From the Laboratory of Cellular and Molecular Biology, NIA-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224
Received for publication, January 9, 2003, and in revised form, January 21, 2003
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ABSTRACT |
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Although epidemiological studies have long
established that inorganic arsenic is a potent human carcinogen, the
underlying mechanisms are still poorly understood. Recent studies
suggest that inorganic arsenic may act as a tumor promoter by
perturbing key signaling transduction pathways. We have shown
previously that arsenite can potently activate the mitogen-activated
protein kinase cascades and induce the expression of
proliferation-associated genes, including proto-oncogenes
c-jun and c-fos. In order to elucidate further
the molecular mechanisms underlying its tumor-promoting properties, we
investigated the signaling events involved in arsenite-mediated induction of c-fos and c-jun. We found that
induction of both c-fos and c-jun by arsenite
can be substantially inhibited by the MEK- selective inhibitor U0126,
suggesting that the ERK pathway is critically involved in their
up-regulation. Interestingly, arsenite dramatically induced the
phosphorylation and acetylation of histone H3 preceding the induction
of mRNAs encoding c-fos and c-jun. Finally,
chromatin immunoprecipitation assays revealed that arsenite treatment
markedly induced the phosphorylation/acetylation of histone H3
associated with the c-fos and c-jun genes
through an ERK-dependent pathway. Our results strongly
suggest that arsenic-triggered alterations in chromatin structure
perturb specific gene transcription, including that of proto-oncogenes
c-jun and c-fos, and may thereby contribute to
the carcinogenic process.
Arsenite, the inorganic trivalent arsenic compound, is a potent
human carcinogen with significant worldwide exposure through natural
contamination of food and drinking water (1). Epidemiological studies
have shown that chronic arsenic exposure is associated with an
increased incidence of skin, lung, and bladder cancers (1, 2). At the
present arsenic regulatory standard of 50 ppb in drinking water, issued
by the United States Environmental Protection Agency in 1942, the
cancer risk due to arsenic contamination is estimated to be comparable
with that of environmental tobacco smoke or radon in homes (3). A
recent analysis conducted by the National Academy of Sciences indicated
that the risk of lung and bladder cancer from arsenic in drinking water
is several times higher than that estimated by the Environmental
Protection Agency when it developed its 10-ppb standard (projected to
go into effect in 2006). According to the new analysis, at the arsenic
standard of 10 ppb in drinking water, the combined lifetime risk of
lung and bladder cancer is between 30 and 37 per 10,000 people (4). In
fact, ~350,000 individuals in the United States drink water containing arsenic levels higher than 50 ppb (3). In many developing countries, arsenic contamination in drinking water can be as high as
1,800 ppb and represents a very serious threat to public health (1,
5).
Although epidemiological studies have long established that arsenite is
potently carcinogenic, the underlying mechanisms remain poorly
understood. Two plausible models have been proposed to explain the
carcinogenic actions of arsenic. According to the first and more
controversial model, arsenite would cause genetic abnormalities
affecting chromosome structure without directly interacting with DNA
(6-8). In the second model, arsenite is proposed to act as a tumor
promoter (6, 9). The latter hypothesis is supported by the findings
that arsenite can induce anchorage independence in human diploid
fibroblasts (10), enhance cell proliferation in bladder epithelium in
mice (11), and potentiate the mutagenic effects of short wavelength
ultraviolet (UVC) radiation in cultured cells (12-14). Consistent with
the notion that arsenite acts on signaling pathways that regulate cell
proliferation, a number of proliferation-associated genes, notably the
proto-oncogenes c-fos and c-jun, have been found
to be induced rapidly in response to arsenite (9, 11, 15). Both
proto-oncogenes encode components of the mitogen-inducible
immediate-early transcription factor AP-1 and have been implicated in
promoting cell proliferation by affecting G1 to S-phase
progression. Moreover, it is well established that the abnormal
expression of both c-fos and c-jun can induce cell transformation (16). Recently, c-jun induction in
response to proliferative stimuli was shown further to be critical for the down-regulation of both the tumor suppressor p53 and the
cyclin-dependent kinase inhibitor p21/WAF-1/CIP1 (17).
Therefore, perturbation of the expression of c-fos and
c-jun by arsenic compounds is likely to play an important
role in carcinogenesis.
The signal transduction pathways that regulate c-fos and
c-jun transcription have been thoroughly investigated. The
mitogen-activated protein
(MAP)1 kinase pathway plays a
crucial role in the induction of both c-fos and
c-jun in response to many extracellular stimuli, such as
growth factors and cellular stress (18, 19). Recent studies (20, 21)
have indicated that growth factors can stimulate nucleosomal histone H3
phosphorylation/acetylation at the loci of both c-fos and
c-jun through a MAP kinase-dependent mechanism, thus providing an important link between the signaling events initiated
at the cell membrane and the modulation of gene transcription in the
nucleus. Like growth factors, arsenite can also activate various MAP
kinase cascades, including those that culminate in the activation of
the extracellular signal-regulated kinase (ERK), c-Jun N-terminal
kinase, and p38 (22). ERK activation by arsenite is primarily mediated
by the epidermal growth factor receptor and the oncoprotein Shc (15).
