From the Laboratory of Food and Biodynamics, In the present study, we studied the
signal transduction mechanism that is involved in the expression of
c-Jun protein evident after exposure of rat liver epithelial RL34 cells
to the major end product of oxidized fatty acid metabolism,
4-hydroxy-2-nonenal (HNE). HNE treatment of the cells resulted in
depletion of intracellular glutathione (GSH) and in the formation of
protein-bound HNE in plasma membrane. In addition, HNE strongly induced
intracellular peroxide production, suggesting that HNE exerted
oxidative stress on the cells. Potent expression of c-Jun occurred
within 30 min of HNE treatment, which was accompanied by a
time-dependent increase in activator protein-1 (AP-1) DNA
binding activity. We found that HNE caused an immediate increase in
tyrosine phosphorylation in RL34 cells. In addition, HNE strongly
induced phosphorylation of c-Jun N-terminal kinases (JNK) and p38
mitogen-activated protein kinases and also moderately induced
phosphorylation of extracellular signal-regulated kinases. The
phosphorylation of JNK was accompanied by a rapid and transient
increase in JNK and p38 activities, whereas changes in the activity of
extracellular signal-regulated kinase were scarcely observed. GSH
depletion by
L-buthionine-S,R-sulfoximine, a specific inhibitor of GSH biosynthesis, only slightly enhanced peroxide production and JNK activation, suggesting that HNE exerted these effects independent of GSH depletion. This and the findings that
(i) HNE strongly induced intracellular peroxide production, (ii)
HNE-induced JNK activation was inhibited by pretreatment of the cells
with a thiol antioxidant, N-acetylcysteine, and (iii) H2O2 significantly activated JNK support the
hypothesis that pro-oxidants play a crucial role in the HNE-induced
activation of stress signaling pathways. In addition, we found that,
among the inhibitors of tyrosine kinases, cyclooxygenase, and
Ca2+ influx, only quercetin exerted a significant
inhibitory effect on HNE-induced JNK activation. In light of the
JNK-dependent induction of c-jun transcription
and the AP-1-induced transcription of xenobiotic-metabolizing enzymes,
these data may show a potential critical role for JNK in the induction
of a cellular defense program against toxic products generated from
lipid peroxidation.
Lipid peroxidation in tissue and in tissue fractions represents a
degradative process, which is the consequence of the production and the
propagation of free radical reactions primarily involving membrane
polyunsaturated fatty acids and has been implicated in the pathogenesis
of numerous diseases including atherosclerosis, diabetes, cancer, and
rheumatoid arthritis as well as in drug-associated toxicity,
postischemic reoxygenation injury, and aging (1). The peroxidative
breakdown of polyunsaturated fatty acids has also been implicated in
the pathogenesis of many types of liver injury and especially in the
hepatic damage induced by several toxic substances. Among these are the
haloalkanes, carbon tetrachloride, trichlorobromomethane, chloroform,
dibromoethane, and halothane; in addition, paracetamol, bromobenzene,
iron, bipyridyl compounds, allyl alcohol, and in some instances ethanol
have been shown to stimulate lipid peroxidation (2).
There is increasing evidence that aldehydes generated endogenously
during the process of lipid peroxidation are causally involved in most
of the pathophysiological effects associated with oxidative stress in
cells and tissues (3). Compared with free radicals, lipid
peroxidation-derived aldehydes are generally stable and can diffuse
within or even escape from the cell and attack targets far from the
site of the original free radical-initiated event, therefore suggesting
that they are not only end products and remnants of lipid peroxidation
processes but also may act as mediators for the primary free radicals
that initiated lipid peroxidation. Among the lipid peroxidation-derived
aldehydes, 4-hydroxy-2-nonenal (HNE)1 that can be produced
from arachidonic acid, linoleic acid, or their hydroperoxides in
relatively large amounts at concentrations of 10 µM to 5 mM in response to oxidative insults is believed to be
largely responsible for the cytopathological effects observed during
oxidative stress in vivo (Scheme
1) (3). HNE exhibits a wide range of
biological activities including inhibition of protein and DNA
synthesis, inactivation of enzymes, stimulation of phospholipase C,
reduction of gap-junction communication, and stimulation of neutrophil
migration (3). In addition, HNE modulates the expression of various
genes, including c-myc and globin genes (4), procollagen
type I (5) and aldolase reductase (6) genes, c-myb (7), and
transforming growth factor c-Jun is a member of a multiprotein family that has been implicated in
a number of signal transduction pathways associated with cellular
growth, differentiation, neuronal excitation, and cellular stress
(10-14) and is thought to be required for cellular defense against
toxicity (15). Induction of c-jun in response to genotoxic
agents and cellular stress is mediated by two AP-1-like sites in its
promoter that are recognized by c-Jun homodimers (16, 17), activation
transcription factor-2 (ATF-2) homodimers, or, preferentially,
c-Jun/ATF-2 heterodimers (18, 19). The transcriptional activity of
these binding factors is regulated by protein kinases related to the
mitogen-activated protein (MAP) kinase superfamily. To date, at least
three different subtypes of MAP kinases are known (20-22). These are
in turn activated by distinct upstream dual specificity kinases, thus
revealing the existence of protein kinase modules that can be
independently and simultaneously activated. Whereas mitogens and growth
factors lead to activation of protein kinase cascades resulting in
activation of extracellular signal-regulated kinase (ERK) family MAP
kinases, many forms of cellular stress preferentially trigger two
related signaling pathways (23-27). These center on two MAP kinases or Jun N-terminal kinases (JNKs) and p38, also termed stress-activated protein kinase and reactivating kinase, respectively. Targets of
stress-activated protein kinase and JNK include several transcription factors such as c-Jun, JunD, ATF-2, and Elk-1, which become activated after exposure to cellular stresses (28-31).
