Inhibition of proteasome activity is involved in
cobalt-induced apoptosis of human alveolar macrophages
Jun
Araya,
Muneharu
Maruyama,
Akira
Inoue,
Tadashi
Fujita,
Junko
Kawahara,
Kazuhiko
Sassa,
Ryuji
Hayashi,
Yukio
Kawagishi,
Naohiro
Yamashita,
Eiji
Sugiyama, and
Masashi
Kobayashi
First Department of Internal Medicine, Faculty of Medicine,
Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama
930-0194, Japan
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ABSTRACT |
Inhalation of
particulate cobalt has been known to induce interstitial lung disease.
There is growing evidence that apoptosis plays a crucial role
in physiological and pathological settings and that the
ubiquitin-proteasome system is involved in the regulation of
apoptosis. Cadmium, the same transitional heavy metal as
cobalt, has been reported to accumulate ubiquitinated proteins in
neuronal cells. On the basis of these findings, we hypothesized that
cobalt would induce apoptosis in the lung by disturbance of the
ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells
and human alveolar macrophages (AMs) to cobalt chloride
(CoCl2) and examined their apoptosis by DNA
fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride
staining, and Western blot analysis. CoCl2 induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl2 inhibited proteasome activity in U-937 cells.
Cobalt-induced apoptosis was mediated via mitochondrial pathway
because CoCl2 released cytochrome c from
mitochondria. These results suggest that cobalt-induced
apoptosis of AMs may be one of the mechanisms for
cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.
ubiquitin-proteasome system; U-937 cells
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INTRODUCTION |
ALTHOUGH COBALT
(Co) is an essential trace element for human nutrition, recent
clinical, epidemiological, and experimental studies have been
accumulating evidence indicating that the particles of this metal, when
inhaled, may produce an interstitial lung disease termed "hard metal
disease" or "cobalt lung" (32, 33, 47). There is a
report describing the case histories of five diamond polishers with
interstitial lung disease caused by exposure to Co only
(5). It is also reported that Co concentration in the
pulmonary specimen from a patient exposed to hard metal dust was
significantly higher than in control subjects (44).
Although it is not easy to distinguish between hard metal lung disease and idiopathic pulmonary fibrosis (IPF), it is speculated that exposure
to metal dust may be an important factor in the etiology of IPF
(15, 18, 24, 49a). In epidemiological studies, a
significantly higher rate of IPF was, indeed, observed in workers
exposed to metal dust compared with control subjects (18, 49a).
Recently it has been reported that Co activates hypoxia-inducible
factor-1
(HIF-1
), which in turn induces accumulation of p53
through direct association of the two proteins (1, 12, 34). p53 is well known to be involved in the maintenance of genome integrity by means of the inhibition of cell cycle progression or the induction of apoptosis (1). These findings
suggest that Co may have the ability to induce apoptosis in
lung cells and that apoptosis might be involved in the
progression of Co-induced lung disease.
Apoptosis is a highly conserved process that can be triggered
by a wide range of physiological and pathological conditions. Recent
evidence indicates that apoptosis is closely implicated in the
pathophysiology of a variety of lung diseases (9, 26). In
bleomycin-induced pulmonary fibrosis, apoptosis has been
reported to play an essential role in the disease progression
(25). There are several reports that the levels of soluble
Fas ligand in bronchoalveolar lavage (BAL) fluid (BALF) obtained from
acute respiratory distress syndrome (ARDS) patients correlate with
their clinical courses and that the BALF is capable of inducing
apoptosis in human lung epithelial cells (38).
There is growing evidence that the ubiquitin-proteasome system is
involved in the regulation of apoptotic pathway (3, 6, 16,
35, 43). Cadmium, which is the same transitional heavy metal as
Co, has been recently reported to be capable of accumulating ubiquitinated proteins in neuronal cells (8). Moreover,
the ubiquitin-proteasome system is known to act as a regulator of HIF-1
that can be upregulated by Co (14).
In the present study, we analyzed whether Co was capable of inducing
apoptosis in U-937 cells and human alveolar macrophages (AMs).
Furthermore, we tried to elucidate the mechanism of Co-induced apoptosis particularly in association with the
ubiquitin-proteasome system.
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MATERIALS AND METHODS |
Reagents.
Polyacrylamide, cobalt chloride (CoCl2), nickel chloride
(NiCl2), zinc chloride (ZnCl2), and ferrous
chloride (FeCl2) were purchased from Sigma (Tokyo, Japan).
Trypsin-EDTA was obtained from Gibco-BRL (Rockville, MD). The enhanced
chemiluminescence (ECL) detection system was purchased from Amersham
Life Science (Tokyo, Japan).
