukemic ML-1 cells. Specifically, JNKs were rapidly
phosphorylated upon treatment of ML-1 cells with UV and H2O2. UV-induced, but not H2O2-induced, JNK-1 phosphorylation was inhibited
by pretreatment with 4-aminopyridine (4-AP), a K+ channel blocker. 4-AP also blocked UV-induced increase in JNK activity as well as p38
phosphorylation. Immunofluorescent microscopy revealed that phosphorylated JNKs were concentrated at centrosomes in ML-1 cells and
that these proteins underwent rapid subcellular translocation upon UV treatment. Consistently, the subcellular translocation of JNKs induced
by UV was largely blocked by 4-AP. Furthermore, UV-induced JNK activation was blocked by NEM, a sulfhydryl alkylating agent also
affecting K+ current. Both UV- and H2O2-induced JNK activities were inhibited by glutathione, suggesting that the redox status does play an
important role in the activation of JNKs. Taken together, our findings suggest that JNK activation by UVand H2O2 is mediated by distinct yet
overlapping pathways and that K+ channel activity and redox status are differentially required for UV- and H2O2-induced activation of JNKs.
D 2004 Elsevier Inc. All rights reserved.
Keywords: JNKs; UV; Reactive oxygen species; K+ channel; Centrosome
Introduction
Ultraviolet (UV) irradiation induces genotoxic stress,
which frequently results in apoptosis. This response appears
to be mediated by various signaling components [1–3]. At
the cell membrane, UV irradiation elicits rapid clustering and
internalization of membrane receptor molecules for epider-
mal growth factor (EGF), tumor necrosis factor (TNF), and
interleukin-1 (IL-1) [4]. Extensive studies show that UV
irradiation strongly activates some MAP kinase family mem-
0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2004.03.047
* Corresponding authors. Fax: +1-914-594-4726.
E-mail addresses: wei_dai@nymc.edu (W. Dai),
llu@rei.edu (L. Lu).1 These authors contributed equally to the work reported in this study.
bers such as c-JunN-terminal kinases (JNKs) and p38 [5–7].
In addition, UV activates ATR and Chk1 [8,9], two kinases
mediating the DNA damage checkpoint response. One of the
consequences of elevated kinase activities of JNK, p38, and
Chk1 is the activation of transcription factors including p53,
NF-nB, and/or AP-1 by phosphorylation [1,2,10]. JNKs can
also be activated by other stressors such as reactive oxygen
species (ROS) [9,11]. ROS can be produced by a variety of
cells in response to stimulation of mechanical forces, growth
factors, and cytokines. Many studies have shown that oxi-
dants participate in both proliferative and apoptotic responses
[12,13]. Although the molecular basis for divergent
responses to oxidants remains unclear, it is believed that both
the intensity and the context of stimulation play key roles in
determination of the outcome.
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471462
K+ channel activity is widely distributed in the cell
membrane to stabilize cell membrane electrophysiological
properties. In myeloblastic cells, fibroblastic cells, and neu-
rons, cytokines modulate the activity of K+, resulting in cell
proliferation or apoptosis [14,15]. Single-channel recording
using the cell-attached mode reveals that exposure to UV-C
irradiation (45 mJ/cm2) strongly stimulates K+ channel ac-
tivity within 30 s. A quick loss of intracellular K+ ions causes
membrane fluctuations and cell shrinkage, leading to activa-
tion of known surface receptors and cytoplasmic signaling
components. In the previous work, we have shown that
activation of a voltage-gated K+ channel in ML-1 cells by
UV-C irradiation promotes apoptosis. The K+ channel in
these cells is sensitive to 4-aminopyridine (4-AP, IC50 = 80
AM), a specific voltage-gate K+ channel blocker [16]. The
effects of UV irradiation-activated K+ channels are observed
at both whole-cell and single-channel levels. Blockade of K+
channels with a similar low dose of 4-AP almost completely
prevents UV-induced apoptosis and suppresses UV-stimulat-
ed JNK pathway, indicating that UV-activated K+ channels
do mediate apoptosis in ML-1 cells [17]. It has been shown
that the loss of intracellular K+ activates interleukin 1a-
converting enzyme (ICE) [18–20]. Recent studies indicate
that UV irradiation-induced activation of ICE and JNK-1
occurs after the stimulation of K+ channels and to the loss of
intracellular K+. This mechanism has been implicated in
apoptosis in neuronal cells [19]. In addition, suppression of
K+ channel activity protects corneal epithelial cells from UV-
induced DNA fragmentation and cell death. However, this
suppression of K+ channel activity does not prevent corneal
epithelial apoptosis induced by etoposide because it directly
inhibits topoisomerase II activity in the nucleus rather than
affecting UV-induced cell membrane events.