In this study, we find that HNE strongly induces c-Jun expression in
rat liver epithelial RL34 cells. To fully understand the signaling
mechanism that triggered this response, the phosphorylation events that
may lead to the activation of AP-1 transcription factors are
investigated. The study reported here demonstrates that the most
cytotoxic aldehyde, HNE, is a potential inducer of a signaling pathway
that involves the JNK. The identification of the proto-oncoprotein AP-1
as a nuclear target of HNE signaling may provide new clues to the
mutagenic and genotoxic potential of this aldehyde.
Materials--
HNE was prepared by acid treatment (1 mM HCl) of HNE diethylacetal synthesized according to the
procedure of De Montarby et al. (32), followed by
purification with a reversed-phase high protein liquid chromatography.
N-Acetylcysteine (NAC),
L-buthionine-S,R-sulfoximine (BSO),
herbimycin, and genistein were obtained from Sigma. AP-1, AP-2, and
AP-3 consensus double-stranded oligonucleotides were obtained from
Stratagene (La Jolla, CA). Indomethacin, aspirin, and quercetin were
kindly provided by Dr. Y. Morimitsu of Nagoya University Graduate
School of Bioagricultural Sciences.
Cell Culture--
RL34 cells were obtained from the Japanese
Cancer Research Resources Bank (33). The cells were grown as monolayer
cultures in Dulbecco's modified Eagle's medium supplemented with 5%
heat-inactivated fetal bovine serum, penicillin (100 units/ml),
streptomycin (100 µg/ml), L-glutamine (588 µg/ml), and
0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and
5% CO2. Cells postconfluency were exposed to the lipid
peroxidation products in Dulbecco's modified Eagle's medium
containing 5% fetal bovine serum.
Glutathione Assay--
Measurement of GSH in the cells was
performed fluorometrically according to the method of Hissin and Hilf
(34). In brief, the cells incubated with HNE or NAC were washed twice
with PBS (pH 7.0) and extracted with the 25% (w/v) metaphosphoric acid solution containing 5 mM EDTA. After ultracentrifugation
(105,000 × g, 30 min), 1.8 ml of 0.1 M
phosphate solution (pH 8.0) containing 5 mM EDTA and 100 µl of the o-phthalaldehyde solution (1 mg/ml) were added
to the resulting supernatant (100 µl), and then the fluorescence
intensity at 420 nm was determined with activation at 350 nm.
Intracellular Peroxide Production--
Cells were incubated with
50 µM 2',7'- dichlorofluorescin diacetate (DCF-DA)
(dissolved in Me2SO) for 30 min at 37 °C and then
treated with different agents for an additional 30 min at 37 °C. In
the case of BSO, cells were treated with BSO for 6 h at 37 °C
and then incubated with DCF-DA for an additional 30 min at 37 °C.
After chilling on ice, cells were washed with ice-cold PBS, scraped
from the plate, and resuspended at 1 × 106 cells/ml
in PBS containing 10 mM EDTA. The fluorescence intensities of 2',7'- dichlorofluorescein formed by the reaction of DCF-DA with
peroxides of more than 10,000 viable cells from each sample were
analyzed by a flow cytometer (CytoACE 150, JASCO, Tokyo, Japan) with
excitation and emission settings of 488 and 525 nm, respectively. Prior
to data collection, propidium iodide was added to the sample for gating
out dead cells. Experiments were repeated at least twice with similar
results. The data are expressed as one representative histogram.
Western Blotting--
For detection of phosphotyrosine, whole
cell lysates (200 µg of protein) were prepared in lysis buffer (1%
Nonidet P-40, 20 mM Tris (pH 8.0), 137 mM NaCl,
10% glycerol, 1 mM Na3VO4, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 0.15 unit/ml aprotinin).
Phosphotyrosine was monitored by SDS-PAGE and immunoblotting of 40 µg
of whole cell extracts with the anti-phosphotyrosine monoclonal
antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY). For
detection of c-Jun, nuclear proteins were prepared as described
previously (35). c-Jun was detected by SDS-PAGE and immunoblotting of
10 µg of nuclear extracts with the anti-c-Jun monoclonal antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The activation of MAP
kinases (JNK, p38, and ERK) was monitored by SDS-PAGE and
immunoblotting of 40 µg of whole cell extracts with highly specific
anti-phospho-(Thr-183/Tyr-185) JNK, anti-phospho-(Thr-180/Tyr-182) p38,
or anti-phospho-(Tyr-204) ERK MAP kinase antibodies (New England
Biolabs, Inc., Beverly, MA). These antibodies are directed against
synthetic phosphopeptides corresponding to residues 179-193 (SFMMT*PY*VVTRYYR), 171-186 (LARHTDDEMT*GY*VATR), and 196-209 (DHTGFLTEY*VATRWC) of JNK, p38, and ERK, respectively. Asterisks represent phosphorylation sites.
