Full text of DOI:10.1093/carcin/21.5.423

Carcinogenesis vol.21 no.7 pp.1423–1432, 2000
Roles of JNK, p38 and ERK mitogen-activated protein kinases in
the growth inhibition and apoptosis induced by cadmium
Show-Mei Chuang, I-Ching Wang and Jia-Ling Yang1
distinct signal transduction pathways that control many aspects
of mammalian cellular physiology, including cell growth,
differentiation and apoptosis (1–4). MAPKs are activated by
dual phosphorylation on Thr and Tyr within the motif Thr-
Glu-Tyr (ERK), Thr-Pro-Tyr (JNK) or Thr-Gly-Tyr (p38) in
subdomain VIII of the catalytic domain (1–4). This phos-
phorylation is mediated by a protein kinase cascade that
consists of a MAPK kinase kinase that phosphorylates and
activates one or more MAPK kinases (MKK) that, in turn,
phosphorylate and activate each MAPK (1–4). JNK is known
to be activated by MKK4 and MKK7 (4). Similarly, p38 is
activated by MKK3, MKK6 and MKK4, whereas ERK is
activated by MKK1 and MKK2 (4). Once activated, MAPKs
regulate gene expression through phosphorylation of down-
stream transcriptional factors. For example, JNKs were
originally identified by their ability to phosphorylate and
activate the c-JUN N-terminal transactivation domain (5),
whereas, ERK phosphorylates an inhibitory C-terminal DNA-
binding domain of c-JUN (6,7).
In general, the ERK cascade is activated by growth factors
and is critical for cell proliferation (8). Conversely, the JNK
and p38 pathways are stimulated by genotoxic agents and
cytokines mediating the stress response, growth arrest and
apoptosis (9–14). However, controversial evidence has indi-
cated that more complex roles of these MAPK pathways exist
to transmit other ultimately distinct cellular effects in different
cell lineages. For example, ERK promotes differentiation in
neuronal cells, T cells and muscle cells (15,16), in contrast to
transmiting mitogenic signals in fibroblasts. On the other hand,
stress-activated JNK and p38 have been associated with
cell survival (17,18), anti-cytotoxicity (19,20), anti-apoptosis
(21,22), transformation (23–25), proliferation (26) and invasion
(27). The complexities of the physiological roles of MAPKs
may be due partially to the duration of MAPK activities
differentially regulating genes in various cell types. While
transient ERK activation leads to proliferation, persistent
activation mediates growth arrest or differentiation signals
(28–30). In contrast, transient JNK and p38 induction could
provide a survival signal, whereas persistent activation induces
apoptosis (31,32).
Molecular Carcinogenesis Laboratory, Department of Life Sciences,
National Tsing Hua University, Hsinchu 300, Taiwan, Republic of China
1To whom correspondence should be addressed
Email: jlyang@life.nthu.edu.tw
Cadmium (Cd), a human carcinogen, can induce apoptosis
in various cell types. Three major mitogen-activated protein
kinases (MAPKs), c-JUN N-terminal kinase (JNK), p38
and extracellular signal-regulated kinase (ERK), have been
shown to regulate apoptosis. In this study we explore the
ability of Cd to activate JNK, p38 and ERK, including their
effects on Cd-mediated growth inhibition and apoptosis in
a human non-small cell lung carcinoma cell line, CL3. The
kinase activity of JNK was induced dose-dependently by
30–160 µM CdCl2. High cytotoxic doses of Cd (130–160
µM) markedly activated p38, but low Cd doses did not.
Conversely, the activities of ERK1 and ERK2 were
decreased by low cytotoxic doses of Cd (≤80 µM) and
moderately activated by high Cd doses. Low cytotoxic
doses of Cd transiently activated JNK and simultaneously
reduced ERK activity, whereas high cytotoxic doses of Cd
persistently activated JNK and p38. PD98059, an inhibitor
of ERK upstream activators MAPK kinase (MKK) 1 and
MKK2, greatly enhanced cytotoxicity and apoptosis in cells
treated with low Cd doses. In contrast, SB202190, an
inhibitor of p38, decreased the cytotoxicity and apoptosis
induced by high Cd doses. Transient expression of a
dominant negative form of JNK1, but not that of JNK2,
significantly increased the viability and prevented apoptosis
of Cd-treated cells. However, expression of wild-type JNK1
did not affect viability and apoptosis of Cd-treated cells.
Transfection of wild-type JNK2 or p38 enhanced apoptosis
of cells exposed to low Cd doses but did not affect those
exposed to high Cd doses. The JNK activity stimulated by
low Cd doses was partially suppressed by expression of a
dominant negative form of MKK7, but not a dominant
negative form of MKK4, indicating that MKK7 is involved
in JNK activation by Cd. Together, the results of this study
suggest that JNK and p38 cooperatively participate in
apoptosis induced by Cd and that the decreased ERK
signal induced by low Cd doses contributes to growth
inhibition or apoptosis.