Although extensive studies have elucidated the molecular
mechanisms that mediate the activation of JNKs, it remains
unclear whether activation of JNKs by various stress signals
involves different pathways. To determine whether JNK
activation by different genotoxic stressors was mediated
by the same cellular components, we measured activities
and subcellular localization of JNKs in ML-1 cells exposed
to either UV irradiation or H2O2. We observed that JNK
activation by UVand H2O2 is mediated by distinct pathways
and that K+ channel activity is required for UV-induced, but
not H2O2-induced, activation of JNKs. Moreover, redox
status of the cell can affect both UV- and H2O2-induced
activation of JNKs.
Fig. 1. Activation of a 4-AP-sensitive K+ current by UV irradiation. Whole-
cell currents were measured after exposure of ML-1 cells to UV light (60 J/
m2) in the presence or absence of 4-AP (1 mM). The membrane potential
was depolarized from a holding potential of � 60 to 0 mV. Currents were
normalized as I/Ic where Ic and I represent amplitudes of the K+ current
measured at 0 mV before and after UV light and H2O2 stimulation,
respectively. Data were obtained from four independent experiments and
plotted as means with standard error bars. Asterisks represent the significant
difference ( P < 0.01).
Experimental procedures
Materials
Rabbit polyclonal antibodies against JNKs and p38 as
well as to the activated (phosphorylated) versions of these
proteins were obtained from Cell Signaling Technology
(Beverly, MA, USA). Mouse monoclonal antibodies against
human g-tubulin, the protease inhibitor cocktail, and 4-
aminopyridine (4-AP) were purchased from Sigma (MO,
USA). H2O2 was purchased from Yuanda Peroxides Co. Ltd.
(Shanghai, China). ML-1 and U937 cell lines, both myelo-
blastic, were obtained from American Type Cell Collection
(Manassas, VA, USA). The labeling kit for DNA strand
breaking was from Phoenix Flow Systems, Inc. (San Diego,
CA, USA). Okadaic acid and sodium orthovanadate were
purchased from Calbiochem (San Diego, CA, USA) and
[g32P]-ATP was from New England Nuclear (MA, USA).
Cell culture and treatments
ML-1 and U937 cells were cultured in RPMI1640 con-
taining 7.5% heat-inactivated fetal bovine serum (PAA
Laboratories, Linz, Austria) containing antibiotics (100
Ag/ml penicillin and 50 Ag/ml streptomycin sulfate) in a
humidified incubator at 37jC with 5% CO2. For UV
irradiation experiments, cells were exposed to UV-C at an
intensity of 60 J/m2 in a UV cross-linker (UVP Products,
Upland, CA, USA) for 10, 30, 60, 120, and 240 min (unless
otherwise specified). Cells (3 � 105/ml) were also exposed
to H2O2 at 0.3 mM and 1 mM in a serum-depleted medium
for various times. In some experiments, ML-1 cells (3 �105/ml) were treated with 4-AP, a K+-channel blocker, at
different concentrations for 30 min before UV or H2O2
treatment. ML-1 cells were also treated with glutathione
(1 mM), N-acetylmaleimide (0.1 mM), dimethylsulfoxide
(0.2%), or mannitol (2 mM) for 30 min before UV or H2O2
treatment.
ell Research 297 (2004) 461–471 463
Patch clamp studies
For whole-cell K+ current recording, the nystatin-perfo-
rated-patch technique was used. Pipettes with a resistance of
3–4 mV when filled with 150 mM KCl solution were
manufactured with a two-stage puller (PP-83, Narishige).