In this report, we have extended our early studies on the carcinogenic
mechanisms of arsenite and have investigated the chromatin
modifications occurring at the nucleosomes of c-fos and
c-jun following arsenite stimulation. We report that
arsenite induces rapid phosphorylation and acetylation of histone H3,
events that precede the induction of both c-fos and
c-jun in normal human diploid fibroblasts. By using
chromatin immunoprecipitation (ChIP) assays, we demonstrate that the
arsenite-triggered phosphoacetylation of histone H3 on the
c-fos and c-jun chromatin is primarily mediated
by the ERK MAP kinase cascade. Our results support the notion
that arsenic compounds can induce chromatin remodeling leading to the
altered expression of genes which, as illustrated by proto-oncogenes
c-fos and c-jun, may contribute to their
carcinogenic effects.
Cell Culture and Treatment--
Early passage WI-38 human
diploid fibroblast cells were cultured in minimum essential medium
(Invitrogen) supplemented with 10% fetal bovine serum (HyClone, Logan,
UT). Subconfluent cultures were rendered quiescent by incubating the
cells in minimum essential medium containing 0.5% fetal bovine serum
for 36 h prior to treatment. Sodium arsenite (Sigma) and
trichostatin A (TSA, Sigma) were directly added to the medium. U0126
(Promega, Madison, WI), SB203580 (Sigma), SB202190 (Calbiochem),
PD169316 (Calbiochem) and p38 MAP kinase inhibitor (p38 INH,
Calbiochem) were dissolved in dimethyl sulfoxide and added to the
medium to a final concentration of 10 µM 15 min prior to treatment.
Northern Blot Analysis--
Total RNA was isolated with STAT-60
(Tel-Test B, Friendswood, TX) and Northern blot analysis performed as
described previously (23). Messenger RNAs encoding c-fos and
c-jun were detected using probes derived from either the
human EST clone AI684077 or a cDNA fragment of human
c-jun kindly provided by J. Woodgett (see Ref. 24),
respectively. To monitor differences in loading and transfer among
samples, blots were stripped and rehybridized with an end-labeled
oligonucleotide complementary to the 18 S rRNA.
Antibodies, Western Blotting, and Immunofluorescence--
To
analyze the phosphorylation status of various MAP kinases, Western
blotting was performed as described previously (23) using 20 µg of
total cell lysate. Rabbit polyclonal antibodies recognizing
phospho-p38, total p38, and phospho-p44/p42 ERK MAP kinases were
from Cell Signaling (Beverly, MA). A mouse monoclonal antibody
against p44/p42 ERK was from Transduction Laboratories (Lexington, KY).
To detect the modification of histone H3, crude histone proteins were
extracted using sulfuric acid (23) and then analyzed by Western
blotting using rabbit polyclonal antibodies recognizing either
phospho-histone H3 (Ser(P)-10) (Cell Signaling), phosphoacetyl-histone
H3 (Ser(P)-10/Ac-Lys-14, Upstate Biotechnology, Inc., Lake Placid, NY),
phospho-histone H2A.X (p-Ser-139, Upstate Biotechnology, Inc.),
acetyl-histone H3 (Ac-Lys-14, Upstate Biotechnology, Inc.),
acetyl-histone H3 (Ac-Lys-9,14, Upstate Biotechnology, Inc.), or a
rabbit antibody recognizing total histone H3 (Cell Signaling).
For the immunofluorescence studies, Alexa 488-conjugated goat
anti-rabbit and Alexa 568-conjugated goat anti-mouse secondary antibodies (Molecular Probes, Eugene, OR) were used according to the
manufacturer's specifications. Phospho-histone H3 was detected using a
rabbit polyclonal antibody specifically recognizing phosphorylated (Ser-10) histone H3 (Cell Signaling). Total histone H3 was detected using a mouse monoclonal antibody (25). DAPI staining was performed as
described previously (26). Samples were visualized by either fluorescence microscopy (Carl Zeiss, New York) or confocal microscopy (Zeiss LSM-410 inverted confocal microscope equipped with a 63× NA 1.4 oil immersion objective). The confocal pinhole was set to obtain a
spatial resolution of 0.4 µm in the horizontal plane and 1 µm in
the axial dimension. Image processing and presentation were done using
MataMorph 4.6.3 software (Universal Imaging, Inc., West Chester, PA).
ERK, MAPKAPK2/3, and MSK1 Activity Assays--
MSK1
was immunoprecipitated from cell lysates using a polyclonal antibody
(Upstate Biotechnology, Inc.), and its activity was determined by
assessing the phosphorylation of a synthetic peptide (Crosstide) in the
presence of [ Chromatin Immunoprecipitation (ChIP) and Real Time
PCR--
Chromatin was cross-linked using formaldehyde and broken down
to fragments with an average size of ~2 kbp through brief sonication according to Clayton et al. (20). The resulting chromatin
solution was divided equally into four fractions that were subsequently used for isolating input DNA (fraction 1) or performing ChIP
assays (fractions 2-4). Chromatin solutions were first incubated for 2 h at 4 °C with 10 µl of rabbit pre-immune serum or with 10 µl of purified antibody specifically recognizing phospho-histone H3
(Ser(P)-10) or 40 µl of purified antibody specifically recognizing phospho-acetylhistone H3 (Ser(P)-10/Ac-Lys-14, Upstate Biotechnology, Inc.). Bovine serum albumin (final concentration 200 µg/ml),
sonicated
Real time PCR was performed using SYBR® Green PCR Master Mix and
GeneAmp® 5700 Sequencing Detection System (Applied Biosystems, Foster
City, CA) using the following parameters: 5 min at 50 °C, 10 min at
95 °C and then 40 cycles of denaturation (95 °C, 30 s),
annealing (60 °C, 30 s), and extension (72 °C, 30 s).