Kinase Assays--
Whole cell lysates were prepared in lysis
buffer (20 mM Tris (pH 7.5) 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
Gel Shift Assay--
Nuclear proteins were prepared as
previously reported (35). Binding reactions were 22-µl volumes
containing 5 µg of nuclear proteins, 3 µg of poly(dI-dC), and
[
In some experiments, 5 µg of nuclear extracts was incubated with 5 µg of highly specific anti-c-Jun, anti-Jun B, anti-Jun D, anti-c-Fos,
or anti-Fra-1 antibodies (Santa Cruz Biotechnology) at 4 °C for
1 h and then further incubated with 32P-labeled DNA
oligonucleotide for another 30 min. The subsequent supershift complexes
were resolved in a 4.5% nondenaturing acrylamide gel and
electrophoresed at room temperature as already described.
Immunocytochemistry--
For immunocytochemistry, cells were
fixed overnight in PBS containing 42% paraformaldehyde and 0.2%
picric acid at 4 OC. Membranes were permeabilized by
exposing the fixed cells to PBS containing 0.3% Triton X-100. The
cells were then incubated sequentially in PBS solutions containing
blocking serum (5% normal swine serum) and primary antibody. The cells
were then incubated for 1 h in the presence of fluorescein
isothiocyanate-labeled antirabbit igG (Dako), rinsed with PBS
containing 0.3% Triton X-100, and covered with anti-fade solution.
Primary antibody that recognizes the HNE-protein epitopes was raised by
immunizing New Zealand White rabbits with HNE-treated KLH (37). Images
of cellular immunofluorescence were aquired using a confocal laser
scanning microscope (FLUOROVIEW, Olympus Optical Co., Ltd., Tokyo) with a × 40 objective (488-nm excitation and 518-nm emission).
HNE Is a Potential Inducer of Oxidative Stress--
Because HNE is
a potent electrophile, we first examined the effect of HNE treatment on
the change in the cellular redox status. As shown in Fig.
1A, a significant decrease in
GSH levels was observed after the first 2 h of the HNE treatment.
Interestingly, the amount of GSH began to recover and rose to above the
basal level at 24 h, indicating that the cell responded to the GSH
depletion. Because GSH is important in metabolism and enzyme regulation
as well as detoxification of cytotoxic materials, the GSH depletion may
lead to an increase in the susceptibility of the cell to insult by HNE.
Indeed, the GSH depletion was associated with the HNE modification of
cellular proteins. As shown in Fig. 1B, the protein-bound HNE was mainly detected in the plasma membrane at 30 min
(b), and subsequently, the localization gradually
shifted to the cytosol (c and d).
We also found that, in addition to GSH depletion and protein
modification, HNE induced intracellular production of pro-oxidants. The
production of pro-oxidants was determined using a peroxide-sensitive fluorescent probe, DCF-DA. This chemical is freely permeable to cells.
Once inside the cells, it is hydrolyzed to DCF and trapped intracellularly. In the presence of peroxides, especialy
H2O2, DCF is oxidized to fluorescent
2',7'-dichlorofluorescein, which can then be readily detected using
fluorescence-activated cell sorting. To validate the assay, an
experiment with H2O2 was performed. As shown in
Fig. 2, treatment of the cells with
H2O2 (100 µM) caused an increase
in fluorescence intensity as compared with control. Treatment with BSO,
a specific inhibitor of GSH biosynthesis, exerted only a slight
increase in peroxide production, despite a considerable reduction of
intracellular GSH level. In contrast, treatment with HNE (20 and 40 µM) strongly enhanced the peroxide-activated fluorescence
intensity of DCF. When these findings are taken together, it is evident
that the lipid peroxidation end product HNE is a potential inducer of
intracellular oxidative stress.
HNE Induces c-Jun Expression and AP-1 DNA Binding Activity--
As
the earliest events, HNE induced oxidative stress in the cells (Figs. 1
and 2). It was anticipated that this event could be the first signal
that might be highly relevant to the onset of the stress response
represented by the induction of immediate early genes. This was also
suggested by the significant recovery of GSH levels after the
depletion. Hence, we examined HNE for the effect on the expression of
products of immediate early genes in RL34 cells. As shown in Fig.
3A, HNE dramatically
stimulated the expression of c-Jun protein in a
time-dependent manner. A maximum 10-fold increase in c-Jun
expression in response to HNE was observed at 45 min after initiation
of treatment, and this response persisted for at least 8 h. It is
noted that HNE had no effect on the level of a wide variety of
proteins, except c-Jun. Those failing to respond included other
transcription factors responsive to various signals, such as c-Fos,
JunB, JunD, Sp1, NF-1, and p53 (data not shown). These data suggest
that induction of transcription factors by HNE is highly specific to
c-Jun.