Cd is a ubiquitous environmental toxicant that has been
evaluated as a human carcinogen (33). The main sources of
human exposure to Cd are tobacco smoke, food and industrial
pollution. Cd is absorbed by inhalation and ingestion and has
a biological half-life Ͼ25 years. For cigarette smoke, up to
60% of Cd deposited in the lung is absorbed (34). In experi-
mental animals Cd compounds have been shown to induce
tumors in lung, testes, prostate, the hematopoietic system and
at injection sites (33,35). Cd compounds have been shown to
induce morphological transformations, chromosomal aberra-
tions and gene mutations in cultured mammalian cells (36–
40). Cd is highly reactive with sulfhydryl groups of proteins
and can substitute for zinc in certain enzymes (41–43). Cd has
been shown to induce mRNA levels of several genes such as
Introduction
Three major mammalian mitogen-activated protein kinases
(MAPKs), extracellular signal-regulated kinase (ERK), c-JUN
N-terminal kinase (JNK) and p38 kinase, are regulated by
Abbreviations: ERK, extracellular signal-regulated kinase; FITC, fluorescein
isothiocyanate; GST, glutathione S-transferase; JNK, c-JUN N-terminal kinase;
MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; MKP, MAPK
phosphatase; MTT, 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium
bromide; PBS, phosphate-buffered saline.
© Oxford University Press
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S.-M.Chuang, I-C.Wang and J.-L.Yang
24 h. The cells were treated with 500 µg/ml 3-(4,5-dimethyl-thiazol-2-yl)
2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co., St Louis,
MO) and cultured for 3 h in a CO2 incubator. Cells having functional
mitochondrial succinate dehydrogenase can convert MTT to formazan that
generates a blue color when dissolved in dimethyl sulfoxide (59). The intensity
was measured using a reader for enzyme-linked immunosorbent assay and an
absorption wavelength of 565 nm.
c-jun (44), c-myc (44), c-fos (45), metallothionein genes (46)
and heme oxygenase (47,48). Cd has been reported to activate
p38 and ERK in 9L rat brain tumor cells (49) and to activate
JNK in LLC-PK1 porcine renal epithelial cells (50). Moreover,
Cd promotes apoptosis in various cells (49,51–53), but the
roles of MAPKs in Cd-mediated growth arrest and apoptosis
have not been explored. In this study we have investigated the
ability of Cd to induce AP-1 DNA-binding activity, c-jun
expression and the activities of JNK, p38 and ERK in a human
lung adenocarcinoma cell line, CL3. The roles of these three
MAPKs in growth arrest and apoptosis induced by Cd were
investigated using PD98059, a specific inhibitor of MKK1 and
MKK2 (ERK upstream kinases), and SB202190, a specific
inhibitor of p38, or by transient transfection assay to block
activation of JNK. The results have suggested that p38 activated
persistently by high cytotoxic doses of Cd induces an apoptotic
signal, while decreased ERK activity induced by low cytotoxic
doses of Cd contributes to growth arrest and apoptosis. The
transfection results have suggested that JNK1 is more important
than JNK2 in growth inhibition and apoptosis of Cd-treated
cells. Additionally, activation of JNK by Cd is mediated
through an MKK7-dependent and MKK4-independent
pathway.
Apoptosis
The annexin V–fluorescein isothiocyanate (FITC) binding assay was adopted
for determination of apoptosis. Briefly, 2ϫ105 cells were washed twice with
PBS and suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl
and 2.5 mM CaCl2). The cells were stained with annexin V–FITC (1 ng/µl)
(Medical and Biological Laboratories Ltd, Japan) and propidium iodide
(5 ng/µl) for 15 min in the dark. The stained cells were analyzed using flow
cytometry and the CellQuest program (Becton Dickinson). Apoptotic cells were
detected as those stained with annexin V–FITC but not with propidium iodide.
Whole cell extract preparation
Cells were lysed in a buffer containing 20 mM HEPES at pH 7.6, 75 mM
NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.1% Triton X-100, 0.1 mM Na3VO4,
50 mM NaF, 0.5 µg/ml leupeptin, 1 µg/ml aprotinin, and 100 µg/ml 4-(2-
aminoethyl)benzenesulfonyl fluoride. The cell lysate was rotated at 4°C for
30 min, centrifuged at 10 000 r.p.m. for 10 min and the precipitates discarded.
Protein concentrations were determined by the BCA protein assay kit (Pierce,
Rockford, IL) using bovine serum albumin as a standard.
Kinase assay
The JNK activity assay was performed using glutathione S-transferase (GST)–
cJun(1–79) (kindly provided by Dr M.Karin) as substrate according to the
procedure described by Hibi et al. (5) with modifications. Briefly, 50–200 µg
of protein prepared from cell extracts were mixed with 5 µg of GST–cJun
(1–79) and 20 µl of glutathione–Sepharose 4B suspension (Amersham Pharma-
cia Biotech, Arlington Heights, IL) at 4°C for 3 h then centrifuged. After
three washes in HEPES binding buffer (20 mM HEPES, pH 7.6, 50 mM
NaCl, 2.5 mM MgCl2, 0.1 mM EDTA and 0.05% Triton X-100) and one
wash in kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl2, 2 mM DTT
and 0.1 mM Na3VO4), the pelleted beads were resuspended in 30 µl of kinase
buffer containing 20 µM ATP and 2 µCi [γ-32P]ATP (6000 Ci/mmol;
Amersham, Pharmacia Biotech). The reaction was performed at room temper-
ature for 30 min and terminated by washing with HEPES binding buffer.
Phosphorylated proteins were eluted with 30 µl of 1.5ϫ Laemmli sample
buffer, boiled for 5 min and resolved on 10% SDS–polyacrylamide gels. The
relative radioactivity was quantitated using a computing densitometer equipped
with the ImagQuant analysis program (Molecular Dynamics, Sunnyvale, CA).
The in-gel kinase assay was conducted by resolving 50 µg of total proteins
on a 10% SDS–polyacrylamide gel containing 40 µg/ml GST–cJun(1–79)
fusion protein as described by Hibi et al. (5).