Carbobenzoxyl-L-leucyl-leucyl-L-leucinal
(MG-132), Z-Val-Ala-Asp(OMe)-CH2F (zVAD-fmk), and
Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (zLEHD-fmk) were
obtained from Calbiochem (La Jolla, CA). Substrates for 26S
proteasome, Suc-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (LLVY-MCA) and
Z-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA), were purchased from
Peptide Institute (Osaka, Japan). Hydrogen peroxide
(H2O2) was from Wako Pure Chemical Industries
(Osaka, Japan).
Cell culture.
U-937 cells derived from a human histiocytic lymphoma were purchased
from American Type Culture Collection (Manassas, VA). The cells were
grown in 100-mm tissue culture dishes in a humidified, 5%
CO2-95% air atmosphere. The culture medium was RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) containing 10% heat-inactivated fetal calf serum (Gibco-BRL), 2 mM L-glutamine, 50 IU/ml
penicillin, and 50 µg/ml streptomycin (complete medium).
Human AMs were obtained from healthy subjects by fiber-optic
bronchoscopy with BAL as described previously (37, 39).
Briefly, the upper airways were anesthetized with 2% lidocaine, and a
fiber-optic bronchoscope (BF-1T200; Olympus, Tokyo, Japan) was inserted
into the tracheobronchial tree. The bronchoscope was placed in a wedged position in a subsegmental bronchus of either right middle lobe or
lingula, where three 50-ml aliquots of sterile saline solution were
consecutively injected and then immediately aspirated. The BALF was
filtered through sterile gauze and centrifuged at 400 g for
10 min. The cells were washed twice with Hanks' balanced salt solution
and then suspended in complete medium. The cells were allowed to adhere
to tissue culture dishes for 1 h at 37°C. Nonadherent cells were
removed by rigorous washing of the dishes with warm medium. The
remaining adherent cells were checked microscopically and found to
consist of >90% AMs. Informed written consent was obtained from all
the participants before fiber-optic bronchoscopy.
Measurement of cell viability.
Cells were treated at the indicated concentrations of CoCl2
for the indicated times before harvest. Then, the cells were collected with trypsin-EDTA. Cell number and viability were determined with a
conventional hemocytometer by trypan blue exclusion.
Western blot analysis.
For detection of Bcl-2 (Transduction Laboratories, Lexington, KY), Bax
(Transduction Laboratories), poly(ADP-ribose) polymerase (PARP; Santa
Cruz, Santa Cruz, CA), ubiquitin (DAKO, Carpenteria, CA), and
cytochrome c (PharMingen, San Diego, CA) proteins, we carried out Western blot analysis. After the treatment with
CoCl2, the cells were washed twice with ice-cold PBS and
lysed with Triton X-based lysis buffer. The concentration of total
cellular protein was measured by Bio-Rad protein assay (Bio-Rad,
Richmond, CA). Thirty micrograms each of the protein preparations were
separated by electrophoresis on a 10% gradient SDS-polyacrylamide gel
and electrically transferred to nitrocellulose membrane. After blockade of nonspecific binding sites with 5% skim milk, blots were incubated overnight at 4°C with each specific antibody. The membranes were washed twice with PBS-Tween 80 and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The filters were
again washed with PBS-Tween 80 and developed with an ECL Western
blotting detection system (Amersham Life Science) at room temperature
before being exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY).
Detection of cytochrome c was performed as previously
described (43). U-937 cells were lysed in buffer
A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, and 250 mM sucrose) and homogenized with
a Dounce homogenizer. Homogenates were centrifuged twice at 750 g for 10 min at 4°C, and supernatants were centrifuged at
10,000 g for 15 min at 4°C. The resulting mitochondrial
pellets were resuspended in buffer A, and supernatants were
further centrifuged at 100,000 g for 1 h at 4°C. The
remaining supernatants (cytosolic fraction) were separated by
electrophoresis on a 12.5% gradient SDS-polyacrylamide gel.
Determination of DNA fragmentation.
After treatment, cells were collected and washed with PBS. The cells
were lysed in 100 µl of cell lysis buffer (10 mM Tris · HCl,
pH 7.4, 10 mM EDTA, pH 8.0, and 0.5% Triton X-100). After centrifugation, the supernatant was treated with 2 µl of RNase A (20 mg/ml) for 30 min at 37°C and then incubated with 2 µl of proteinase K (20 mg/ml) for 30 min at 37°C. DNA in the supernatant was precipitated overnight by the addition of 20 µl 5 M NaCl and 120 µl isopropanol. After centrifugation, the DNA pellet was dissolved in
Tris-EDTA buffer followed by electrophoresis on a 2% agarose gel. The
agarose gel was stained with ethidium bromide, and the resulting DNA
fragmentation pattern was revealed by ultraviolet illumination.
4',6-diamidino-2'-phenylindol dihydrochloride staining.