J. Gao et al. / Experimental C
Fig. 2. K+ channel activity is necessary for UV- but not H2O2-induced apoptosis. (
times in the presence or absence of 4-AP (1 mM). TUNEL staining as described in
cell death. Green staining denotes the cells undergoing apoptosis. (B) Quantificati
H2O2 in the presence or absence of 4-AP. Data (mean F SE) were summarized
The pipette tip was filled with a nystatin-free solution
containing 140 mM KCl, 2 mM MgCl2, 0.5 mM CaCl2, 2
mM ATP, 0.05 mM GTP, 1 mM EGTA, and 10 mM HEPES
(pH 7.2). Pipette was back-filled with the same solution
with 200 Ag/ml of nystatin. The bath solution was composed
of 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 10 mM HEPES
A) ML-1 cells were exposed to UV (60 J/m2) or H2O2 (0.3 mM) for various
Materials and methods was performed to visualize the extent of programmed
on of percent ML-1 cell death at various times after stimulation with UVor
from three independent experiments.
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471464
(pH 7.4). An Axopatch 200A patch-clamp amplifier was
used to measure whole-cell current. Data were collected and
analyzed with pCLAMP software (Axon Instruments, Inc.).
Channel activity was determined as a fraction of currents
measured in UVor H2O2 in the presence or absence of 4-AP
(I) and in control conditions (Ic). All experiments were
performed at room temperature (21–23jC). Results were
analyzed by the Student’s t test.
TUNEL assay
ML-1 cells cultured in 100-mm dishes were treated with
4-AP or vehicle for 5 min before exposure to UV-C for 5
min. Cells harvested at 5, 30, 60, and 180 min post UV
exposure were spun onto slides through cytocentrifuge.
After fixing in methanol, cells on slides were rinsed three
times in PBS and subjected to DNA strand-breaking label-
ing via the APO-BrdU procedure according to the protocol
provided by the supplier. BrdU incorporated into the 3VOHgroups via the action of terminal deoxynucleotidyl trans-
Fig. 3. UVand H2O2 can both induce JNK phosphorylation. ML-1 cells were expo
Cell extracts were prepared and equal amounts of cell lysates were blotted for th
migrated faster than pJNKp46 band was also detected by the phospho-JNK anti
antibody), we designated this band as nonspecific (NS). As a loading control, the s
The experiment was repeated for at least three times and identical results were o
ferease was recognized by the anti-BrdU monoclonal anti-
body conjugated with FITC. The specific signals were
detected by fluorescent microscopy. Apoptotic cells (green)
in each treatment group were scored, and statistical signif-
icance of difference in the apoptotic rate between cells
treated with or without 4-AP was determined using the
Student’s t test.
Western blot analysis
ML-1 cells (4 � 106) were lysed in 160 Al of a lysis
buffer (20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 1.5 mM
MgCl2, 2 mM EDTA, 10 mM sodium pyrophosphate, 25
mM h-glycerophosphate, 10% glycerol, 1% Triton X-100,
1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl
fluoride) supplemented with 1 � protease inhibitor cocktail
and 10 AM okadaic acid. After incubation on ice for 10
min, cell lysates were then precleared by centrifugation at
13,000 � g for 25 min. Equal amounts of cell lysates (20
Ag) were fractionated by SDS-PAGE followed by electro-
sed to either UV (A and B) or H2O2 (C and D) for different lengths of time.
e phosphorylation status of JNK-1 and JNK-2. One prominent protein that
body. As its status is unknown (failed to be detected by the general JNK
ame blots were also blotted for all forms of JNKs using a pan-JNK antibody.
btained.