Materials and methods
Cell culture
Cell line CL3, established from a non-small cell lung carcinoma tumor of a
60-year-old male patient in Taiwan (54), was provided by Dr P.C. Yang
(Department of Internal Medicine and Clinical Pathology, National Taiwan
University Hospital, Taipei, Taiwan). Cells were cultured in RPMI 1640
medium (Gibco Life Technologies, Grand Island, NY) supplemented with
sodium bicarbonate (2.2% w/v),
L-glutamine (0.03% w/v), penicillin
(100 U/ml), streptomycin (100 µg/ml) and fetal calf serum (10%). Cells were
maintained at 37°C in a humidified incubator containing 5% CO2 in air.
Transfection
Plasmids containing dominant negative forms of JNK1 (pCMV-JNK1-APF;
JNK1DN) or JNK2 (pSR-JNK2-APF; JNK2DN), MKK4 (JNKK[K116R];
MKK4KR) or wild-type forms of JNK1 and JNK2 were kindly provided by
Dr M. Karin (University of California, San Diego, CA) (5,55,56). Plasmids
containing a dominant negative form of MKK7 (MKK7A) and the wild-type
form of p38 were gifts from Dr J. Han (The Scripps Research Institute, La
Jolla, CA) (57,58). Cells (4ϫ105) were plated in a 60 mm dish 1 day before
transfection. Plasmids (5 µg) were transfected into CL3 cells by calcium
phosphate co-precipitation. After incubation for 6 h, the cells were washed
with phosphate-buffered saline (PBS) and cultured in complete medium for
24 h, followed by maintenance in serum-free medium for 16–18 h. The cells
were then subjected to Cd treatment.
Western blot analysis
Cellular protein (20–50 µg) was loaded onto 10% SDS–polyacrylamide
gels. The protein bands were then transferred electrophoretically to PVDF
membranes (Micron Separations Inc., Westborough, MA). Membranes were
probed with primary antibody, followed by a horseradish peroxidase-conjug-
ated second antibody (Bio-Rad, Hercules, CA). Phospho-specific antibodies
for p38 (9211) and ERK (9101) were purchased from New England BioLabs
Inc. (Beverly, MA). Anti-JNK1 antibody (G151-666) was purchased from
Pharmingen (San Diego, CA). Anti-ERK2 (C-14) and anti-p38 (C-20) antibod-
ies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Antibody reactions were detected using the enhanced chemiluminescence
detection procedure according to the manufacturer’s recommendations (Amer-
sham, Pharmacia Biotech).
Treatment
Cells in exponential growth were plated at a density of 1ϫ106 cells/100 mm
dish and cultured in serum-free medium for 16–18 h before Cd treatment.
Cadmium chloride (Merck, Darmstadt, Germany) was dissolved in MilliQ
purified water (Millipore, Bedford, MA). Cells were treated with CdCl2 for
0.5–12 h in serum-free medium. In experiments to determine the effects of
PD98059 (Calbiochem, San Diego, CA) and SB202190 (Calbiochem) on
Cd-induced MAPKs, cell growth inhibition, cytotoxicity and apoptosis, cells
were treated with these kinase inhibitors for 1 h and then co-exposed to CdCl2
for 3 h. At the end of treatment, the drug-containing medium was removed
and the cells were washed twice with PBS.
Isolation and analysis of RNA
Total RNA (15 µg), isolated by a guanidium isothiocyanate/phenol/chloroform
extraction procedure (60), was subjected to electrophoresis in a 1% agarose–
2.2 M formaldehyde gel, transferred to nylon membranes and hybridized to
32P-labeled DNA probes prepared by rediprime random primer labeling
(Amersham Pharmacia Biotech) with a specific activity of Ͼ1.5ϫ108 c.p.m..
Hybridization was performed overnight at 42°C in a solution containing 50%
formamide, 6ϫ SSC (1ϫ SSC ϭ 180 mM NaCl, 10 mM sodium citrate, pH
8.0), 5ϫ Denhardt’s reagent, 0.5% SDS and 100 µg/ml salmon sperm DNA.
Membranes were washed to a stringency of 0.1ϫ SSC, 0.1% SDS for 10 min
at 65°C and exposed to X-ray films.
Cell growth
After treatment, the cells were cultured in medium containing 10% serum for
0–3 days before trypsinization. A portion of cells were mixed with 0.4%
trypan blue (Gibco Life Technologies) for 15 min and the number of unstained
cells were determined using a hemocytometer.
Viability assay
Cells were seeded in 96-well plates at a density of 5ϫ103 cells/well and
cultured in serum-free medium for 16–18 h before CdCl2 treatment. The cells
were treated with CdCl2 for 3 h in the presence or absence of kinase inhibitors.
After treatment, the cells were cultured in medium containing 10% serum for
Electrophoretic mobility shift assay
Nuclear protein was extracted as described by Andrews and Faller (61).
Binding reactions were performed at room temperature with 20 µl of a mixture
containing 6 µg nuclear protein, 10 mM Tris–HCl, pH 7.6, 3% glycerol,
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Roles of three MAPKs in Cd-induced apoptosis
50 mM KCl, 2.25 mM MgCl2, 0.125% NP-40, 2 µg bovine serum albumin
and 1 µg poly(dI·dC). After incubation for 20 min, 1 ng 32P-labeled
oligonucleotides (~20 000 c.p.m.) was added and incubated at room temper-
ature for another 30 min. The DNA–protein complexes were resolved on 6%
non-denaturing polyacrylamide gels at 4°C, dried and visualized by auto-
radiography. For competition experiments, a 100-fold excess of unlabeled
oligonucleotides was added to the reaction mixture before adding the labeled
probe. The sequences of the oligonucleotides used, specific for the AP-1
binding site and that containing a point mutation, were 5Ј-CGATCGCAT-
GACTCACTCAAT-3Ј and 5Ј-CGATCGCAgGACTCACTCAAT-3Ј, respect-
ively (the AP-1 binding site is underlined and the point mutation is in lower
case; only one strand is shown).