Primer pair GGTCTGCTTCCACGCTTTGCACTGAATTAG and
AGCGCCTTTTTACCCTTGTACGGAAACTG was used to amplify a 172-bp region in
the c-fos promoter. Primer pair
GGGTTGACTGGTAGCAGATAAGTGTTGAG and TCTGGGCAGTTAGAGAGAAGGTGAAAAG was used
to amplify a 200-bp fragment in the human c-jun promoter. Primer pair GGCAAGGTGAACGTGGATGAAGTTGGTG and
GGAGTGGACAGATCCCCAAAGGACTCAAAG was used to amplify a 237-bp
region in the human
After SYBR Green PCR amplification, data acquisition and subsequent
data analyses were performed using the GeneAmp 5700 sequence detection
system (version 1.3). For all the amplifications described in this
paper, the threshold value of the Arsenite Stimulates Histone H3
Phosphorylation/Acetylation and Activates MSK1--
It has
been shown that in mouse fibroblasts, expression of several oncogenes,
including Ha-RasT24, v-mos, and
v-fes, leads to enhanced phosphorylation of histone H3 (28).
Likewise, stimulation of cells with three well established tumor
promoters, TPA, okadaic acid, and UVC, also increased histone H3
phosphorylation (28-30). Recent studies (20, 28, 31, 32) have also
indicated that phosphorylation of histone H3 is coupled to its
acetylation, and such modifications are critical for the induction of
immediate-early genes in response to extracellular stimuli. To address
whether arsenite triggers histone H3 modifications, histone H3
phosphorylation in normal human lung fibroblast WI-38 cells was
examined by immunofluorescence, using an antibody that recognizes
phospho-histone H3 (Ser(P)-10). Compared with control cells, where the
level of phospho-histone H3 was low, arsenite triggered a rapid
increase of phospho-histone H3 in the nucleus. As shown in Fig.
1A, the levels of
phospho-histone H3 after a 60-min treatment with arsenite were
comparable with those seen after treatment with a combination of EGF
and anisomycin, shown previously (20) to enhance potently histone H3
phosphorylation.
EGF-stimulated histone H3 phosphorylation is reportedly restricted to a
small subset of nucleosomes that are associated with active gene
transcription (20, 21, 31, 33, 34). Likewise, in Drosophila
salivary glands, heat shock significantly stimulates histone H3
phosphorylation at a few loci where the heat shock protein genes are
located (35). To investigate whether arsenite stimulates
phosphorylation of histone H3 throughout the entire nucleus or only at
a few loci, confocal microscopy was performed. A mouse monoclonal
antibody recognizing both phosphorylated and unphosphorylated histone
H3 was used to visualize all histone H3 proteins (Fig. 1B).
In unstimulated cells, only a few loci were intensely stained by the
phospho-histone H3 antibody (Fig. 1B). By contrast, in cells
stimulated with EGF + anisomycin, phospho-histone H3 was markedly
elevated and displayed a punctate staining pattern in the nucleus (Fig.
1B). As seen in the EGF + anisomycin treatment group,
arsenite stimulation also resulted in a dramatic increase in
phospho-histone H3, revealing a similar punctate staining pattern in
the nucleus (Fig. 1B). The marked difference between the
staining patterns of phospho-histone H3 and total histone H3 in
arsenite-stimulated cells strongly suggests that arsenite induces the
phosphorylation of histone H3 at a subset of nuclear loci (Fig.
1B).
To examine further the effect of arsenite on histone H3 modification,
histone proteins were extracted with sulfuric acid from either
untreated or arsenite-treated cells. Histone H3 modification was
examined by Western blotting using antibodies specific for either
phospho-histone H3 (Ser(P)-10) or dually modified histone H3
(Ser(P)-10/Ac-Lys-14). Arsenite treatment led to increased histone H3
phosphorylation in a time-dependent manner. This
modification was visible within 10 min after the addition of arsenite
into the medium and continued to increase over the period studied (Fig. 2A). Phosphoacetyl-histone H3
increased in a time-dependent fashion with kinetics similar
to that of histone H3 phosphorylation (Fig. 2A), supporting
the notion that phosphorylation and acetylation of histone H3 are
tightly coupled events (21). In contrast, bulk levels of acetylated H3
did not significantly change after arsenite treatment (Fig.
2A), presumably because acetylated H3 is generally
associated with actively transcribing chromatin, whereas
arsenite-regulated genes constitute only a small subset of the genome,
so arsenite treatment did not greatly affect total levels of acetylated
H3, whereas TSA induced hyperacetylation of histone H3 (Fig.