Fig. 4. K+ channel activity is involved in UV- but not H2O2-induced JNK
phosphorylation. ML-1 cells were exposed to UV (A, 60 J/m2) or H2O2 (B,
0.3 mM) for 60 min in the presence of various concentrations of 4-AP. Cell
extracts were prepared and equal amounts of cell lysates were blotted for
the phosphorylation status of JNK-1. As a loading control, the same blots
were also blotted for all forms of JNKs. The experiment was performed at
least three different times and representative results were shown.
Fig. 5. UV induces p38 phosphorylation that can be blocked by 4-AP
treatment. ML-1 cells were exposed to UV (60 J/m2) for the indicated
periods of time in the absence (A) or for 1 h in the presence (B) of different
concentrations of 4-AP. Cell extracts were prepared from the treated cells
and Western blots were probed using antibodies against either phosphor-
ylated or total p38 proteins. The experiment was repeated for three times as
before.
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471 465
transferring to nitrocellulose membrane. After blocking
with 5% nonfat milk in Tris-buffered saline containing
0.1% Tween 20 (TBST), the nitrocellulose membranes
were incubated with primary antibodies in TBST contain-
ing 5% nonfat milk overnight at 4jC. After washing three
times with TBST, the membranes were then incubated with
a goat anti-rabbit immunoglobulin G (IgGs) conjugated
with horse radish peroxidase (1:2000). Specific signals
were detected with enhanced chemiluminescent procedure
using a Supersignal West Pico Kit (Pierce, Rockford, IL,
USA).
Fluorescence microscopy
Localization of JNKs was determined by double immu-
nofluorescence analysis of a centrosomal marker. ML-1
cells spun onto culture slides via a Cytospin were fixed in
methanol for 5 min at room temperature. Fixed cells were
treated with 0.1% Triton X-100 in PBS for 5 min on ice,
and then washed three times with ice-cold PBS. After
blocking with 2.0% bovine serum albumin (BSA) in PBS
for 15 min, cells were incubated for 1 h with rabbit
polyclonal antibodies to JNKs and a mouse monoclonal
antibody to g-tubulin in 2% BSA solution. After the
primary antibody incubation, cells were washed three times
with PBS and then incubated with Rhodamine-Red-X-
conjugated anti-mouse IgGs and/or fluorescein isothiocya-
nate (FITC)-conjugated anti-rabbit IgGs (Jackson Immuno
Research) for 1 h in the dark. Fluorescence microscopy was
performed on a Nikon microscope and images were cap-
tured using a digital camera (Optronics) using Optronics
MagFire, Image-Pro Plus softwares. Statistical significance
of difference in the percentage of cells with diffused (non-
centrosomal) JNKs between cells treated with UV and cells
treated with UV plus 4-AP was determined by the Student’s
t test.
Kinase assays
Immunocomplex kinase assays were performed essen-
tially as described Ref. [19]. In brief, ML-1 cells treated
with UV or H2O2 for various times were lysed as
described above. An equal amount of cell lysates (0.5
mg) was subjected to immunoprecipitation with antibodies
to JNKs. The resulting precipitates were resuspended in a
kinase buffer as described [5]. The kinase reaction was
initiated by the addition of [g-32P]ATP (2 ACi) and ATF-
2. After incubation for 30 min at 37jC, the reaction
mixtures were analyzed by SDS-PAGE followed by im-
munoblotting for JNKs and by autoradiography for phos-
pho-ATF-2. Each assay was repeated for at least three
times and parallel results were obtained. Changes of
kinase activity were presented as a fraction of band
densities (as arbitrary units) of treated cells over the
control cells on X-ray films.
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471466
Results
We have previously shown that genotoxic stressors such
as UV irradiation and ROS exposure induce apoptosis in
established cell lines [5,12,17]. We have also shown that K+
channels are involved in UV-induced apoptosis [5,21]. To
determine whether K+ channel activity is an essential part of
apoptosis induced by various genotoxic stresses, we ex-
posed ML-1 cells to UV light or H2O2 in the presence or
absence of 4-AP. Effects of UV irradiation or H2O2 on K+
activity were measured using the nystatin-perforated whole-
cell patch clamp. The whole-cell current was recorded that
resulted from depolarization of the membrane potential
Fig. 6. Subcellular localization of JNKs before and after exposure to UV irradiation
tubulin (red). DNA was stained with DAPI (blue). Stained cells were examined
different periods of time in the absence or presence of 4-AP (1 mM) were stained
fluorescence microscopy. (C) ML-1 cells were exposed to UV light for various tim
discrete staining (dot) of centrosomal JNKs was scored using a fluorescent micros
represent the average of three independent experiments.