Results
Growth rate and viability of Cd-treated CL3 cells
The effects of Cd on the cellular growth rate and viability of
human lung adenocarcinoma CL3 cells were examined in
order to understand Cd cytotoxicity. Cells in exponential
growth were cultured in serum-free medium for 18 h before
they were exposed to various CdCl2 concentrations for 3 h.
The cells were maintained in culture in complete medium for
0–3 days and the numbers of viable cells were determined by
the trypan blue exclusion assay. Figure 1A shows that the
growth rates of CL3 cells treated with 30 or 60 µM Cd were
not significantly different from untreated cells, whereas 130
or 160 µM Cd markedly inhibited cell growth (P Ͻ 0.01).
Although the number of viable cells was decreased by 80 µM
Cd (P Ͻ 0.05), these cells could proliferate at a normal rate
2–3 days after treatment (Figure 1A). The relative survival of
cells treated with Cd was determined by the MTT assay.
Approximately 70 and 5% of cells survived after exposure to
80 and 160 µM Cd, respectively (Figure 1B).
Induction of c-jun expression and AP-1 DNA-binding ability
by Cd in CL3 cells
Fig. 1. Growth rate and viability of CdCl2-treated CL3 cells. (A) Cells
(1ϫ105) in exponential growth were plated and cultured for 24 h in
complete medium followed by serum starvation for 18 h. The cells were
then treated with Cd in serum-free medium for 3 h, washed with PBS and
cultured in medium containing 10% serum for 0–3 days. Cells were
trypsinized, stained with trypan blue and the numbers of viable cells were
determined with a hemocytometer. (B) The viability of cells was determined
by MTT assay 1 day after exposure to Cd. Results were obtained from three
or four experiments and the bar represents SEM. *P Ͻ 0.05 and **P Ͻ
0.01 using Student’s t-test for the comparison between untreated and Cd-
treated cells.
CL3 cells were treated with CdCl2 in serum-free medium for
3 h and subjected to northern blot analysis to examine the
ability of Cd to induce c-jun mRNA. Figure 2A shows that
Cd (50–150 µM) markedly induced c-jun mRNA above the
background level. The induced c-jun expression may sub-
sequently enhance the amount of AP-1 transcription factor and
its DNA-binding activity (2). The nuclear proteins of Cd-
treated and untreated CL3 cells were then extracted to examine
AP-1 DNA-binding activity using the electrophoretic mobility
shift assay. The nuclear extract obtained from untreated control
cells had little AP-1 DNA-binding activity, whereas it was
markedly induced by 100–200 µM Cd in CL3 cells (Figure
2B). Competition experiments showed that the induced AP-1
transcription factor interacts with a specific DNA AP-1-binding
sequence but does not interact with an AP-1 site containing a
point mutation (Figure 2B). These results clearly show that
Cd in the absence of serum can induce c-jun mRNA levels
and AP-1 DNA-binding activity in CL3 cells.
rylation sites of the catalytic domain. CL3 cells in exponential
growth were serum starved for 18 h prior to CdCl2 treatment
for 3 h. Western blot analyses showed that the amounts of
JNK, p38 and ERK proteins in Cd-treated cells were the same
as in untreated cells (Figure 3), whereas activation of these
MAPKs was markedly altered by Cd in a dose-dependent
manner (Figure 3). JNK activity was increased 2-fold by
30 µM Cd and dramatically increased by 60–160 µM Cd
(Figure 3A). Cd concentrations of 130–160 µM markedly
activated p38 but lower Cd concentrations (15–80 µM) did
not increase it (Figure 3B). In contrast, ERK activity was
decreased by 15–80 µM Cd and increased 4-fold at higher Cd
concentrations (130–160 µM) (Figure 3C). In-gel kinase assays
showed that both JNK1 and JNK2 activities were induced in
cells exposed to 100 µM Cd for 3 h (Figure 3D). The induced
kinase activities were higher than that induced by 40 J/m2
UVC, a well-known JNK activator (5; Figure 3D).