2A). To confirm the specificity of the phosphoacetyl-H3 antibody on Western blots, lysates from TSA- and EGF + anisomycin-treated cells were included as positive controls for both
phospho-histone H3 and phosphoacetyl-H3 antibodies (Fig.
2A), and only acetylation of histone H3 induced by TSA was
not recognized by the anti-phosphoacetyl-H3 antibody. These
results demonstrate the presence of H3 histone that is dually modified
by phosphorylation and acetylation (at Ser-10 and Lys-14, respectively)
in arsenite-treated cells. Furthermore, to investigate whether
arsenite-induced histone phosphorylation is specific for H3, a rabbit
polyclonal antibody recognizing phospho-H1 (Upstate Biotechnology,
Inc.) and a mouse monoclonal antibody recognizing histone phospho-H2A.X
(phosphorylated at Ser-139, Upstate Biotechnology, Inc.) were used for
Western blot analysis. Staurosporine-treated Jurkat cells (0.5 µM, 4 h) were included as positive control of
phospho-H2A.X. Arsenite treatment did not have any effect on
either histone H1 phosphorylation (data not shown) or histone H2A
phosphorylation at Ser-139 (Fig. 2A).
Because arsenite is a well known activator of MAP kinases ERK and p38,
we sought to assess whether activity of MAP kinases was required for
the observed histone H3 modifications. Phosphoacetylation of histone H3
in arsenite-treated cells was moderately inhibited in cells that were
pretreated with either the MEK inhibitor U0126 or the p38 inhibitor
SB203580 (Fig. 2B). Combination of both U0126 and SB203580
resulted in a more significant inhibition of histone modifications
(Fig. 2B). These observations suggest that
arsenite-triggered MAP kinase activation contributes to the
implementation of histone H3 modifications.
Protein kinase MSK1 lies downstream of p38 and ERK and can be activated
by both mitogenic stimulation and stress (36). Recent studies (32)
suggest that MSK1 may play an important role in mediating the
phosphorylation of histone H3, thus serving as a link between the MAP
kinase cascades and nucleosomal alterations. To examine whether MSK1
could be involved in histone H3 phosphorylation, we examined the
kinetics of MSK1 activation following arsenite treatment. MSK1 was
immunoprecipitated and assayed using a synthetic peptide (Crosstide) as
substrate (36). Arsenite significantly activated MSK1 in a
time-dependent manner (Fig. 2C), but this activation was significantly inhibited in cells pretreated with either
U0126, SB203580, or a combination of both inhibitors, with the combined
inhibitory treatment showing the greatest effect (Fig. 2D).
The similarity between the kinetics of histone H3 phosphorylation and
MSK1 activation as well as the comparable susceptibility profiles toward the two MAP kinase inhibitors support the hypothesis that MSK1
plays a significant role in modulating histone H3 modification in
response to arsenite.
In Human Diploid Fibroblasts, Arsenite Induces c-fos and c-jun
Expression through Mechanisms Partially Dependent on the ERK
Pathway--
To investigate whether histone H3 modification in
response to arsenite may play a role in the induction of
proliferation-associated genes, we first examined the effect of
arsenite on c-fos and c-jun expression in WI-38
cells by Northern blotting (Fig.
3A). Basal c-fos
and c-jun mRNA levels were very low in unstimulated
cells, and arsenite treatment potently increased their abundance in a time- and dose-dependent fashion (Fig. 3A). The
kinetics of induction of mRNAs encoding c-fos and
c-jun was delayed in comparison with that of histone H3
modification (compare Figs. 1 and 2 with Fig. 3).
The effects of arsenite on various MAP kinases in WI-38 cells were
examined by Western blot analysis and immunocomplex kinase assays.
Arsenite treatment resulted in a transient activation of the ERK
MAP kinases, as indicated by the time-dependent increase in
phosphorylated ERK (Fig. 3B). This observation was confirmed by immunocomplex kinase assays, showing that ERK activity reached peak
levels at about 10 min, rapidly declining thereafter (Fig. 3B). In contrast to the early and transient ERK activation,
p38 activation was delayed, and its activity was sustained throughout the period examined (Fig. 3B). This observation was
confirmed by immunocomplex kinase assays, showing that MAPKAPK2/3
activity was sustained throughout the period examined (Fig.
3B).
To investigate the role of ERK and p38 MAP kinases in the induction of
c-fos and c-jun, inhibitors U0126 and SB203580
were used, respectively. U0126 completely abolished
phosphorylation of the ERK MAP kinases (Fig.
4A) but had no effect on p38
activation by arsenite (Fig. 4B). Interestingly, SB203580
significantly augmented ERK activity induced by arsenite (Fig.
4A), suggesting that the p38 pathway antagonized the ERK
pathway. The inhibition of the p38 pathway by SB203580 was confirmed by
the marked attenuation of arsenite-induced phosphorylation of HSP27
(Fig. 4B) and by the dramatic inhibition of
arsenite-stimulated MAPKAPK2/3 activity (Fig. 4B). U0126
substantially inhibited c-fos and c-jun mRNA induction by arsenite, reducing c-fos mRNA levels by
greater than 70% and c-jun mRNA levels by ~50% (Fig.