We performed nuclear staining with 4',6-diamidino-2'-phenylindol
dihydrochloride (DAPI; Boehringer Mannheim Biochemicals, Indianapolis,
IN). Harvested cells were washed with PBS and stained with
DAPI-methanol (10 µg/ml) at room temperature in the dark. Then the
treated cells were washed twice with distilled water and seeded on a
glass slide. The glass slide was viewed and photographed with a
fluorescence microscope (Nikon, Tokyo, Japan).
Proteasome activity assay.
Proteasome activity was analyzed as previously described (43, 45,
56). Harvested cells were lysed with proteasome lysis buffer (20 mM Tris · HCl, pH 7.2, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 5 mM ATP, 20% glycerol, and 0.04% Nonidet P-40) by repeated pipetting, followed by the incubation on ice for 20 min. Cell lysates were centrifuged at 16,000 g at 4°C for 10 min, and the
supernatant (30 µg of each of the protein preparations) was added to
proteasome activity assay buffer (50 mM HEPES, pH 8.0, 5 mM EGTA).
Substrate for 26S proteasome, 50 µM LLVY-MCA or 50 µM ZLLL-MCA, was
added to the mixture and incubated at 37°C for 30 min. We stopped the reaction by adding cold distilled water, and the reaction mixture was
placed on ice for at least 10 min. The intensity of fluorescence of
each reaction mixture was measured by fluorescence spectrophotometry at
355-nm excitatory and 460-nm emission wavelengths.
Electron spin resonance-spin trapping.
The procedures were performed as previously described
(55). The reaction mixtures contained 100 mM
5,5-dimethyl-1-pyrroline 1-oxide (DMPO; Labotec, Tokyo, Japan), 1 mM
H2O2, and 1 mM CoCl2 with or
without either DMSO (1%), dimethylthiourea (DMTU, 10 mM), or catalase
(1,000 U/ml) in 0.1 M PBS, pH 7.4. By 9.425-GHz field modulation with a
0.1-mT amplitude using a microwave power of 4 mW, electron spin
resonance (ESR) spectra of the reaction mixture in a quarts-flat cell
were recorded with ESR (RFR-30; Radical Research, Tokyo, Japan) at room
temperature. The yields of spin adduct were determined using a stable
nitroxide radical, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy, as a
standard. We determined a calibration curve by plotting the product of
the peak-to-peak derivative amplitude and the square of the width at
maximum slope of the signal vs. different concentrations of the
standard nitroxide radical.
Statistical analysis.
We repeated each type of experiment at least three times and confirmed
that similar data were obtained. All values are presented as means ± SD. Comparisons were made with one-way ANOVA with Fisher's post hoc
test. A P value of <0.05 was considered to be of
statistical significance.
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RESULTS |
CoCl2 induces apoptosis in U-937 cells.
In a number of studies on the toxicity of occupationally inhaled dust,
such as crystalline silica, AMs were recognized not only as the first
target of inhaled toxic substances but also as cells that play a
critical role in orchestrating the multiple events leading to fibrotic
changes (53). Although it has been shown that Co and
Co-containing dust are cytotoxic for AMs (46), accurate
mechanisms of toxicity have not been elucidated. We chose to utilize
U-937 cells as a model of AMs to begin to study Co-induced cytotoxicity. This histiocytic cell line was first isolated by Sundstrom and Nilsson (54) and was lysozyme positive and
weakly phagocytic, two characteristics compatible with
monocyte/macrophage-lineage cells, including AMs.
CoCl2-induced cell death in U-937 cells in a dose- and
time-dependent manner (Fig. 1,
A and B). Incubation with 1,000 µM of
CoCl2 for 24 h reduced the cell viability to ~50%.
To determine whether or not Co-induced cytotoxicity against U-937 cells
had the characteristics of apoptosis, we performed DNA
fragmentation assay and nuclear staining with DAPI. DNA fragmentation assay demonstrated that incubation with CoCl2 for 8 h
induced U-937 cells to show clear DNA ladder formation in a
dose-dependent manner (Fig.
2A). Using nuclear staining
with DAPI, we easily observed characteristic apoptotic features,
such as cell shrinkage, surface blebbing, and nuclear condensation, in
the CoCl2-exposed U-937 cells (Fig. 2B). In
contrast, no morphological changes were observed in control cells. In
the apoptotic pathway, caspase-3 activation is observed at the
common death-effector phase. Then we evaluated the activation of
caspase-3 by Western blot analysis of PARP, which is known as a
substrate for caspase-3. CoCl2 induced PARP cleavage in
U-937 cells in a dose-dependent manner as shown in Fig.
3.

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Fig. 1.
Effect of CoCl2 on the cell viability in U-937 cells.
A: U-937 cells were treated for 24 h with increasing
concentrations of CoCl2 (100-1,000 µM). Then the
cell viability was determined with a conventional hemocytometer by
trypan blue exclusion. Results are means ± SD obtained from 3 independent experiments. B: U-937 cells were cultured for
the indicated times in the presence ( ) and absence
( ) of CoCl2 (1,000 µM). Then the cell
viability was determined with a conventional hemocytometer by trypan
blue exclusion. Results are means ± SD obtained from 3 independent experiments.