Cd alters the activities of JNK, p38 and ERK in CL3 cells
The transcription of c-jun is regulated by binding of a c-JUN/
ATF-2 heterodimer to the promoter (2). Both c-JUN and ATF-
2 are activated by the JNK pathway; ATF-2 is also activated
by the p38 pathway (2,4). Moreover, AP-1 activation is
correlated with the ERK pathway (2). We therefore examined
the ability of Cd to induce JNK, p38 and ERK activities in
serum-starved CL3 cells. JNK activity in CL3 cells was
determined using [γ-32P]ATP and a specific JNK substrate,
GST–cJUN(1–79). Activation of p38 and ERK were deter-
mined using specific antibodies recognizing the dual phospho-
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S.-M.Chuang, I-C.Wang and J.-L.Yang
Fig. 3. Dose-dependent activation of JNK and p38 and alteration in ERK
activity by CdCl2 in CL3 cells. (A) Cells were treated with Cd (0–160 µM)
in serum-free medium for 3 h. The proteins (200 µg) prepared from the
whole cell extract were assayed for JNK activity using GST–cJUN(1–79) as
substrate. The same cell extracts obtained from Cd-treated CL3 cells were
examined for amounts of dual phosphorylated p38 and ERK using phospho-
specific p38 (B) and phospho-specific p44/p42 MAPK antibodies (C),
respectively. The lower panels in (A)–(C) represent western blot analyses of
JNK, p38 and ERK2 protein levels. The relative intensity was quantitated
using a computing densitometer equipped with the ImagQuant analysis
program. The numbers in the middle of (A)–(C) indicate the relative
activities of kinases calculated from the average of three experiments. (D)
In-gel kinase analysis of JNK in Cd-treated (100 µM, 3 h) or UV-irradiated
(40 J/m2, 254 nm) CL3 cells. The whole cell extract of UV-irradiated cells
were isolated 30 min after treatment. The fluence rate of UV was measured
with a UVX radiometer (UVP Inc.).
Fig. 2. Induction of c-jun expression and AP-1 DNA-binding activity by
CdCl2 in CL3 cells. Cells were treated with Cd in serum-free medium for
3 h. Total RNA isolation and assay of AP-1 DNA-binding activity were
performed as described in Materials and methods. (A) Northern blot
analysis shows that the expression of c-jun mRNA was induced by 50, 100
and 150 µM Cd (lanes 2–4). Expression of 18S rRNA was analyzed to
serve as an internal control. (B) Electrophoretic mobility shift assay shows
that the nuclear extract obtained from untreated control cells (lane 1) or
cells treated with 10–50 µM Cd (lanes 2 and 3) had little AP-1 DNA-
binding activity, whereas it was markedly induced by 100–200 µM Cd
(lanes 4 and 5). Competition experiments were carried out by adding a 100-
fold excess of non-isotope-labeled wild-type AP-1 oligonucleotide (lane 6)
or oligonucleotides having a point mutation (lane 7) to the reaction mixture
containing the nuclear sample of lane 4 before adding the isotope-labeled
probe. Autoradiographs shown are from a representative experiment, which
was repeated three times with comparable results.
Figure 4 shows the activities of these MAPKs in a time
course Cd treatment. CL3 cells in exponential growth were
serum starved for 18 h and exposed to 80 µM Cd for 0.5–
12 h. The maximum activity of JNK induced by 80 µM Cd
in CL3 cells was observed after a 3 h treatment, and decreased
after a longer period (6–12 h) of Cd exposure (Figure 4). In
contrast, p38 activity increased when the cells were exposed
to 80 µM Cd for 6–12 h in a time-dependent manner (Figure
4). ERK activity abated during Cd exposure for 0.5–3 h
and increased above the control levels by 6–12 h treatment
(Figure 4).
Fig. 4. Time-dependent activation of JNK and p38 and reduction of ERK by
low doses of CdCl2 in CL3 cells. Cells were treated with 80 µM Cd in
serum-free medium for 0.5–12 h. Proteins (50 µg) prepared from whole cell
extract were assayed for JNK activity using GST–cJUN(1–79). The ERK
and p38 activities were determined by phospho-specific antibodies. The
relative activities of kinases shown are calculated from the average of three
experiments.
To further investigate the stability of Cd-activated JNK and
p38, Cd-treated CL3 cells were washed with PBS, cultured in
Cd-free medium for another 0, 1 or 8 h and then assayed for
JNK and p38 activities. Figure 5 shows that the JNK activity
induced by 80 µM Cd markedly decreased with increased
incubation time post-treatment, whereas JNK activity induced
by 150 µM Cd remained at high levels when the metal was
removed from the medium for 8 h (Figure 5). The results
suggest that Cd at low doses transiently activates JNK, whereas
high doses of Cd persistently activate it. High doses of Cd
also persistently activate p38, whereas low Cd doses do not
increase it (Figure 5).
Fig. 5. Duration of the activation of JNK and p38 by CdCl2 in CL3 cells.
Cells were treated with Cd (80–150 µM) in serum-free medium for 3 h,
washed with PBS and cultured continually in RPMI 1640 medium
containing 10% serum for 0, 1 and 8 h. The relative activities of kinases
shown are calculated from the average of three experiments.
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Roles of three MAPKs in Cd-induced apoptosis
Fig. 7. SB202190 attenuates, whereas PD98059 potentiates CdCl2-induced
apoptosis in CL3 cells. (A) PD98059 (50 µM) or SB202190 (10 µM) was
added to cells 1 h before Cd treatment for 3 h. Cells undergoing apoptosis
were examined at 8 h post-exposure to Cd using the annexin V–FITC
binding assay. Results were obtained from five or six experiments and bars
represent SEMs. *P Ͻ 0.05 and **P Ͻ 0.01 using Student’s t-test for
comparison between Cd-treated cells and those exposed to the same doses
of Cd in the presence of PD98059 or SB202190. (B) SB202190 or
PD98059 did not alter Cd-induced JNK activity. Cells were exposed to Cd
in the presence or absence of kinase inhibitors as in (A) and the whole cell
extract was isolated for determination of JNK activity. Each lane
Fig. 6. Effects of SB202190 and PD98059 on the growth rate and viability
of CdCl2-treated CL3 cells. PD98059 (50 µM) or SB202190 (10 µM) was
added to cells 1 h before Cd treatment for 3 h. The growth rate (A) and
viability (B) were determined as described in Figure 1. Results were
obtained from between three or five experiments and bars represent SEMs.