from a holding potential of �60 to 0 mV. Normalized K+
currents in the presence or absence of 1 mM 4-AP were
determined by a fraction of UV-induced current versus
control current amplitudes (I/Ic) where I and Ic represent
currents measured with and without stimulations, respec-
tively. Upon exposure of ML-1 cells to UV light, the
amplitude of the K+ current was markedly increased. The
UV-evoked K+ current was sensitive to 4-AP as it was
completely blocked by this compound (Fig. 1). However,
H2O2 treatment failed to activate K+ channel as compared
with the untreated control (Fig. 1), suggesting a difference
in K+ requirement in ML-1 cells upon exposure to various
genotoxic stresses.
. (A) ML-1 cells were double-stained with antibodies to JNKs (green) and g-
by fluorescent microscopy. (B) ML-1 cells exposed to UV (60 J/m2) for
with the JNK antibody. The subcellular location of JNKs was examined by
es in the presence or absence of 4-AP. The percent of ML-1 cells without
cope. At least two hundred cells from each treatment were examined. Data
Fig. 7. K+ current is necessary for UV-induced JNK activation.ML-1 (A) and
U937 (B) cells were exposed to UV (60 J/m2) in the presence or absence of 4-
AP (1 mM). Cell extracts were prepared and equal amounts of cell lysates
were immunoprecipitated with the antibody to JNK. The JNK immunopre-
cipitates were used for in vitro kinase assays using recombinant ATF-2 as
substrate as described in Materials and methods. After reaction, the mixture
was fractionated on denaturing polyacrylamide gel followed by autoradiog-
raphy. Immunoprecipitation efficiency was also determined by blotting with
the antibody to JNKs. JNK activities were quantified by densitometric
scanning. Data represent the average of three independent experiments.
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471 467
To determine whether K+ channel activity is essential for
ROS-induced apoptosis, we treated ML-1 cells with H2O2
or UV for various times in the presence or absence of 4-AP.
TUNEL assays revealed that ML-1 cells underwent apopto-
sis in a time-dependent manner (Fig. 2A). By 3 h post UV
exposure, nearly 50% of ML-1 cells were apoptotic (Fig.
2B). In contrast, no significant apoptosis was detected when
UV-irradiated ML-1 cells were pretreated with the K+
blocker 4-AP (Figs. 2A and B). H2O2 treatment also
induced rapid apoptosis, and pretreatment of ML-1 cells
with 4-AP did not obviously affect the rate of cell death
(Figs. 2A and B), suggesting that K+ channel activity is not
involved in apoptotic response initiated by H2O2.
Transcription factors such as AP-1, NF-nB, and p53 are
rapidly activated upon exposure of cells to genotoxic
stresses [22,23]. As JNKs are known to directly regulate
these transcription factors, we measured phosphorylation
status of JNKs in ML-1 cells exposed to UV for various
times. We observed that both JNK-1 (p54) and JNK-2 (p46)
were rapidly phosphorylated upon exposure to UV at a dose
of 60 J/m2, and that a higher dose of UV treatment (300 J/
m2) apparently neither boosted nor shortened the induction
of JNK phosphorylation (Figs. 3A and B). Although the
protein level of JNK-2 was much lower than that of JNK-1,
its phosphorylation level was comparable to that of JNK-1.
Upon H2O2 exposure, phosphorylation of JNKs was in-
creased in a manner similar to that induced by UV (Figs. 3C
and D). Again, the peak phosphorylation of JNKs was
around 1 h post exposure. It was noted that JNK phospho-
specific antibody detected a band that migrated faster than
the JNK-2. As it was not detected by the pan-JNK antibody,
we designated it as a nonspecific signal (NS). Nonetheless,
activation of JNK-1 by both UV and H2O2 was very
consistent in various experiments.