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Fig. 2.
Effect of CoCl2 on the apoptosis of U-937
cells. A: DNA fragmentation induced by CoCl2.
U-937 cells were incubated with the increasing concentrations of
CoCl2 (100-1,000 µM) or ZnCl2 (1 mM) for
8 h. Then the genomic DNA was extracted from the treated cells,
electrophoresed on a 2% agarose gel, and visualized by ethidium
bromide staining. B: morphological changes in U-937 cells
treated with CoCl2. After incubation with CoCl2
(1,000 µM) for 8 h, the cells were stained with
4',6-diamidino-2'-phenylindol dihydrochloride (10 µg/ml) at 37°C
for 30 min. Then the treated cells were washed twice with distilled
water, seeded on a glass slide, and photographed with a fluorescence
microscope.
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Fig. 3.
Western blot analysis of apoptosis-related
proteins in U-937 cells treated with CoCl2. U-937 cells
were incubated with CoCl2 for 8 h, and then the cell
lysates were analyzed for poly(ADP-ribose)polymerase (PARP), Bcl-2,
and Bax proteins by Western blotting. The protein preparations (30 µg
each) were separated by electrophoresis on a 10% gradient
SDS-polyacrylamide gel and blotted. Immunoblotting was performed with
each specific monoclonal antibody.
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CoCl2 accumulates ubiquitinated proteins via inhibition
of proteasome activity in U-937 cells.
CoCl2 accumulated ubiquitinated proteins in U-937 cells in
parallel with the formation of the DNA ladder (Fig.
4). To determine whether the inhibition
of proteasome activity increases ubiquitinated proteins in U-937 cells,
we treated the cells with a proteasome inhibitor MG-132. As shown in
Fig. 5, A and B,
MG-132 accumulated ubiquitinated proteins and induced apoptosis
in U-937 cells. Addition of CoCl2 to MG-132 had no additive
or synergistic effect on the accumulation of ubiquitinated proteins
(Fig. 5A). These findings suggest that CoCl2 may
accumulate ubiquitinated proteins in U-937 cells through the inhibition
of proteasome activity.

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Fig. 4.
Western blot analysis of ubiquitinated proteins in U-937
cells. U-937 cells were incubated with CoCl2 for 8 h,
and then the cell lysates were analyzed for ubiquitinated proteins by
Western blotting.
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Fig. 5.
Effect of a proteasome inhibitor on CoCl2-induced
accumulation of ubiquitinated proteins and DNA fragmentation in U-937
cells. A: Western blot of ubiquitinated proteins. U-937
cells were incubated with CoCl2 and/or a proteasome
inhibitor, MG-132, for 8 h, and then the cell lysates were
analyzed for ubiquitinated proteins by Western blotting. B:
DNA fragmentation was analyzed after incubation with CoCl2
and/or MG-132 for 8 h.
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To rule out the possibility that Co-induced accumulation of
ubiquitinated proteins might merely be a consequence of
apoptosis, we examined the effect of CoCl2 on the
accumulation of ubiquitinated proteins in U-937 cells on condition that
apoptosis should not proceed in the presence of zVAD-fmk, a
broad caspase inhibitor. Although treatment with zVAD-fmk almost
completely suppressed CoCl2-induced DNA fragmentation, no
apparent decrease in the accumulation of ubiquitinated proteins was
observed (Fig. 6, A and
B). This indicates that the accumulation of ubiquitinated
proteins occurs before the activation of caspases in the process of
Co-induced apoptosis.

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Fig. 6.
Effect of Z-Val-Ala-Asp(OMe)-CH2F (zVAD-fmk),
a broad caspase inhibitor, on CoCl2-induced accumulation of
ubiquitinated proteins and DNA fragmentation in U-937 cells.
A: Western blot of ubiquitinated proteins. U-937 cells were
incubated with CoCl2 and/or zVAD-fmk for 6 h, and then
the cell lysates were analyzed for ubiquitinated proteins by Western
blotting. B: DNA fragmentation was analyzed after incubation
with CoCl2 and/or zVAD-fmk for 6 h.
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To directly verify the inhibitory effect of CoCl2 on the
function of 26S proteasome complex to degrade ubiquitinated proteins, we measured the effect of CoCl2 on the proteasome activity
in U-937 cells using peptide substrates for 26S proteasome (43, 45, 56). CoCl2 inhibited proteasome activity in a
dose-dependent manner in proportion to the accumulation of
ubiquitinated proteins (Fig. 7).
NiCl2, which is known as a toxic hard metal, also inhibited proteasome activity (Fig. 7). The results indicate that the inhibition of proteasome activity is involved in Co-induced accumulation of
ubiquitinated proteins.

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Fig. 7.