4C). In contrast, the p38 inhibitor SB203580 neither
inhibited c-fos expression nor affected c-jun
induction by arsenite (Fig. 4C). Interestingly, SB203580
actually further enhanced arsenite-mediated induction of
c-fos (Fig. 4C), consistently with the
observation that SB203580 enhanced ERK activation by arsenite (Fig.
4A). The combined effect of both U0126 and SB203580 on
c-fos and c-jun mRNA induction by arsenite
was similar to that of U0126 alone (Fig. 4C). Taken
together, these results indicate that ERK plays a prominent role in the induction of c-fos and c-jun expression by
arsenite in normal human fibroblasts.
The observation that SB203580 significantly augmented arsenite-induced
ERK activity was confirmed by immunocomplex kinase assays using MBP as
a substrate (Fig. 5A), and by
using a panel of p38 inhibitors (SB202190, PD169316, and p38 INH),
which also augmented arsenite-induced ERK activity (Fig.
5A). Moreover, like SB203580, the other p38 inhibitors were
also capable of further enhancing arsenite-mediated induction of
c-fos but did not affect arsenite-mediated induction of
c-jun (Fig. 5B). These observations further
suggest that the p38 pathway antagonizes the ERK pathway and
ERK-mediated c-fos induction by arsenite in normal human
fibroblasts.
Transcriptional Induction of c-fos and c-jun Is Associated with
Phosphoacetylation of Histone H3 at the c-fos and c-jun Chromatin
Sites--
To examine whether arsenite stimulates histone H3
modification at the c-fos and c-jun loci, control
and arsenite-treated cells were treated with formaldehyde to cross-link
chromatin and genomic DNA. Following solubilization with SDS and
sonication, ChIP assays were carried out using antibodies that
specifically recognize either phospho-histone H3 or
phosphoacetyl-histone H3. Genomic DNA present in the immunoprecipitates
was extracted and analyzed by real time PCR using primers specific for
the amplification of the genes of interest. Real time PCR assays
revealed a considerable increase in the levels of phosphorylated
histone H3 on the c-fos chromatin after arsenite treatment,
reducing the CT value (which represents the number of PCR
cycles required to reach a threshold set arbitrarily) from 33.1 to
29.4, which corresponds to a 9.8-fold increase in the amount of
phospho-histone H3 (Fig. 6A).
Consistent with the notion that acetylation and phosphorylation of
histone H3 are tightly coupled events, arsenite treatment also resulted in a substantial increase in the levels of phosphoacetyl-histone H3 on
the c-fos chromatin, reducing CT from 32.2 to
28.7, which corresponds to a 8.6-fold increase in the amount of
phosphoacetyl-histone H3 (Fig. 6A). Interestingly, U0126
alone substantially inhibited both phosphorylation and
phosphoacetylation of histone H3 at the c-fos chromatin.
SB203580 alone had no significant effect on histone H3 modification at
the c-fos locus (Fig. 6A). Combination of
SB203580 with U0126 did not further reduce histone H3 modification at
the c-fos locus, underscoring the notion that p38 does not
play a significant role in arsenite-induced c-fos expression
in these cells. The specificity of this assay was demonstrated both by the absence of c-fos amplification in the mock
immunoprecipitation samples (Fig. 6, B and C,
Ab
Similarly, histone H3 modification at the c-jun locus was
examined. In response to arsenite treatment, the levels of
phosphorylated and phosphoacetylated histone H3 on the c-jun
chromatin increased 14.9- and 6.9-fold, respectively (Fig.
7A). U0126 strongly inhibited these modifications at the c-jun locus, whereas SB203580 had
no such effect (Fig. 7A). Moreover, pre-immune serum did not
bring down any c-jun chromatin, as indicated by the absence
of c-jun amplification from the mock ChIP samples (Fig.
7B, Ab Epidemiological studies have clearly established that elevated
arsenic levels in drinking water is associated with an increase in the
incidence of cancer of the skin, lung, bladder, kidney, nasal passages,
liver, and prostate (4). Although the carcinogenic mechanisms of
arsenic are not fully understood, recent studies (3, 6, 9, 10, 15)
strongly suggest that arsenic may exert its carcinogenic effects by
perturbing key signal transduction pathways. In this regard, the
c-fos and c-jun proto-oncogenes have been shown
to be inducible by arsenite treatment, both in vitro and
in vivo (9, 11, 15). We have shown previously that arsenite
treatment can activate various MAP kinase cascades and that
arsenite-triggered ERK activation is initiated through the EGF receptor
and mediated by oncoproteins Shc and Ras (15, 22). To gain insight into
downstream mechanisms of action of this carcinogen, we have examined
arsenite-triggered signaling events at the chromatin level. We found
that arsenite treatment caused rapid phosphoacetylation of histone H3,
which preceded the induction of c-fos and c-jun.
By using pharmacological agents that selectively inhibit either the ERK
or the p38 MAP kinase cascades, we demonstrate that the ERK pathway
plays a primary role in mediating the induction of c-fos and
c-jun by arsenite. Through ChIP and quantitative real time
PCR analyses, we show that arsenite treatment enhances histone H3
phosphoacetylation at the loci of both c-fos and
c-jun via an ERK-dependent mechanism. Our
results provide a plausible mechanistic link between the
arsenite-triggered activation of oncogenic events occurring at the cell
surface and the enhanced gene transcription in the nucleus. We propose
that arsenite promotes carcinogenesis, at least in part, by causing specific alterations in chromatin structure that lead to the increased transcription of critical proliferative genes.