To determine whether JNK activation by UV and H2O2
was affected by K+ channel activity, ML-1 cells were
pretreated with various concentrations of 4-AP before ex-
posure to UV or H2O2. Whereas phosphorylation of JNK1
induced by UV was significantly inhibited by 4-AP at a
concentration higher than 0.5 mM, its phosphorylation
induced by H2O2 was largely unaffected by 4-AP even at
a 2 mM concentration (Figs. 4A and B). We focused on the
activation of JNK1 in this series of experiments as its
phosphorylation was more consistent in our assays. The
phosphorylation of p38 was also greatly induced by UV
irradiation and the peak phosphorylation was around 2
h after UV treatment (Fig. 5A). Likewise, 4-AP significantly
inhibited UV-induced phosphorylation of p38 (Fig. 5B)
although the level of inhibition was not nearly as complete
as that of JNKs.
JNKs are primarily localized in the cytosol. A significant
fraction of activated JNKs is thought to be localized in the
nucleus where they target substrates such as c-Jun. A recent
study showed that JNKs are present in the cell in both soluble
and insoluble forms [24]. Whereas soluble JNKs are present
in the cytoplasm, insoluble JNKs are localized to the
centrosomes [24]. We examined the subcellular localization
of JNKs in ML-1 cells via fluorescent microscopy. A low
level of JNK staining was detectable throughout the cyto-
plasm of ML-1 cells and JNK signals detected as a bright dot
were present in each cell (Fig. 6A, arrow). Immunostaining
with g-tubulin, a centrosome-specific protein, confirmed that
JNKs were concentrated at the centrosomes (Fig. 6A). We
next examined the subcellular localization of JNKs in ML-1
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471468
cells exposed to UV for various times. Five minutes post UV
exposure, JNK staining that started to disperse no longer
confined to the centrosomes (Fig. 6B). The dissipation of
JNKs throughout the cell persisted after UV treatment for 30
min or longer (Figs. 6B and C). On the other hand, when
cells were pretreated with 4-AP, centrosomal JNKs were not
significantly dispersed and JNKs remained at the centro-
somes or the centrosomal region in most ML-1 cells (Figs.
6B and C). In addition, little nuclear JNK signals were
detected 30 min after UV treatment (Fig. 6B).
The enhanced phosphorylation status and subcellular
translocation of JNKs strongly suggest that K+ channel is
required for the activation of JNKs. We directly measured
JNK activities in ML-1 cells exposed to UV in the presence
or absence of 4-AP. Immunocomplex kinase assays revealed
that UV treatment strongly activated JNKs and that pre-
treatment with 4-AP completely abolished the activation
(Fig. 7A), again indicating the requirement of K+ channel in
Fig. 8. Redox status plays an important role in the activation of JNKs upon UV irra
(60 J/m2, C and D) in the presence or absence of glutathione (GSH), N-acetylm
amounts of cell lysates were immunoprecipitated with antibody to JNK. The JNK
vitro substrate as described in Materials and methods. GSH and NEM but not DM
activities were quantified by densitometric scanning. Data represent the average o
JNK activation by UV. The requirement of the K+ channel
activity in JNK activation was also confirmed in U937 cells
(Fig. 7B). We then asked whether the redox status or
sulfhydryl group was involved in regulating JNK activation
by H2O2 and UV. ML-1 cells were pretreated with reducing
agents before exposure to H2O2 as well as to UV. JNK
activity was increased upon H2O2 exposure that peaked
around 1 h in ML-1 cells, and treatment with glutathione
(GSH), a potent reducing agent, significantly abolished
H2O2-induced activation of JNKs (Figs. 8A and B). Pre-
treatment of the cells with DMSO and mannitol, the latter
being a hydroxyl radical scavenger, did not affect JNK
activity (Fig. 8B). To determine the effect of reducing agents
on JNK activation by UV, ML-1 cells were pretreated with
GSH and N-acetylmaleimide (NEM), the latter being a
strong sulfhydryl alkylating agent that also affects K+
current in vivo, before UV irradiation. Immunocomplex
kinase assays revealed that NEM completely inhibited
diation. ML-1 cells were exposed to either H2O2 (0.3 mM, A and B) or UV
aleimide (NEM), dimethyl sulfoxide (DMSO), or Mannitol (Man). Equal
immunoprecipitates were analyzed for kinase activities using ATP-2 as in
SO and Man selectively block H2O2- and UV-induced JNK activation. JNK
f three independent experiments.