Effect of CoCl2 on proteasome activity. U-937
cells were incubated with CoCl2, MG-132, or
NiCl2 for 6 h and lysed with proteasome lysis buffer.
The cell lysates were centrifuged at 16,000 g for 10 min,
and the protein concentrations in the resulting supernatants were
measured. The protein preparations (30 µg each) were added to
proteasome activity buffer.
Suc-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide-MCA or
Z-Leu-Leu-Leu-4-methylcoumaryl-7-amide-MCA substrate for 26S proteasome
(50 µM) was added to the mixture and incubated at 37°C for 30 min.
The fluorescence intensities of reaction mixtures were measured by
fluorescence spectrophotometry at 380-nm excitatory and 440-nm emission
wavelengths. Significant difference from control samples:
*P < 0.05; **P < 0.001.
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Effects of various metals on accumulation of ubiquitinated proteins
and apoptosis in U-937 cells.
To further elucidate the association between Co-induced inhibition of
proteasome activity and its proapoptotic effect, we analyzed the
effect of NiCl2, which also has the ability to inhibit proteasome activity (Fig. 7), on the accumulation of ubiqutinated proteins and on apoptosis in U-937 cells. As expected,
NiCl2 accumulated ubiquitinated proteins in parallel with
the formation of the DNA ladder (Fig. 8,
A and B). In addition to CoCl2 and
NiCl2, we performed the same experiments on
ZnCl2, an antiapoptotic metal (10), and
FeCl2, a transitional metal capable of generating reactive oxygen species (ROS) by Fenton reaction (22). As shown in
Fig. 9, A and B,
neither ZnCl2 nor FeCl2 accumulated
ubiquitinated proteins and induced DNA ladder formation. These results
imply that metal-induced inhibition of proteasome activity is closely related to its proapoptotic effect.

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Fig. 8.
Effect of NiCl2 on accumulation of ubiquitinated
proteins and DNA fragmentation in U-937 cells. A: Western
blot of ubiquitinated proteins. U-937 cells were incubated with
NiCl2 for 6 h, and then the cell lysates were analyzed
for ubiquitinated proteins by Western blotting. B: DNA
fragmentation was analyzed after incubation with NiCl2 for
6 h.
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Fig. 9.
Effect of various metals on accumulation of ubiquitinated
proteins and DNA fragmentation in U-937 cells. A: Western
blot of ubiquitinated proteins. U-937 cells were incubated with various
metals at the indicated concentrations for 6 h, and then the cell
lysates were analyzed for ubiquitinated proteins by Western blotting.
B: DNA fragmentation was analyzed after incubation with
various metals at the indicated concentrations for 6 h.
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CoCl2-induced apoptosis is mediated via
mitochondrial pathway in U-937 cells.
Proteasome inhibitors have been reported to induce apoptosis
via a mitochondrial pathway (43). The fundamental events
involved in mitochondria-mediated apoptosis in mammalian cells
include release of cytochrome c from the intermitochondrial
membrane space to the cytosol, the formation of apoptosome (a
high-molecular-weight complex of cytochrome
c-Apaf-1-procaspase-9), caspase-9 activation, caspase-3
activation, and cleavage of key cellular proteins such as PARP. To
determine whether CoCl2 would induce apoptosis via mitochondrial pathway in U-937 cells, we analyzed the two steps crucial
to mitochondrial apoptotic signaling: the release of cytochrome c from mitochondria and caspase-9 activation. Cell
fractionation studies demonstrated that CoCl2 induced
mitochondrial cytochrome c release into the cytosol similar
to the phenomenon seen in proteasome inhibitor-induced
apoptosis (Fig.
10A). To confirm that
caspase-9 activation mediates Co-induced apoptosis, we used
zLEHD-fmk, a specific inhibitor of caspase-9. We found that treatment
with zLEHD-fmk obviously inhibited Co-induced DNA fragmentation in U-937 cells (Fig. 10B). These results indicate that
CoCl2 induces the mitochondrial pathway of
apoptosis signaling in U-937 cells.

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Fig. 10.
A: effect of CoCl2 on the
mitochondrial release of cytochrome c in U-937 cells. U-937
cells were incubated in the presence or absence of 1,000 µM
CoCl2 for 6 h and fractionated into mitochondrial and
cytosolic fractions as described in MATERIALS AND METHODS.
Western blot analysis of cytochrome c in the cell fraction
was then performed. B: effect of
Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (zLEHD-fmk), a specific
caspase-9 inhibitor, on Co-induced DNA fragmentation in U-937 cells.
DNA fragmentation was analyzed after incubation with CoCl2
in the presence or absence of zLEHD-fmk (100 µg/ml) for 6 h.
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ROS are not involved in CoCl2-induced accumulation of
ubiquitinated proteins and apoptosis in U-937 cells.