Since c-Jun is the major constituent of the transcriptional factor AP-1
and HNE increased the expression of this proto-oncogene, we tested the
effect of HNE on AP-1 activation in RL34 cells. AP-1 activity was
measured by its ability to bind to the
12-O-tetradecanoylphorbol-13-acetate response element (TRE).
Nuclear extracts of HNE-treated cells displayed 3-5-fold greater DNA
binding activity than extracts of untreated cells, as determined by gel
shift assay (Fig. 3B). Almost maximal increase in AP-1·TRE
binding activity in response to HNE treatment was observed by 1 h,
and these responses were persistent for at least 2 h. To identify
the factors that make up HNE-inducible AP-1 activity, highly specific
antibodies directed against individual AP-1 proteins were evaluated for
the ability to deplete nuclear extracts of DNA binding activity. The
extract used for this analysis was isolated from RL34 cells treated
with HNE for 1 h. Antibodies against c-Jun, JunB, and JunD
supershifted the HNE-induced AP-1·TRE complex, and anti-Fra-1
antibody significantly reduced the AP-1 binding activity (Fig.
3C), whereas the antibody against c-Fos had little effect.
These data suggest that c-Jun, JunB, JunD, and Fra-1 may be important
components responsible for AP-1 transactivation by HNE. Moreover, the
addition of AP-2 or AP-3 consensus cold oligonucleotide had no effect
on HNE-induced AP-1-TRE binding; however, the addition of excess cold
AP-1 oligonucleotide probe to the reaction mixture abolished AP-1
binding to 32P-labeled TRE (data not shown). This
observation clearly indicates that the AP-1-DNA binding activity
observed in the nuclear extracts of HNE-treated cells is specific.
Induction of Tyrosine Phosphorylation by HNE--
To understand
the signaling mechanism that triggered this response, we examined the
phosphorylation events that may lead to the activation of AP-1
transcription factors. To investigate whether HNE can activate cellular
protein kinases, the cells incubated with 25 µM HNE and
tyrosine phosphorylation of cellular proteins were determined by
Western blot analysis with anti-phosphotyrosine antibody. As shown in
Fig. 4, several proteins with apparent
molecular mass of 32, 38, 42, 57, and 90 kDa were the dominant proteins phosphorylated after incubation with HNE. The increase in tyrosine phosphorylation was detectable within 15 min during HNE treatment to at
least 2 h after the treatment. The early increase in tyrosine phosphorylation led us to hypothesize that the activation of
protein-tyrosine kinases is probably one of the upstream signals of the
HNE-induced c-Jun expression in the cells. It is also noted that HNE
induced an immediate dephosphorylation of a major
tyrosine-phosphorylated 60-kDa protein. This may also represent the
earliest event that leads to the induction of c-Jun (Fig. 3).
A number of transcription factors such as c-Jun and c-Fos have been
shown to be phosphorylated in vitro by distinct members of
the MAP kinase families triggered by a large variety of extracellular stresses. MAP kinases are important cellular signaling components that
convert various extracellular signals into intracellular responses
through serial phosphorylation cascades. At the present time, three
distinct but parallel MAP kinase cascades have been identified in
mammalian cells. It has been established that the activation of MAP
kinases culminates in the phosphorylation of downstream cytosolic and
nuclear substrates, ultimately leading to changes in gene expression.
Based on the findings that HNE is a potential inducer of c-Jun
expression, a possible involvement of MAP kinases in the HNE-induced
c-Jun expression was examined. To determine the possibility that HNE
treatment of the cells results in the phosphorylation of MAP kinases,
whole cell lysates were probed with the antibodies specific for
phosphorylated JNK, p38, and ERK. As shown in Fig.
5, HNE potently induced phosphorylation of p46 and p54 JNK (A) and p38 (B) and moderately
induced phosphorylation of ERK (ERK1 and ERK2) (C). The
HNE-induced phosphorylation of JNK and p38 reached a maximum within 30 min and returned to the basal levels at 8 h.
HNE Activates JNK and p38 but Not ERK--
To verify that the HNE
treatment of the cells resulted in a functional increase in JNK, p38,
and ERK activities, we assayed for their activities in the lysates of
the cells. As shown in Fig. 6,
A and B, the activation of JNK and p38 by HNE
began within 30 min and continued for at least 24 h. The
activation of ERK by HNE was examined, but no significant change in the
substrate phosphorylation was observed (Fig. 5C). The
HNE-induced activation of JNK was scarcely affected by actinomycin D,
an inhibitor of RNA synthesis, and cycloheximide, an inhibitor of
protein sysnthesis (data not shown), indicating that the HNE-mediated
JNK activation is not the result of the activation of gene
expression.
The HNE-induced JNK activation was significantly inhibited by a thiol
antioxidant NAC (Fig. 7, A and
B), whereas BSO that caused a considerable reduction of GSH
level only slightly activated JNK (Fig. 7C). This and the
observation that GSH depletion by treatment with BSO resulted in a
slight increase in intracellular peroxide level (Fig. 2) suggest that
the HNE-induced JNK activation may be independent of GSH depletion,
whereas the findings that HNE strongly induced intracellular peroxide
production (Fig. 2) and H2O2 treatment of the
cells significantly activated JNK (Fig. 7D) suggest the
involvement of intracellular peroxide production in the JNK
activation.