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471 469
UV-induced JNK activities in vitro (Fig. 8C). Consistently,
the reducing agent GSH also blocked the kinase activity
induced by UV (Fig. 8D), indicating that redox status does
play an important role in the activation of JNKs upon H2O2
or UV irradiation.
Discussion
In the present study, we determined whether JNK acti-
vation by different genotoxic stressors was mediated by the
same cellular components by measuring activities and
subcellular localization of JNKs in ML-1 cells exposed to
either UV irradiation or H2O2. Our results suggest that JNK
activation by UV and H2O2 is mediated by distinct yet
overlapping pathways and that K+ channel activity and
redox status are differentially required for UV- and H2O2-
induced activation of JNKs.
We found that exposure of ML-1 cells to UV and H2O2
rapidly induces phosphorylation and activation of JNKs.
Moreover, UV-induced phosphorylation, activation and sub-
cellular translocation of JNKs are inhibited by 4-AP, indi-
cating that K+ channel activity is essential for the JNK
functional activation in vivo. Indeed, UV irradiation acti-
vates a 4-AP-sensitive K current. Interestingly, H2O2 also
induces phosphorylation and activation of JNKs in a kinet-
ics similar to that induced by UV. However, JNK activation
by H2O2 does not activate K currents and is not affected by
4-AP, suggesting that there exist distinct pathways of JNK
activation that can be distinguished by K+ channel depen-
dence. Although the exact nature of these molecular path-
ways remains unclear, UV light directly irradiates the
surface membrane of the cell, which may trigger the
activation of K+ channels in the membrane, an essential
early process in the signaling cascade that leads to JNK
activation. The increased K+ channel activity may result in a
change in intracellular K+ concentration and a change in cell
volume, leading to activation of stress response proteins
such as JNKs and p38. On the other hand, H2O2 is thought
to be able to penetrate through the cell surface membrane,
bypassing the requirement for K+ channel and directly
activating downstream molecules in the JNK activation
cascade. As we have shown, UV irradiation induces an
activation of p38, which is consistent with previous studies
in other cell types [25,26]. Relative to JNK/SAPK activa-
tion, activation of p38 in ML-1 cells in response to UV
irradiation was much less sensitive to suppression of K+
channels by 4-AP (>1.0 mM, Fig. 5B). It remains unknown
as to what is the effect of UV-induced p38 activation on the
survival of ML-1 cells. At present, we cannot exclude that
there may be potential side effects of 4-AP directly on JNK
activities or indirectly on its signaling pathway other than
blocking K+ channel.
Numerous studies reveal that K+ channel activity is
affected by apoptotic inducers including reactive oxygen
species [27], Fas ligand and TNF [28], and anticancer drugs
[29]. There is growing evidence showing that K+ channel
activities are probably involved in programmed cell death.
Neurons undergoing apoptosis exhibited an upregulation of
outward K+ currents. This enhancement of outward K+
current, induced by serum deprivation and staurosporine,
can be prevented by the K+ channel blockers 4-AP and
TEA. It has also been observed that the K+ channel opener
cromakalim induces neuronal apoptosis [30]. In addition,
the K+ channel blocker 4-AP inhibits the shrinkage of
human eosinophils undergoing apoptosis induced by cyto-
kine withdrawal [31], and a combination of two K+ channel
blockers, TEA and 4-AP, inhibited IL-1b release from LPS-
stimulated monocytes [32]. Thus, blockade of UV irradia-
tion-induced activation of K+ channels by 4-AP in ML-1
cells provides further evidence that inhibition of possible K+
efflux, the consequent membrane hyperpolarization, and
decrease in cell volume prevent activating a particular
signaling system leading to cell apoptosis.