One of the pathophysiological mechanisms of Co is postulated to be its
ability to produce ROS (27, 28, 31-33, 41). It has
been reported that cadmium-mediated accumulation of ubiquitinated proteins is enhanced by its oxidative stress (8). Although the implication of ROS in the ubiquitin-proteasome system has been
thoroughly investigated (19, 51, 52), it remains yet to be
fully determined. We therefore analyzed the effect of
H2O2 on accumulation of ubiquitinated proteins
and apoptosis in U-937 cells because of its general use as a
representative ROS. Compared with the effect of CoCl2,
exposure to 100 µM H2O2 induced slight accumulation of ubiquitinated proteins and DNA fragmentation in U-937
cells (Fig. 11, A and
B). When the concentration of H2O2 was raised to 1 mM, there was no proportionate increase in the extent
of the accumulation of ubiquitinated proteins and of apoptosis (Fig. 11, A and B). Then we analyzed the effects
of a variety of antioxidants on Co-induced accumulation of
ubiquitinated proteins and apoptosis in U-937 cells. First, we
examined the ability of a variety of antioxidants (1% dimethyl
sulfoxide, 10 mM DMTU, and 1,000 U/ml catalase) to inhibit the
generation of ROS by a Fenton-type reaction of the mixture of
CoCl2 and H2O2 with the ESR-spin
trapping (22). Each of the antioxidants effectively eliminated the hydroxyl radical produced by the mixture (Fig. 12). On the basis of these results,
U-937 cells were pretreated for 30 min with each of the antioxidants at
a concentration sufficient to inhibit ROS production, and then
CoCl2 was added to the culture medium. None of these
antioxidants suppressed Co-induced accumulation of ubiquitinated
proteins and DNA fragmentation (Fig.
13, A and B).
These findings indicate that ROS are not implicated in Co-induced accumulation of ubiquitinated proteins and apoptosis in U-937 cells.

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Fig. 11.
Effect of H2O2 on accumulation of
ubiquitinated proteins and DNA fragmentation in U-937 cells.
A: Western blot of ubiquitinated proteins in U-937 cells.
The cells were incubated with the increasing concentrations of
H2O2 (10-1,000 µM) for 8 h. Then
the cell lysates were analyzed for ubiquitinated proteins by Western
blotting. B: DNA fragmentation was analyzed after incubation
with H2O2 for 8 h.
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Fig. 12.
Effect of a variety of antioxidants on reactive oxygen
species (ROS) formation by a Fenton-type reaction of CoCl2
and H2O2. The production of ROS was measured
with the method of electron spin resonance (ESR)-spin trapping as
described in MATERIALS AND METHODS. Hydroxyl radical
formation was measured by 5,5-dimethyl-1-pyrroline 1-oxide (DMPO)-OH
ESR signal (y-axis). ESR spectra were obtained from the
reaction mixtures containing 1 mM CoCl2 and 1 mM
H2O2 with or without DMSO (1%),
dimethylthiourea (DMTU, 10 mM), or catalase (1,000 U/ml).
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Fig. 13.
Effect of antioxidants on CoCl2-induced accumulation
of ubiquitinated proteins and DNA fragmentation in U-937 cells.
A: Western blot of ubiquitinated proteins in U-937 cells.
The cells were exposed to CoCl2 with or without coexposure
to DMSO (1%), DMTU (10 mM), or catalase (1,000 U/ml). Then the cell
lysates were analyzed for ubiquitinated proteins. B: DNA
fragmentation was analyzed after incubation with CoCl2 with
or without DMSO (1%), DMTU (10 mM), or catalase (1,000 U/ml).
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CoCl2 accumulates ubiquitinated proteins and induces
apoptosis in human AMs.
Next we analyzed the effect of CoCl2 on human AMs.
Consistent with the data of U-937 cells, CoCl2 accumulated
ubiquitinated proteins and induced apoptosis as confirmed by
DNA fragmentation (Fig. 14,
A and B) and DAPI staining (data not shown).

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Fig. 14.
Effect of CoCl2 on accumulation of
ubiquitinated proteins and DNA fragmentation in human alveolar
macrophages (AMs). A: Western blot analysis of ubiquitinated
proteins in human AMs. Human AMs were incubated with the indicated
concentrations of CoCl2 for 8 h, and then the cell
lysates were analyzed for ubiquitinated proteins. B: DNA
fragmentation was analyzed after incubation with CoCl2 for
8 h.
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CoCl2 has no effect on the levels of Bcl-2 and Bax
proteins.
A number of investigators have reported that Bcl-2 family members are
intimately involved in apoptosis (4). Recently,
several proteins of the Bcl-2 family, such as Bcl-2 and Bax, have been shown to be specifically degraded by means of the ubiquitin-dependent proteasome complex (29). We therefore evaluated the effect
of CoCl2 on the expression of these Bcl-2 family members,
Bcl-2 and Bax, in U-937 cells and human AMs. CoCl2 did not
modulate the levels of Bcl-2 and Bax in U-937 cells and human AMs
(Figs. 3 and 15).