Quercetin Is a Specific Inhibitor for the HNE-induced JNK
Activation--
Because tyrosine kinases have been shown to be
involved in a variety of cellular responses, such as growth factors,
oxidative stress, and G protein-coupled signaling cascades (38-40),
the effect of tyrosine kinase inhibitors upon HNE-induced JNK
activation was tested. As shown in Fig.
8, both herbimycin and genistein that
exhibit specific inhibitory activity against src family
tyrosine kinases were ineffective; however, the HNE-induced JNK
activation was significantly inhibited by quercetin. The results
suggest that the src family tyrosine kinases may not be
involved in HNE-induced JNK activation and that HNE may activate JNK
via activation of protein kinase C, phosphorylase kinase,
Na+,K+-ATPase, and/or
Ca2+,Mg2+-ATPase, which have been reported to
be inhibited by quercetin (41).
On the other hand, HNE was previously reported to stimulate the
Ca2+ influx in rat hepatocytes, leading to the activation
of phospholipase C (42, 43). To evaluate the contributions of
Ca2+-mediated cell signaling on JNK activation by HNE,
cells were treated with the GdCl3, which is known to block the activity
of store-operated Ca2+ channels in the hepatocyte plasma
membrane, or phospholipase C inhibitor U73122, followed by stimulation
with HNE; however, neither of the inhibitors significantly influenced
HNE-induced JNK activity (data not shown). The cyclooxygenase
inhibitors indomethacin and aspirin were also ineffective (data not
shown), suggesting that eicosanoid biosynthesis is not involved in the
HNE-induced JNK activation.
In the present study, we have found that HNE is a potential source
of intracellular pro-oxidants. As far as we know, this is the first
report that clearly demonstrates the intracellular peroxide production
by HNE. This finding is consistent with previous studies showing that
HNE itself induces lipid peroxidation, as indicated by increased levels
of malondialdehyde (44). It is therefore likely that, in addition to
GSH depletion and protein modification, the lipid peroxidation end
product HNE exerts an increased intracellular peroxide production that
further potentiates lipid peroxidation in the cells, while the detailed
mechanism for HNE-induced peroxide production is currently unknown.
The major finding of this study is that HNE appears to be a potential
inducer of stress signaling pathways. The first experimental evidence
that suggested the involvement of HNE-induced signal transduction was
the detection of potent and transient expression of transcription
factor c-Jun (Fig. 3). It was assumed that activation of protein
kinases induced by HNE might cause activation of the signaling pathway,
which phosphorylated and activated c-Jun. Being the major component of
the AP-1 transcription factor, c-Jun, once activated, can subsequently
activate the transcription of several genes. The fact that the
c-jun gene itself also contains an AP-1 element in its
promoter indicates that phosphorylation of c-Jun allows signals in this
pathway to be amplified. First, phosphorylation of c-Jun activates the
transcription of c-jun per se, increasing the amount of
available c-Jun for the formation of the AP-1 complex. Second,
phosphorylation of c-Jun also increases the activity of the newly
formed AP-1 factors, allowing the AP-1 transcriptional activity to be
further activated. Activation of AP-1 is regulated by complex
mechanisms which consist of distinct effects on the preexisting AP-1
complex or on the de novo synthesis of AP-1 subunits.
It has been revealed that ERKs, which are primarily activated in
response to growth factor stimulation, are implicated in processes of
cellular proliferation (45-48) and differentiation (49, 50), whereas
JNK and p38 MAP kinases are characterized by their strong response to
cellular stresses such as UV light (51), osmotic stress (25), DNA
damaging agents (52), and proinflammatory cytokines (53). Activation of
JNK has also been implicated in inducing apoptosis in response to
growth factor withdrawal and other environmental stimuli (54, 55) and
in stimulating cell proliferation and transformation (56). We found in
the present study that a different kind of stress, HNE, also stimulated
JNK and p38 (Fig. 6A), which might be directly responsible for the induction of c-Jun expression. In addition to the large body of
data demonstrating stimulation of cell signaling, our present findings
add HNE to a growing list of chemicals that can trigger stress
signaling pathways mediated by JNK and p38 MAP kinases.
It has been shown that the activation of JNK and p38 is dependent on an
upstream tyrosine/threonine kinase. One potential upstream kinase
responsible for their phosphorylations is MAP kinase kinase 4, which
itself must be serine/threonine-phosphorylated for activity (57, 58)
and activates both JNK and p38 (58, 59); however, our preliminary
experiment demonstrated that HNE does not induce phosphorylation of MAP
kinase kinase 4 (data not shown). This implies that HNE acts through a
different upstream regulatory element than MAP kinase kinase 4. A
similar MAP kinase kinase 4-independent activation of JNK has also been
seen in human glomerular mesangial cells treated with diamide or
hydrogen peroxide (60). Thus, it remains to be determined whether HNE
activates JNK via a known pathway or through an alternative signaling cascade.