*P Ͻ 0.01 using Student’s t-test for comparison between Cd-treated cells
and those exposed to the same doses of Cd in the presence of PD98059 or
SB202190.
corresponds to the treatment as labeled in (A). The relative activities of
kinases shown are calculated from the average of three experiments.
administrating 10 µM SB202190 with 130 µM Cd markedly
reduced the frequency of apoptotic cells (P Ͻ 0.01; Figure
7A). In contrast, 50 µM PD98059 co-administration enhanced
apoptotic cell induction by 80 µM Cd (P Ͻ 0.05; Figure 7A).
Additionally, co-administrating PD98059 or SB202190 with
Cd did not affect the JNK activity induced by Cd (Figure 7B).
PD98059 or SB202190 alone neither increased the number of
apoptotic cells nor JNK activity above untreated control cells.
The results suggest that the p38 signal pathway, persistently
activated by high doses of Cd, participates in cytotoxicity and
apoptosis. Also, decreased ERK signaling by low doses of Cd
contributes to growth arrest or apoptosis.
Opposing roles of ERK and p38 in Cd-induced cytotoxicity
and apoptosis
To inspect the roles of ERK and p38 signaling pathways in
Cd cytotoxicity, CL3 cells were exposed to Cd in the presence
of the kinase inhibitors PD98059 and SB202190, specific for
MKK1/2 (ERK upstream kinases) and p38, respectively. The
numbers of viable cells were determined using the trypan blue
exclusion assay. Figure 6A shows that PD98059 (50 µM)
decreased the viability of cells exposed to 80 µM Cd (P Ͻ
0.01, day 3), whereas SB202190 (10 µM) potentiated the
numbers of viable cells in populations treated with 130 µM
Cd (P Ͻ 0.01, days 2 and 3). The effects of these kinase
inhibitors on Cd-induced cytotoxicity were also determined by
MTT assay. The results confirmed that PD98059 greatly
enhanced Cd cytotoxicity (P Ͻ 0.01, 80 and 120 µM), while
SB202190 inhibited the cytotoxicity induced by high doses of
Cd (P Ͻ 0.01, 160 µM; Figure 6B). These results indicate that
ERK and p38 have opposing roles in Cd-mediated cytotoxicity.
The annexin V–FITC binding assay was adopted to deter-
mine the role of apoptosis in Cd-mediated cell death. Figure
7A shows that Cd induced significant numbers of apoptotic
cells in a dose-dependent manner, ~40% of cells undergoing
apoptosis 8 h post-exposure to 130 µM Cd for 3 h. Co-
Roles of JNK1 and JNK2 in cell growth and apoptosis of
Cd-treated CL3 cells
Recent evidence indicates that individual JNK isoforms may
transmit distinct signals to regulate protective and cell death-
inducing pathways. For example, expression of JNK1 but not
JNK2 restores the ability of yeast lacking HOG1 (a p38
homolog) to grow in hypertonic medium (62). Expression of
dominant negative JNK1 (JNK1DN) but not dominant negative
JNK2 (JNK2DN) markedly increased the resistance of small
cell lung cancer cells to UV-induced apoptosis (63). Therefore,
we investigated the effect of Cd-induced JNK1 and JNK2 on
cell growth and apoptosis by transfection of CL3 cells with
vectors containing JNK1DN, JNK2DN or the wild-type forms
of JNK1 and JNK2. Cells transfected with 5 µg of vectors
were allowed 2 days for expression and then treated with Cd
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S.-M.Chuang, I-C.Wang and J.-L.Yang
Fig. 8. Transient transfection assay to determine the effects of JNK1 and
JNK2 on the growth rate of CdCl2-treated CL3 cells. Cells (4ϫ105) were
plated in a 60 mm dish 1 day before transfection. Vectors containing
JNK1DN, JNK2DN, JNK1 and JNK2 (5 µg each) were co-precipitated with
calcium phosphate and transfected into CL3 cells. After incubation for 6 h,
the cells were washed in PBS and resuspended in complete medium for 24
h, followed by sustenance in serum-free medium for 16–18 h. The cells
were then treated with 80 or 120 µM Cd for 3 h. (A) The kinase activity of
JNK was determined immediately after Cd treatment. The relative activities
of kinases shown are calculated from the average of three experiments. (B)
The numbers of viable cells were determined 24 h after Cd treatment by the
trypan blue exclusion assay. Data were obtained from three or four
independent experiments and bars represent SEMs. The statistical analysis
compares cells containing JNK1DN treated with Cd and those containing
pcDNA3 exposed to the same dose of Cd using Student’s t-test (*P Ͻ 0.05
and **P Ͻ 0.01).
Fig. 9. Effects of JNK1, JNK2 and p38 on apoptosis induced by CdCl2 in
CL3 cells. Transient transfection and expression of JNK1DN, JNK2DN,
JNK1, JNK2 or p38 (5 µg each) in cells followed by Cd treatment were
performed as described in Figure 8. Cells undergoing apoptosis were
examined 24 and 8 h post-exposure to 80 and 120 µM Cd, respectively,
using the annexin V–FITC binding assay. Results were obtained from
between four or six experiments and bars represent SEMs. The statistical
analyses compare cells containing JNK1DN, JNK2 or p38 treated with Cd
and those containing pcDNA3 exposed to the same dose of Cd using
Student’s t-test (*P Ͻ 0.05 and **P Ͻ 0.01).