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|
Fig. 15.
Western blot analysis of apoptosis-related
proteins in AMs treated with CoCl2. Human AMs were
incubated with CoCl2 for 8 h, and then the cell
lysates were analyzed for Bcl-2 and Bax proteins by Western blotting.
|
|
Cadmium chloride has been recently reported to decrease the levels of
Bcl-XL and Bid in U-937 cells (30). Hence, we
determined the effect of CoCl2 on the levels of
Bcl-XL and Bid in U-937 cells. Similar to Bcl-2 and Bax,
the levels of Bcl-XL and Bid did not vary with
CoCl2 exposure in U-937 cells (data not shown).
These observations imply that the expression levels of the Bcl-2 family
proteins are not involved in the Co-induced apoptosis in
monocyte/macrophage-lineage cells.
 |
DISCUSSION |
Occupational exposure to Co has been associated with the
development of various lung diseases such as hard metal lung disease and bronchial asthma (32, 33). Hard metal lung disease
caused by Co is a fibrosis characterized by desquamative and giant cell interstitial pneumonitis (47). It has been postulated that
several biochemical mechanisms may be implicated in Co-induced lung
injury: production of inflammatory cytokines and growth factors,
allergic or autoimmune-like reaction, and generation of ROS
(32). However, the detailed cellular mechanism involved in
the progression of Co-induced lung injury has not been well understood.
Apoptosis is a fundamental form of cell death, and a growing
body of recent literature supports its important roles in the pathophysiology of a variety of pulmonary disorders, including interstitial lung fibrosis, pulmonary emphysema, ARDS, and bronchial asthma (9). However, the contribution of apoptosis
to hard metal lung disease and its cellular mechanisms have not been
fully elucidated.
Our results have shown that CoCl2 induces the accumulation
of ubiquitinated proteins and apoptosis in U-937 cells as well as in human AMs. We also demonstrated that a potent proteasome inhibitor, MG-132, induced apoptosis in the same cells and that a broad caspase inhibitor, zVAD-fmk, was able to effectively suppress Co-induced apoptosis but not the accumulation of ubiquitinated proteins. These findings indicate that Co ions may induce
apoptosis in monocyte/macrophage-lineage cells, at least
partly, via the inhibition of proteasome activity. Indeed, we confirmed
that CoCl2 inhibited proteasome activity in U-937 cells
(Fig. 7). Although the inhibitory effect of CoCl2 (1,000 µM) on proteasome activity was not as potent as that of MG-132 (100 µM), the expression of ubiquitinated proteins in U-937 cells exposed
to CoCl2 was comparable to that in MG-132-treated cells.
There is growing evidence that ubiquitination is a reversible process
and deubiquitination, reversal of this modification by deubiquitinating
enzymes, is recognized as an important regulatory step. These suggest
that Co ions may have another function to accumulate ubiquitinated
proteins, such as the inhibition of deubiquitinating proteases.
Furthermore, we have shown that CoCl2 induced the release
of cytochrome c from mitochondria to the cytosol and that
Co-induced apoptosis was obviously inhibited by zLEHD-fmk, a
specific caspase-9 inhibitor in U-937 cells (Fig. 10B).
These findings indicate that, similar to other reagents that accumulate
ubiquitinated proteins (e.g., MG-132), Co ions induce apoptosis
through mitochondria-dependent pathway in U-937 cells.
Because numerous important regulatory proteins, such as p53, Bcl-2
family proteins, and cyclins, are the substrates of proteasome, it
seems that the inhibition of degradation of these proteins results in
their accumulation, thereby leading to apoptosis
(35). A prime candidate for a regulator of
apoptosis is p53. CoCl2 has been recently reported
to induce HIF-1
, which in turn stabilizes p53 (1).
Indeed, we have observed the accumulation of ubiquitinated p53 in A549
cells exposed to CoCl2 (data not shown). However, we have
clearly shown that both CoCl2 and MG-132 induced
apoptosis in p53-negative U-937 cells, indicating that the p53
was not involved in Co-induced apoptosis in our study.