Oxidative stress has been reported to be involved in the activation of
MAP kinases (60-62). Although the primary target of HNE is still
unknown, there is evidence that the intracellular level of GSH,
regulating the redox state of the cell (63), may be an important sensor
for the initiation of the cellular response to various compounds. In
fact, intracellular GSH levels of RL34 cells were readily reduced by
treatment with HNE in a time-dependent manner (Fig.
1A). These data led to the assumption that intracellular GSH
is a critical parameter for the stress signaling cascade induced by
HNE. However, BSO itself did not so effectively enhance intracellular peroxide production (Fig. 2) and only slightly activated JNK (Fig. 7C). These data suggest that the HNE-induced JNK activation
may not simply result from GSH depletion alone, whereas the results that HNE strongly induced intracellular peroxide production (Fig. 2)
and the HNE-induced JNK activation was significantly blocked by a thiol
antioxidant NAC (Figs. 7, A and B) suggest the
involvement of pro-oxidants on JNK activation. This was also supported
by the result that H2O2 treatment of the cells
resulted in a significant activation of JNK (Fig. 7D). Taken
together, it is proposed that pro-oxdidants such as peroxides are
involved in the HNE-induced activation of stress signaling pathways.
It is noteworthy that the HNE-induced JNK activation was significantly
inhibited by quercetin (Fig. 8), which is one of most widely
distributed bioflavonoids in the plant kingdom and is a component of
most edible fruits and vegetables. It has been demonstrated that
quercetin inhibits the growth of malignant cells through various
mechanisms: inhibition of glycolysis, macromolecule synthesis and
enzymes, freezing cell cycle, and interaction with estrogen type II
binding sites (41). In addition, the flavone inhibits the induction of
heat shock proteins and thermotolerance without affecting other protein
synthesis. The exact mechanisms responsible for the inhibitory effect
of quercetin on HNE-induced JNK activation is not yet thoroughly
understood, but it is possible that quercetin exerts the activity by
inhibiting enzymes involved in the HNE-induced stress signaling
pathways. In this regard, it is notable that quercetin is a potential
inhibitor of protein kinase C (41). This and the evidence that protein
kinase C is activated by H2O2 (64) suggest that
HNE-induced peroxide production and the consequential activation of
protein kinase C and/or other protein kinases may contribute to the
activation of the stress signaling pathways (Scheme
2).
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
1 gene (8). HNE is a potent alkylating
agent that reacts with a variety of nucleophilic sites in DNA and
protein, generating various types of adducts (3), leading to the
assumption that they cause a specific cellular stress response. This
program may include transcriptional induction of proto-oncogenes,
including c-jun. In fact, it has been demonstrated that the
lipid hydroperoxide, a precursor of HNE, triggers cellular signaling
pathways, leading to activation of activator protein-1 (AP-1) (9).
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Scheme 1.
A proposed mechanism for the formation of HNE
during peroxidation of arachidonate. Like other -6-polyunsaturated
fatty acids, arachidonate is susceptible to free radical reaction at
bis-allyl sites, e.g. C-8, to form a free radical
intermediate, which further reacts with molecular oxygen to generate
hydroperoxide intermediates. After intramolecular cyclization followed
by oxidative C-C cleavages, HNE is produced. R, CH3(CH2)4-.
R', -(CH2)3-COOH.
EXPERIMENTAL PROCEDURES
-glycerophosphate, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin). For ERK assay,
the lysates were incubated for 4 h at 4 °C with protein A-Sepharose plus 1 µg of anti-phospho-(Tyr-204)-p44/p42 ERK antibody (New England Biolabs), which recognizes activated ERK. The
immunoprecipitate was washed as previously outlined and resuspended in
50 µl of kinase buffer containing 200 µM ATP and 2 µg
GST-Elk1 fusion protein (New England Biolabs). The reactions were
terminated after 30 min by the addition of Laemmli sample buffer (36),
and the phosphorylation of GST-Elk1 was examined after SDS-PAGE and
immunoblotting with anti-phospho-(Ser-383)-Elk1 antibody (New England
BioLabs) by autoradiography. JNK activity was precipitated from whole
cell lysates by incubating with 2 µg of GST-c-Jun-(1-89) fusion
protein/GSH-Sepharose beads overnight at 4 °C (New England Biolabs).
The beads were then washed twice in lysis buffer and twice in kinase
buffer (25 mM Tris (pH 7.5), 5 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM
MgCl2) and resuspended in 50 µl of kinase buffer with 100 µM ATP for 30 min at 30 °C. The solid-phase kinase reaction was terminated by the addition of Laemmli sample buffer, and
the phosphorylation of GST-c-Jun on Ser-63 was examined after SDS-PAGE
and immunoblotting with anti-phospho-(Ser-63) c-Jun antibody (New
England BioLabs). For p38 assay, the lysates were incubated with
phosphorylated heat and acid-stable protein regulated by insulin
(PHAS-I) and [
-32P]ATP for 10 min at 30 °C. The
kinase reaction was terminated by the addition of Laemmli sample
buffer, and the phosphorylation of PHAS-I was examined after
SDS-PAGE.
