INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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).

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).

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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

View larger version (16K): [in this window] [in a new window]   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.

View larger version (33K): [in this window] [in a new window]   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.

View larger version (44K): [in this window] [in a new window]   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.

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.

View larger version (37K): [in this window] [in a new window]   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.

View larger version (50K): [in this window] [in a new window]   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.

View larger version (67K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   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.

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.

View larger version (38K): [in this window] [in a new window]   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.

View larger version (60K): [in this window] [in a new window]   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.

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.

View larger version (34K): [in this window] [in a new window]   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.

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.

View larger version (49K): [in this window] [in a new window]   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.

View larger version (22K): [in this window] [in a new window]   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).

View larger version (43K): [in this window] [in a new window]   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).

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).

View larger version (56K): [in this window] [in a new window]   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.

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).

View larger version (35K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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-X_L, can inhibit apoptosis, whereas others, such as Bax and Bid, promote cell death (4). The overexpression of Bcl-2 and Bcl-X_L 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-X_L, 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.