UV irradiation also causes oxidative stress [33]. Pretreat-
ment of ML-1 cells with GSH and NEM prevents the
activation of JNKs by UV, suggesting that UV may trigger
an oxidative stress on cell surface membrane, an event before
the activation of K+ channel. It is also possible that K+
channel activity can be directly affected by the oxidative
stress. For example, disulfide bond formation, largely influ-
enced by the redox status, could be an essential part that
regulates the K+ channel molecules. It has been shown that
the antioxidant agent GSH plays a central part in the cellular
defense against UV radiation and that an increase in GSH
concentration may provide photoprotection in many cell
types [34]. We have demonstrated that GSH blocks UV-
induced JNK activation in ML-1 cells in a concentration
range of 5–15 mM (data not shown). A moderate concen-
tration of GSH prevents UV- and H2O2-induced JNK acti-
vation in ML-1 cells, the target(s) of which appears to lie at
the very upstream of the signaling cascade. Moreover, NEM,
a protective alkylating agent of sulfhydryl group only [35],
prevents UV-induced JNK activation without affecting the
effect of H2O2-induced JNK activation. It suggests that NEM
may interact with sulfhydryl group in the channel protein to
inhibit UV-induced changes of the channel conformation.
The mechanism of NEM effect is quite different from the
effect of 4-AP. 4-AP is a specific blocker of voltage-depen-
dent K+ channels and it binds to the pore region of the
channel to inhibit K+ ion fluxes [36]. Free radical scavengers
DMSO and mannitol are also used to study whether UV
induced K+ channel activation and downstream JNK activa-
tion. The membrane permeable and impermeant scavengers,
respectively, did not affect UV-induced response in ML-1
cells, supporting the notion that a conformational change of
the channel protein in response to UV irradiation may be
required to activate the channel.
A direct consequence of enhanced K+ channel activity is
a change in the cell volume due to water loss, which
inevitably affects the cytoskeletons of the cell. Centrosome,
the major microtubule organization center of the cell, may
J. Gao et al. / Experimental Cell Research 297 (2004) 461–471470
function to sense cellular (e.g., microtubule) stresses. Reg-
ulatory molecules that reside at the centrosome can be
activated because the microtubule stress initiated by the cell
volume changes. This notion is supported by several lines of
existing evidence: (i) JNKs are localized to the centrosomes,
and UV irradiation causes their rapid subcellular transloca-
tion; (ii) UV-induced subcellular translocation is largely
blocked by 4-AP treatment; (iii) nocodazole, an agent that
depolymerizes microtubules, also activates MEKK1 [37], an
upstream activator of JNKs, and p38 [38]. An alternative
explanation for JNK subcellular localization at the centro-
somes is that the centrosome serves as an intracellular
transportation depot. Signaling molecules move rapidly
along the tracks of microtubules and are modified (e.g.,
phosphorylation) at the centrosome so that the signals on the
cell surface membrane can be directed to various organelles
including the nucleus. The fact that many signaling protein
kinases, such as MEK1, ERKs, and Plks [39] (unpublished
data), and checkpoint proteins, including p53 and BRCAs
[40], are found at the centrosomes supports this possibility
although it remains to be elucidated as to the exact role of
JNKs as centrosomal proteins.
Acknowledgments
We thank the members in the joint laboratory of Drs. Lu,
Dai, and Xu for helpful discussions. We are also grateful to
Drs. Guoqiang Chen and Ying Jin for the use of equipment,
and Xueyu Chen for assistance. This work was supported in
part by a start-up fund from the SSMU, a core support from
the Chinese Academy of Sciences, a One Hundred Talent
Grant from the Chinese Academy of Sciences (M.X.), a
National Key Program (973) for Basic Research of China
grant (NO2002CB512805), and a grant from E-Institute of
Shanghai Municipal Education Commission.
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