-32P]ATP-labeled oligonucleotide probe (2 × 104 cpm). After 30 min, the DNA-protein complexes were
resolved in a 6% nondenaturing acrylamide gel and electrophoresed at
room temperature. The gel was dried and exposed to Kodak film at
80 °C overnight. The oligonucleotide probes used were as follows: AP-1, 5'-CTAGTGATGAGTCAGCCGGATC-3'; AP-2,
5'-GATCGAACTGACCGCCCGCGGCCCGT-3'; AP-3,
5'-CTAGTGGGACTTTCCACAGATC-3'.
RESULTS
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Fig. 1.
HNE-induced redox alteration and formation of
protein-bound HNE in RL34 cells. A, effect of HNE
treatment on intracellular GSH levels. The cells were treated with 25 µM HNE for different time intervals as indicated.
Intracellular GSH levels were fluorometrically measured as described
under "Experimental Procedures." B, immunocytochemical
detection of protein-bound HNE. a, untreated control;
b, 30 min after HNE treatment; c, 1 h after
HNE treatment; d, 4 h after HNE treatment. The cells
were fixed in 2% paraformaldehyde and 0.2% picric acid and
immunostained with polyclonal antibodies that specifically recognize
protein-bound HNE. Images of cellular immunofluorescence were aquired
using a confocal laser scanning microscope.
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Fig. 2.
Changes in intracellular peroxide level after
exposure of RL34 cells to exogenous H2O2, BSO,
or HNE. RL34 cells were incubated with the dye dichlorofluorescein
diacetate (DCF-DA, 50 µM) for 30 min and then treated
with 100 µM H2O2, 20 µM HNE, or 40 µM HNE for 30 min. In the
case of BSO, cells were treated with 100 µM BSO for
6 h and then incubated with DCF-DA for 30 min. Cells were washed
with PBS and resuspended in PBS containing 10 mM EDTA. The
fluorescence intensity of more than 10,000 cells was analyzed using a
flow cytometer.
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Fig. 3.
HNE induces c-Jun expression and AP-1 DNA
binding activity. RL34 cells were treated with 25 µM
HNE for different time intervals as indicated. A, an equal
amount of protein (40 µg) from nuclear extracts and analyzed by
Western blotting with anti-c-Jun monoclonal antibody. B,
nuclear proteins (5 µg) isolated from untreated control and
HNE-treated cells were incubated with 32P-labeled AP-1
oligonucleotides and separated by 4.5% acrylamide gel electrophoresis.
C, nuclear proteins (5 µg) isolated from the cells treated
with HNE for 1 h were first incubated with antibodies to c-Jun,
JunB, JunD, c-Fos, or Fra-1 for 1 h at 4 OC, and
32P-labeled AP-1 oligonucleotides were then added into each
reaction and separated by 4.5% acrylamide gel electrophoresis.
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Fig. 4.
HNE-induced tyrosine phosphorylation.
RL34 cells were treated with 25 µM HNE for different time
intervals as indicated. An equal amount of protein (10 µg) from the
cell lysates was analyzed by Western blotting with anti-phosphotyrosine
antibodies. M, markers.
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Fig. 5.
Phosphorylation of JNK (A), p38
(B), and ERK (C). RL34 cells were treated
with 25 µM HNE for different time intervals as indicated.
Phosphorylation of ERK (A), JNK (B), and p38
(C) was monitored by Western blotting of 410 µg of whole
cell extracts with highly specific anti-phospho-(Thr-183/Tyr-185) JNK,
anti-phospho-(Thr-180/Tyr-182) p38, or anti-phospho-(Tyr-204) ERK MAP
kinase antibodies.
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Fig. 6.
Activation of JNK and p38, but not ERK, by
HNE. RL34 cells were treated with 25 µM HNE for
different time intervals as indicated. Cell lysis and kinase assays
were performed as described under "Experimental Procedures."
A, JNK assay, showing the phosphorylation of GST-c-Jun.
B, p38 assay, showing the phosphorylation of PHAS-I.
C, ERK assay, showing the phosphorylation of
GST-Elk-1.
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Fig. 7.
Effect of NAC on HNE-induced JNK activation
and H2O2-induced transient activation of
JNK. A, effect of NAC treatment on intracellular GSH
levels. RL34 cells were incubated with 0-15 mM NAC for
24 h, and intracellular GSH levels were fluorometrically measured
as described under "Experimental Procedures." B, effect
of NAC pretreatment on the HNE-induced JNK activation. The cells
incubated with 0-15 mM NAC for 24 h were treated with
25 µM HNE for 1 h. C, effect of NAC and
BSO on JNK activation. The cells were incubated with 10 mM
NAC or 1 mM BSO for 6 h. D, effect of
H2O2 on JNK activation. The cells were
incubated with 40 µM H2O2 for
different time intervals as indicated. In B-D, JNK activity
in cell lysates was determined by an immunocomplex kinase assay as
described under "Experimental Procedures" using GST-c-Jun as the
substrate.
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Fig. 8.
Effect of inhibitors on HNE-induced JNK
activation. RL34 cells were incubated with inhibitors for 30 min
and then treated with 25 µM HNE for 1 h. JNK
activity in cell lysates was determined by an immunocomplex kinase
assay as described under "Experimental Procedures" using GST-c-Jun
as the substrate.