for 3 h in serum-free medium. Figure 8A shows that transient
expression of JNK1DN and JNK2DN blocked the activity of
JNK induced by Cd. JNK1DN was more efficient than JNK2DN
in arresting activation of JNK by Cd, suggesting that Cd
activated JNK1 more than JNK2. Transient expression of
JNK1DN or JNK2DN did not affect the activities of p38 and
ERK induced by Cd (data not shown). The effects of Cd-
activated JNK1 and JNK2 on cell growth inhibition and
apoptosis were determined using the trypan blue exclusion
and annexin V–FITC binding assays. The numbers of viable
cells in Cd-treated populations were significantly increased by
expression of JNK1DN in CL3 cells (80 µM Cd, P Ͻ 0.05;
120 µM Cd, P Ͻ 0.01), whereas JNK2DN had no effect
(Figure 8B). Similarly, JNK1DN significantly abated Cd-
induced apoptosis but JNK2DN did not (Figure 9). Conversely,
transfection of wild-type JNK1 into CL3 cells influenced
neither growth inhibition nor apoptosis caused by Cd (Figures
8B and 9). In contrast, transfection of wild-type JNK2 or p38
could enhance apoptosis induced by low Cd doses in CL3
cells (P Ͻ 0.05; Figure 9). These results suggest that JNK1
in combination with JNK2 and p38 transmit a signal for Cd-
induced growth inhibition and apoptosis.
Fig. 10. Effects of MKK7 and MKK4 on activation of JNK by CdCl2 in
CL3 cells. Cells were transfected with kinase-defective MKK7A or
MKK4KR (5 µg each) and exposed to Cd as described in Figure 8. The
relative activities of JNK shown are calculated from the average of nine
experiments.
MKK7 but not MKK4 is involved in Cd induction of JNK activity
Both MKK4 and MKK7 are reported to be upstream activators
of JNKs by various stimuli (64–68). To determine the effect
of these MKKs on Cd-induced JNK activity, we transfected
CL3 cells with a dominant negative form of MKK4 (MKK4KR)
or MKK7 (MKK7A). The results showed that 35% of the
JNK activity stimulated by Cd (80 µM) was suppressed by
expression of MKK7A in CL3 cells (Figure 10). In contrast,
1428

Roles of three MAPKs in Cd-induced apoptosis
expression of MKK4KR enhanced Cd-induced JNK activity
~2-fold (Figure 10). The ability of MKK7A to suppress Cd-
induced JNK activity, however, was not observed in cells
treated with 130 µM Cd (data not shown).
MKPs are encoded by immediate early genes and induced
in response to environmental stresses and growth factor stimu-
lation to selectively down-regulate MAPK activities (69). For
example, the cytosolic M3/M6 MKP is highly specific for
JNK and p38 inactivation, while the association of MKP-3
with ERK in the cytosol markedly stimulates ERK dephospho-
rylation and inactivation (72–74). Activation of the JNK
pathway in NIH 3T3 fibroblasts could result in MKP-1 gene
expression and ERK inactivation, suggesting the existence of
cross-talk between the JNK and ERK signaling pathways (75).
However, Cd did not affect the total amounts of MKP-1 in
CL3 cells (S.-M.Chuang, unpublished data), suggesting that
MKP-1 may not be involved in the regulation of MAPK
activities in Cd-treated cells. Whether Cd affects expression
of MKPs deserves further investigation.
Proteins that interact and suppress MAPKs may also block
these signaling pathways. For example, GST π was recently
identified as associated with JNK and suppressed its kinase
activity (71). The anti-apoptotic effect of thioredoxin is reported
to be associated with inhibition of ASK1, a MAPK kinase
kinase that activates JNK and p38 (70). Both GST and
thioredoxin play important roles in detoxification of reactive
oxygen species and the balance of cellular redox levels (76,77).
The genotoxicity of Cd is correlated with intracellular reactive
oxygen species (39,78,79). Cd may down-regulate the functions
of GST π and thioredoxin, thereby elevating JNK and p38
activities. Moreover, the scaffold or adaptor proteins that
interact with multiple components of a specific signal cascade
can influence activation and function of distinct kinases. For
example, the adaptor protein JIP-1 (JNK interacting protein
1), which specifically binds to JNK, is required for JNK
activation (80), however, JIP-1 overexpression causes cyto-
plasmic retention of JNK and subsequently suppresses JNK
activity to phosphorylate nuclear transcriptional factors (80,81).
It will be important to explore the mechanism by which Cd
activation of JNK and p38 is mediated by its capability to
block cellular MAPK inhibitors.
Discussion
In the present study we have examined the ability of CdCl2
to activate JNK, p38 and ERK and the effects of these MAPKs
on Cd-mediated growth arrest and apoptosis in CL3 human lung
adenocarcinoma cells. Cd at low cytotoxic doses transiently
activated JNK and concomitantly reduced ERK activity,
whereas high cytotoxic doses of Cd persistently activated JNK
and p38. Co-administration of Cd with specific inhibitors to
block ERK or p38 signaling showed that these pathways have
opposing effects on the growth arrest and apoptosis induced
by Cd. Inhibition of ERK activity by PD98059 accelerated
growth inhibition and apoptosis induced by low Cd doses,
suggesting that ERK is involved in cell proliferation and anti-
apoptosis. Inhibition of p38 activity by SB202190, however,
enhanced cell viability and prevented apoptosis induced by
high Cd doses, suggesting that persistently activated p38
participates in apoptosis. Transient expression of JNK1DN,
but not JNK2DN, in CL3 cells markedly increased cell viability
and prevented apoptosis in Cd-treated populations. On the
other hand, transfection of wild-type JNK2 or p38, but not
JNK1, could enhance apoptosis induced by low Cd doses in
CL3 cells. Low Cd doses did not activate p38 and may activate
JNK1 more than JNK2. This would explain in part why
transfection of wild-type p38 or JNK2 enhanced apoptosis
when the cells were exposed to low Cd doses. The results
shown here suggest that Cd may sequentially activate JNK1,
JNK2 and p38 in a dose-dependent manner to cooperatively
induce apoptosis. Decreased ERK activity may combine with
increased JNK activity to transmit a signal temporally causing
growth arrest by low Cd doses in CL3 cells.