The importance of the ERK pathway in mediating c-fos and
c-jun induction by mitogenic stimuli is well established
(18, 19, 37-39). Our findings that arsenite induces the transcription
of both c-fos and c-jun through mechanisms
primarily involving the ERK pathway reinforce the notion that arsenite
may stimulate carcinogenesis by usurping pathways typically utilized by
growth factors. It should be pointed out that although ERK is involved
in the induction of both c-fos and c-jun, it
appears to be more important for inducing c-fos expression.
This is likely due to the fact that the immediate-early induction of
c-jun in the absence of de novo
protein synthesis is primarily mediated by phosphorylation of AP-1
transcription factor complexes composed of c-Jun and ATF-2, a process
that is primarily accomplished by the c-Jun N-terminal kinase pathway (18, 39, 40). Our results are consistent with the findings of Huang
et al. (41), who used transgenic mice carrying an
AP-1-luciferase reporter construct to show that activation of AP-1 in
mouse epidermal cells by arsenite can be blocked by the MEK inhibitor
PD98059. Interestingly, in WI-38 cells, p38 does not play a positive
role in the induction of either c-fos or c-jun
expression by arsenite (Figs. 3-7), although it does appear to play a
role in the regulation of global histone H3 phosphoacetylation (Fig.
2). Our observations contradict previous reports (42, 43) indicating
that p38 can phosphorylate transcription factors critical for
c-fos induction and is involved in c-fos
induction by anisomycin and UV. This apparent discrepancy can be
explained by the observation that SB203580 augmented ERK activity
stimulated by arsenite (Fig. 3A). A similar augmentation of
ERK activity by SB203580 has been reported (44) in human neutrophils
stimulated with lipopolysaccharide. Likewise, SB203580 has been shown
to potentiate the induction of c-fos and c-jun by
both EGF and TPA (45). Our results, along with those reported by Sheth
et al. (44), suggest that p38 may have a negative effect on
the ERK pathway, although the exact mechanisms involved remain unclear.
The most prominent finding in this study is that arsenite potently
stimulated histone H3 phosphoacetylation specifically at the
c-fos and c-jun chromatin (Figs. 6 and 7).
Acetylation of chromatin-associated histone H3 has long been linked
with increased gene transcription (46, 47). Recent studies (21)
indicate growth factor-mediated histone H3 acetylation is regulated by the ERK pathway and is coupled to histone H3 phosphorylation. A number
of oncoproteins such as H-Ras, v-Fes, and v-Mos, as well as several
well established tumor promoters including TPA, okadaic acid and UVC,
can stimulate histone H3 phosphorylation (28). Histone H3
phosphorylation triggered by 400 µM arsenite is
comparable with that triggered by EGF (Fig. 1A). Enhanced
histone H3 phosphoacetylation can be detected in cells exposed to
arsenic levels as low as 2 µM (although the response is
not as rapid as that seen with higher concentrations),2 a
concentration that is comparable with that measured in underground water in parts of the western United States such as Utah (48). The
substantial increase in global histone H3 phosphoacetylation is likely
to have a profound effect on the overall gene expression profile.
Indeed, the enhanced expression of c-fos and
c-jun triggered by arsenite observed here probably only
represents "the tip of the iceberg," with the expression of other
genes likely regulated by arsenite in a similar fashion. Therefore, we
propose that arsenite stimulates cell proliferation and promotes
carcinogenesis, at least in part, by modulating chromatin structure and
perturbing the expression of key proliferative genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (23). ERK activity was
determined by immune complex kinase assays using myelin basic protein
(MBP, Sigma) as a substrate as described previously (26). MAPKAPK2/3
activity was determined by immunocomplex kinase assays using
recombinant Hsp25 (25-kDa heat-shock protein, Stressgen, Vancouver,
Canada) as a substrate as described previously (27).
phage DNA (5 µg), and protein A-Sepharose beads
(Amersham Biosciences) were added to the solutions and incubated
overnight with gentle rotation at 4 °C. The beads were washed
extensively as described previously (23). The resulting
immunoprecipitates or input chromatin solutions were then incubated
with RNase A (50 µg/ml) for 1 h at 37 °C and then digested
for 16 h using proteinase K (100 µg/ml). The input and
immunoprecipitated chromatin were incubated at 65 °C for
6 h to
reverse the formaldehyde cross-links. The DNA was extracted with
phenol/chloroform, precipitated with ethanol, and dissolved in 30 µl
of water.
-globin gene.
Rn was considered to be
0.5. The PCR cycle where a statistically significant increase in the
Rn was first detected is called the threshold cycle
(CT). Target DNA copy number and CT values are
inversely related. The absolute levels of c-fos and
c-jun in the experimental samples were determined by
extrapolating the CT values from the standard curves of the
genomic DNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Arsenite stimulates histone H3
phosphorylation at serine 10. WI-38 cells were treated with
arsenite (400 µM) for the times indicated, and histone H3
phosphorylation was detected by indirect immunofluorescence.