DISCUSSION
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Scheme 2.
A proposed mechanism for the activation of stress
signaling pathways by HNE.
It has been suggested that the induction of JNK followed by c-Jun
expression may be an important mechanism for controlling the genetic
program which cells use to actively and immediately respond to various
extracellular stimuli by the induction of proteins that are ultimately
responsible for the detoxification of cytotoxic xenobiotics. Previous
studies have shown that the expression of many phase II detoxifying
enzyme genes is regulated by the AP-1 and AP-1-like elements. In early
studies of the stress response system, a wide variety of phase II
detoxifying enzymes inducers was found to be electrophiles. The
transcriptional activation of response genes by these compounds has
been traced to a cis-acting transcriptional enhancer called
the antioxidant response element, which has been detected in the
promoters of various phase II detoxifying enzymes, including rat and
mouse glutathione S-transferase A-1 (65), rat glutathione
S-transferase P-1 (66), and NAD(P)H:quinone oxidoreductase
(67). Indeed, HNE induced a significant increase in specific binding to
the antioxidant response element from the rat glutathione
S-transferase P-1 gene, leading to the induction of gene
expression (68). Moreover, our preliminary experiments have shown that
HNE is a potential inducer of phase II detoxifying enzyme genes
including a rate-limiting enzyme of GSH biosynthesis, -glutamylcysteine
synthetase.2 Therefore, the
HNE-induced gene expression of this enzyme may be responsible for the
recovery and subsequent increase in the GSH levels (Fig.
1A). The rise in GSH and glutathione
S-transferase contents may allow inactivation of HNE and
therefore contribute to the progressive down-regulation of JNK activity
poststimulation (Fig. 6A). Our data suggest that the
induction of JNK is an important mechanism for controlling the genetic
program that cells use to actively and immediately respond to
environmental stimuli by the induction of proteins that are responsible
for the detoxification of cytotoxic xenobiotics.
Alkylating agents including HNE are versatile mutagens and/or
carcinogens. Once formed, they induce diverse aspects of severe cellular stress, including chromosomal aberrations, sister chromatid exchanges, point mutations, and cell killing. These agents react with a
variety of nucleophilic sites in DNA and protein, generating various
types of adducts. Upon reaction with protein, HNE selectively reacts
with the imidazole moiety of histidine residues, the -amino group of
lysine residues, and the sulfhydryl group of cysteine residues
(69-71). The primary products are simple Michael addition-type products, which further undergo cyclization between the aldehyde moiety
and the C-4 position of HNE to form a hemiacetal structure. Although
the primary target molecule resposible for the initiation of
HNE-induced signal transduction remains to be identified, it is assumed
that the induction of stress-activated cell signaling by HNE may be
closely associated with the modification of cellular proteins (Fig.
1B). It has been shown that HNE exogenously added to the
cells or endogenously generated in the cells binds to different proteins and impairs their function; examples include the
Na+,K+-ATPase (72), neuronal glucose
transporter GLUT3 (73), the astrocyte glutamate transporter GLT-1 (44),
and the GTP-binding protein G
q/11 (74). HNE is
relatively stable and can pass among subcellular compartments; thereby,
it has the potential to interact with many different cell proteins
including Tau (75). Furthermore, the presence of protein-bound HNE
in vivo has been assessed in various human tissue samples,
including the human aorta with atherosclerotic lesions (76), nigral
neurons in Parkinson's disease (77), renal cell carcinomas (78),
amyloid deposits in systemic amiloidosis (79), and trophoblast cells of
preeclamptic placentas (80).
In summary, this report demonstrates that the potential protein
alkylating agent HNE strongly induces peroxide production and activates
JNK and p38 MAP kinases in RL34 cells. We have shown that HNE is not
only a potential inducer of tyrosine phosphorylation but also a
specific activator of JNK and p38 cascades, which may phosphorylate and
activate c-Jun. The HNE-mediated activation of c-Jun may eventually
lead to the up-regulation of many AP-1-dependent genes. We
are currently investigating the further upstream cell signaling induced
by HNE. Our future challenge is to define a target molecule that
triggers signal transduction pathways leading to JNK activation and to
define the biological significance of the activation of these stress
signaling pathways mediated by HNE. Studies focusing on these
biochemical steps would extend our understanding of the regulation of
stress signaling cascades stimulated by various reactive products
generated during lipid peroxidation.
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FOOTNOTES |
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* This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences.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: Laboratory of Food and
Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan. Fax: 81-52-789-5741; E-mail: uchidak{at}agr.nagoya-u.ac.jp.
The abbreviations used are: HNE, 4-hydroxy-2-nonenal; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; AP, activator protein; ATF-2, activation transcription factor-2; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; NAC, N-acetylcysteine; BSO, L-buthionine-S,R-sulfoximine; GST, glutathione S-transferase; DCF-DA, 2',7'-dichlorofluorescin diacetate; PHAS-I, phosphorylated heat and acid-stable protein regulated by insulin.
2 K. Uchida, M. Shiraishi, Y. Naito, Y. Torii, Y. Nakamura, and T. Osawa, unpublished data.
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