Separate signaling modules may provide specific responses
of MAPK pathways to different stimuli. For example, MKK1
and MKK2 specifically activate the ERK group, MKK3 and
MKK6 specifically activate p38 and MKK4 activates both p38
and JNK (2). Recently, MKK7/JNKK2 was identified as
phosphorylating and activating JNK, but not p38 or ERK
(67,68). We have demonstrated that JNK activity stimulated
by low Cd doses is suppressed by expression of a dominant
negative form of MKK7. In contrast, expression of a dominant
negative form of MKK4 moderately enhances Cd-induced
JNK activity. It has been shown that JNK activation by various
stimuli is mediated differentially through MKK4 and MKK7.
For example, tumor necrosis factor α and interleukin 1 activate
MKK7 much more than MKK4, suggesting that JNK activation
by these stimuli is primarily through MKK7; anisomycin and
UV activate MKK4 more than MKK7 (64,66). The results of
this study suggest that JNK activation by Cd is at least in
part by activation of its upstream activator MKK7 and is
independent of MKK4 in CL3 cells. Nevertheless, kinase-
defective MKK7A could not completely block JNK activation
by low Cd doses and did not suppress its activation by high
Cd doses. It is possible that Cd may induce JNK upstream
activators other than MKK7 and MKK4; the existence of such
an uncharacterized JNK upstream activator has been observed
in cells exposed to a hyperosmolar environment (66). Alternat-
ively, Cd may destroy a negative control pathway which blocks
specific MAPK phosphatases (MKPs) (69) or inhibitors (70,71).
We have shown here that the expression of c-jun mRNA
was markedly increased in CL3 cells treated with 50–150 µM
Cd for 3 h. JNK was also activated by the same Cd doses.
These findings agree with the notion that JNK phosphorylates
c-JUN/ATF-2 which binds to the promoter of c-jun (2). The
Cd-induced c-jun mRNA would enhance c-JUN levels, thereby
increasing AP-1 DNA-binding activity (2). However, AP-1
DNA-binding activity was induced at somewhat higher doses
of Cd. The failure to observe enhanced AP-1 binding activity
at the low Cd doses that were able to induce JNK kinase
activity and c-jun expression may be due in part to the fact
that the specific AP-1-binding site used here was the promoter
sequence of gadd153. Furthermore, activated JNK is not
always correlated with increased levels of c-jun transcripts
and AP-1 binding activity (82,83).
The cytotoxicity of Cd is highly associated with the levels
of intracellular metallothionein, which sequesters Cd and
alleviates its toxicity (84). Cd can induce expression of
metallothionein in CL3 cells as determined by northern blot
analysis (I.-C.Wang, unpublished data). This may explain why
CL3 cells are more resistant to Cd than those such as Chinese
hamster ovary K1 cells that are deficient for metallothionein
(39). Nevertheless, CdCl2 (4 µM, 3 h, ~30% cytotoxicity) can
also markedly activate JNK in Chinese hamster ovary K1 cells
(S.-M.Chuang, unpublished data), suggesting that metallothio-
nein is not required for JNK activation by Cd. Furthermore,
1429

S.-M.Chuang, I-C.Wang and J.-L.Yang
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CdCl2 (30 µM, 3 h, ~30% cytotoxicity) significantly activates
JNK in HFW normal diploid human fibroblasts (S.-M.Chuang,
unpublished data), indicating that the ability of Cd to activate
JNK is not restricted to CL3 lung adenocarcinoma cells.
Cd is classified as a human carcinogen (33). However,
several reports have shown an ability of Cd to suppress tumor
growth and progression in rodent lung and liver (85,86). Cd
has been shown to induce apoptosis in various cells (49,51–
53). The relationship between apoptosis and tumor suppression
by Cd is not clear (86). Our finding that the JNK and p38
signal pathways induced by Cd participate in growth arrest
and apoptosis of human lung adenocarcinoma cells suggests
a potential role of these MAPKs in Cd-mediated tumor
suppression.
In summary, we have demonstrated here that low cytotoxic
doses of Cd activate JNK, reduce ERK and do not affect p38,
producing convergent signals transiently inhibiting cell growth,
whereas high cytotoxic doses of Cd persistently activate JNK
and p38 to induce apoptosis. JNK activation by Cd is mediated
through a MKK7-dependent and MKK4-independent pathway.
Further investigation into the mechanisms by which Cd alters
the functions of specific regulators of JNK and p38 would
greatly help to elucidate the role of MAPK components in
Cd-mediated apoptosis and carcinogenesis.
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Acknowledgements
The authors would like to thank Dr P.C.Yang for providing the CL3 cells and
Drs M.Karin and J.Han for the expression vectors. This work was supported
by the National Science Council, Republic of China under contract no. NSC87-
2311-B007-032.
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Received January 5, 2000; revised March 23, 2000; accepted March 27, 2000
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