Bcl-2 family proteins have been reported to be important regulators of
apoptosis, and Bcl-2, Bax, and Bid are degraded via the
ubiquitin-proteasome system (29). Members of this family, such as Bcl-2 and Bcl-XL, can inhibit apoptosis,
whereas others, such as Bax and Bid, promote cell death
(4). The overexpression of Bcl-2 and Bcl-XL
has an antiapoptotic effect on proteasome inhibitor-induced
apoptosis (3, 6, 43). It has been also demonstrated that proteasome inhibitor-induced apoptosis in
human M-07e leukemic cells is associated with the cleavage of Bcl-2 (58). We therefore examined the effect of
CoCl2 on the level of four Bcl-2 family proteins, Bcl-2,
Bcl-XL, Bax, and Bid, in U-937 cells. None of these four
proteins varied with CoCl2 exposure. This finding indicates
that the expression level of Bcl-2 family proteins is not involved in
Co-induced apoptosis. Recently, the activation of MAP kinases
and phosphorylation of Bcl-2 family proteins have been shown in
stress-induced apoptosis (13, 57). Indeed, in the
present study, we were able to detect the activation of MAP kinases in
Co-stimulated U-937 cells (data not shown). Hence, the determinant of
Co-induced apoptosis might be not the expression level but the
phosphorylation status of Bcl-2 family proteins. We are now exploring
the involvement of MAP kinase activation and phosphorylation of Bcl-2
family proteins in Co-induced apoptosis (40).
Another possibility is that Co ions might promote protein aggregation,
culminating in the inhibition of the ubiquitin-proteasome system, which
would in turn further increase the production of aggregated protein and
could account for the accumulation of ubiquitinated proteins. In
neurodegenerative disorders, aggregates of unfolded proteins have been
proposed to induce the progressive loss of proteasome activity and
consequent derangement of critical regulatory factors, leading to
apoptosis (2, 20). Moreover, it has been demonstrated that several metals, such as zinc and aluminum,
destabilize protein structure and promote progressive protein
aggregation (36). In contrast to zinc and aluminum, Co
ions have been reported to have no effect on the aggregation of human
-amyloid peptide
-A4 (7, 49). It is not likely that
CoCl2 primarily promotes protein aggregation, which then
inhibits proteasome activity.
In in vitro and in vivo studies (27, 28, 41), Co
ions have been shown to increase the generation of ROS that is closely implicated in apoptosis in a variety of cells
(23). Indeed, Zou et al. (59) demonstrated
that antioxidants significantly block CoCl2-induced
apoptosis in neuronal PC12 cells. Furthermore, ROS has been
recently reported to influence the activity of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme (19, 51, 52). We therefore analyzed the effect of H2O2 as a
representative ROS on accumulation of ubiquitinated proteins and
apoptosis. However, H2O2 induced only
slight accumulation of ubiquitinated proteins and DNA fragmentation
compared with CoCl2. We also examined the effect of
sufficient concentrations of antioxidants, which were confirmed to
eliminate ROS produced by the reaction mixture of Co and
H2O2, on Co-induced accumulation of
ubiquitinated proteins and apoptosis in U-937 cells. Our result
has shown that antioxidants do not inhibit the accumulation of
ubiquitinated proteins or apoptosis in U-937 cells exposed to
Co. The discrepancy between our study and that of Zou et al.
(59) not only may reflect experimental systems but also
may be due to the different cells used. Our finding implies that ROS
may not be implicated in the Co-induced cell death in
monocyte/macrophage-lineage cells. Similar to our findings on
Co-induced apoptosis, it has been recently reported that the activation of HIF-dependent genes by CoCl2 is ROS
independent (48).
In contrast to our findings of the proapoptotic effect of Co
on U-937 cells, this transitional metal has been reported to inhibit
epigallocatechin gallate-induced apoptosis in human oral tumor
cell lines (17). CoCl2 induces HIF-1
and
ROS in most cell types (12, 27). Indeed, we obtained the
finding that CoCl2 increased the expression of HIF-1
in
U-937 cells dramatically (data not shown). HIF-1 is known to contribute
not only to the survival but also to the death of cells through its
transcriptional induction of ubiquitous and cell-type specific genes
(50). Oxidative stress activates several other
transcription factors, such as activator protein-1 and NF-
B, which
induce the expression of an array of characteristic genes including
growth factors and cytokines in each cell type (11).
Similarly, whether proteasome inhibitors induce or inhibit
apoptosis depends upon the concentration and cell type utilized
(42). Together, many factors induced by Co may be involved
in the fate of cells, and their interactions may be so varied and
condition dependent that the stimulus inducing apoptosis in one
cell type inhibits death in another cell type.
In conclusion, CoCl2 induced apoptosis and
accumulated ubiquitinated proteins in U-937 cells and human AMs.
Cobalt-induced inhibition of proteasome activity was involved in this
accumulation of ubiquitinated proteins. The perturbation of the
ubiquitin-proteasome system may be one of the mechanisms of Co-induced
lung injury.
 |
ACKNOWLEDGEMENTS |
The authors acknowledge the thoughtful suggestions and technical
support of Dr. Takashi Kondo (Department of Radiological Science,
Toyama Medical and Pharmaceutical University).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. Maruyama, The First Dept. of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical Univ., 2630 Sugitani, Toyama 930-0194, Japan (E-mail:
mmaruyam-tym{at}umin.ac.jp).
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.
June 5, 2002;10.1152/ajplung.00422.2001
Received 30 October 2001; accepted in final form 17 May 2002.
 |
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