A, time course of histone H3 phosphorylation in
response to arsenite treatment. Upper panels,
immunofluorescent detection of phospho-histone H3 (Ser(P)-10).
Lower panels, DAPI staining of cell nuclei. Cells treated
with a combination of EGF (50 ng/ml) and anisomycin (An) (10 µg/ml) for 60 min were included as a positive control.
B, confocal microscopy of histone H3 phosphorylation.
Unstimulated cells (control), cells treated with arsenite (400 µM) for 60 min, or cells stimulated with EGF (50 ng/ml)
together with anisomycin (10 µg/ml) for 60 min were first incubated
with a rabbit phospho-histone H3 antibody (green signal) and
then with a mouse monoclonal antibody recognizing total histone H3
(red signal). Representative fluorescent images are
shown.
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[in a new window]
Fig. 2.
Arsenite enhances histone H3
phosphoacetylation and activates MSK1. WI-38 cells were treated
with arsenite (400 µM) in the absence or presence of
U0126 or SB203580 for the indicated times. Cells were lysed to extract
crude histone proteins for Western blotting or for assaying MSK1
activity. A, histone H3 phosphoacetylation in cells
treated with arsenite. Western blot analysis was performed on the crude
histone samples using an antibody specifically recognizing
phospho-histone H3 (Ser(P)-10) (1st panel from the
top), phosphoacetyl-histone H3 (Ser(P)-10/Ac-Lys-14)
(2nd panel), acetyl-histone H3 (Ac-Lys-14) (3rd
panel), acetyl-histone H3 (Ac-Lys-9,14) (4th panel),
total histone H3 (5th panel), or phospho-histone H2A.X
(p-Ser-139) (bottom panel). SOP, staurosporine.
TSA (500 ng/ml)- and EGF (50 ng/ml) + anisomycin (An) (10 µg/ml)-treated cells used as positive control. B,
effect of U0126 and SB203580 on the histone H3 phosphoacetylation.
Cells were stimulated with arsenite (400 µM) for 60 min
in the presence or absence of either U0126 (10 µM) or
SB203580 (10 µM) and harvested to do Western blots.
C, kinetics of MSK1 activation in response to arsenite.
MSK1 activity in cell lysates was determined by immunocomplex kinase
assays using Crosstide peptide as a substrate. D,
effects of U0126 and SB203580 on MSK1 activity. Cells were stimulated
with arsenite (400 µM) for 60 min in the presence of
either U0126 (10 µM) or SB203580 (10 µM)
and harvested to assess MSK1 activity. The data in C and
D represent the mean ± S.E. from three independent
experiments, with each determination carried out in duplicate.
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Fig. 3.
The kinetics of arsenite-induced
c-fos and c-jun expression and the
kinetics of ERK and p38 activation by arsenite. A,
time- and dose-dependent induction of c-fos and
c-jun by arsenite. Messenger RNAs encoding c-fos
and c-jun were detected by Northern blotting. Equality of
RNA loading and transfer was assessed by monitoring 18 S rRNA signals.
B, effect of arsenite on ERK and p38 activity. WI-38
cells were stimulated with arsenite for the times indicated. Whole-cell
lysates were analyzed by Western blotting using antibodies against
active ERKs (top panel), total ERK (2nd panel
from the top), active p38 (4th panel), and total
p38 (5th panel). ERK activity was also examined by
immunocomplex kinase assays using MBP as a substrate (3rd
panel from the top). MAPKAPK2/3 activity was examined
by immunocomplex kinase assays using recombinant Hsp25 (heat-shock
protein of 25 kDa) as a substrate (bottom panel).
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[in a new window]
Fig. 4.
The ERK MAP kinase pathway
plays an important role in mediating the induction of both
c-fos and c-jun by arsenite.
A, effect of U0126 and SB203580 on the ERK pathway.
WI-38 cells were treated with arsenite for 10 min in the absence or the
presence of either U0126 (10 µM), SB203580 (10 µM), or both inhibitors. The effect of these inhibitors
on the ERK pathway was analyzed by Western blotting using antibodies
against active ERK (top panel) and total ERK (bottom
panel). B, effect of U0126 and SB203580 on the p38
pathway. WI-38 cells were treated with arsenite for 1 h in the
absence or the presence of either U0126 (10 µM), SB203580
(10 µM), or both inhibitors. The effect of these
inhibitors on the p38 pathway was analyzed by Western blotting using
antibodies against phosphorylated HSP27 (top panel) and
total HSP27 (middle panel) and by immunocomplex kinase
(MAPKAPK2/3) assays using recombinant Hsp25 (heat-shock protein of 25 kDa) as a substrate (bottom panel). C,
effect of U0126 and SB203580 on the induction of c-fos and
c-jun by arsenite. WI-38 cells were treated with arsenite in
the absence or the presence of either U0126 (10 µM),
SB203580 (10 µM), or both inhibitors. Total RNA was
analyzed by Northern blotting. RNA loading was indicated by 18 S
rRNA.
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Fig. 5.
Effect of p38 inhibitors on activation of ERK
and on induction of c-fos and c-jun
caused by arsenite. A, effect of p38
inhibitors on the ERK pathway. WI-38 cells were treated with arsenite
(Ars) for 10 min in the absence or the presence of one of
the following p38 inhibitors: SB202190 (10 µM), PD169316
(10 µM), SB203580 (10 µM), or p38 INH (10 µM). The effect of these inhibitors on the ERK pathway
was analyzed by Western blotting using antibodies against active ERK
(top panel) and total ERK (middle panel) and by
immunocomplex kinase assays using MBP as a substrate (bottom
panel) which are quantitatively analyzed as shown in the graph.
Con, control. B, effect of p38 inhibitors on
the induction of c-fos and c-jun by arsenite.
WI-38 cells were treated with arsenite for 60 min in the absence or the
presence of one of the following p38 inhibitors: SB202190 (10 µM), PD169316 (10 µM), SB203580 (10 µM), or p38 INH (10 µM). Total RNA was
analyzed by Northern blotting. RNA loading was indicated by 18 S
rRNA.
) and by the inability of arsenite to stimulate
histone H3 modification at the transcriptionally inactive
-globin
locus (Fig. 6, B and C). These results support
the view that arsenite promotes chromatin remodeling only at a subset
of genes.
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Fig. 6.
Arsenite stimulates histone H3
phosphoacetylation on the c-fos chromatin.
A, analysis of phosphorylated histone H3 (Ser(P)-10) or
phosphoacetylated histone H3 (Ser(P)-10/Ac-Lys-14) at the
c-fos chromatin by real time PCR after ChIP. WI-38 cells
were treated with 400 µM arsenite (Ars) for
1 h in the presence or absence of either U0126 (U),
SB203580 (SB), or a combination of both inhibitors (each 10 µM). ChIP assays were performed using an antibody
recognizing either phospho-histone H3 ( -Phos-H3),
phosphoacetyl-histone H3 (
-Phos-Ac-H3), or
pre-immune serum (Ab
). DNA recovered from the
antibody-bound fractions as well as the DNA from input chromatin
(Input) were analyzed for the presence of c-fos
promoter and
-globin sequences through real time PCR. Graphs
represent the amplification curves for the c-fos promoter
using the following DNA as templates: top left graph, DNA
isolated using anti-phospho-histone H3 antibody; top right
graph, DNA isolated using anti-phosphoacetyl-histone H3;
middle left graph, input DNA; middle right graph
represents the relative target DNA copy number compared with control.
The cycle numbers to reach a threshold of 0.5 (CT) are
indicated. Final DNA products were separated on 2% agarose gels after
the PCRs in A were completed. DNA was stained with ethidium
bromide. B, final PCR products of c-fos and
-globin amplified from input DNA (Input), DNA
isolated using anti-phospho-histone H3 antibody (Phos-H3) or
pre-immune serum (Ab
). C,
final c-fos and
-globin PCR products amplified from input
DNA (Input), DNA isolated using anti-phosphoacetyl-histone
H3 antibody (Phos-Ac-H3), or pre-immune serum
(Ab
).
). Taken together, our
results indicate that arsenite potently enhances histone H3
modification at both the c-fos and c-jun loci through an ERK MAP kinase-mediated pathway.
View larger version (40K):
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Fig. 7.
Arsenite treatment leads to enhanced histone
H3 phosphoacetylation on the c-jun chromatin in WI-38
cells. A, analysis of the levels of phosphorylated
(Ser(P)-10) or phosphoacetylated (Ser(P)-10/Ac-Lys-14) histone H3 at
the c-jun chromatin by real time PCR after ChIP, carried out
as described in the legend of Fig. 6. Graphs represent the
amplification curves for the c-jun promoter using the
following DNA as templates: top left graph, DNA isolated
using anti-phospho-histone H3 antibody; top right graph, DNA
isolated using anti-phosphoacetyl-histone H3; middle left
graph, input DNA; middle right graph represents the
relative target DNA copy number compared with control. The number of
cycles required to reach a threshold of 0.5 (CT) are
indicated below the graphs. Final DNA products were
separated on 2% agarose gels after the PCRs in A were
completed. DNA was stained with ethidium bromide. Ars,
arsenite; U, U0126; SB, SB203580.
B, final PCR products of c-jun amplified
from input DNA (Input), DNA isolated using
anti-phospho-histone H3 antibody (Phos-H3), or pre-immune
serum (Ab ). C, final
c-jun PCR products amplified from input DNA
(Input), DNA isolated using anti-phosphoacetyl-histone H3
antibody (Phos-Ac-H3), or pre-immune serum
(Ab
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Center for
Developmental Pharmacology and Toxicology, Columbus Children's
Research Institute, Columbus Children's Hospital, Dept. of Pediatrics, the Ohio State University, 700 Children's Dr., Columbus, OH 43205. Tel.: 614-722-2915; Fax: 614-722-2007; E-mail:
liuy@pediatrics.ohio-state.edu.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M300269200
2 J. Li and Y. Liu, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: MAP, mitogen-activated protein; ChIP, chromatin immunoprecipitation; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; MBP, myelin basic protein; TPA, phorbol 12-myristate 13-acetate; TSA, trichostatin A; MEK, MAP kinase/ERK kinase.
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REFERENCES |
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