* Department of Biochemistry and Molecular Biology, Graduate Program in Genetics,
National Food Safety and Toxicology Center, and
Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824-1319
Received May 25, 2004; accepted September 3, 2004
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ABSTRACT |
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Key Words: cobalt; hypoxia; toxicity; BNIP3; metal.
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INTRODUCTION |
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Certain metals are also essential for human health. For example, cobalt plays a critical role in the synthesis of vitamin B12. In contrast, excessive exposure to cobalt is associated with several conditions, including asthma, pneumonia, and hematological abnormalities (Lauwerys and Lison, 1994). In addition, nickel, cobalt, cadmium, and other metals are known or suspected carcinogens (Hayes, 1997
). Despite numerous reports of metal-induced toxicity, the underlying mechanism remains unclear. Studies in various systems have shown that exposure to certain metals, such as cobalt, promotes a response similar to hypoxia. Hypoxia is defined as a state when oxygen tension drops below normal limits and it plays a central role in development and several pathological conditions including stroke, cardiovascular disease, and tumorigenesis (Giaccia et al., 2003
). Due to oxygen's critical role in energy production, organisms have developed a programmed response to hypoxia that increases glucose utilization and stimulates erythropoiesis and angiogenesis to compensate for the decrease in available oxygen (Bunn and Poyton, 1996
; Li et al., 1996
; Maltepe and Simon, 1998
; Semenza et al., 1994
). The hypoxia inducible factors (HIFs) are a family of transcription factors that mediate the response to hypoxia by regulating the expression of genes capable of regulating glycolysis, angiogenesis, and erythropoiesis, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), pyruvate kinase, and many others (Forsythe et al., 1996
; Gleadle and Ratcliffe, 1997
; Krieg et al., 1998
; Sandner et al., 1997
; Wang and Semenza, 1995
).
Prolonged hypoxia can also induce genes involved in cell death (Bruick, 2000; Guo et al., 2001
). Cobalt and nickel can activate hypoxia-mediated signaling pathways aberrantly under normoxia by stabilizing the cytosolic hypoxia inducible factor 1
(HIF1
) (Ho and Bunn, 1996
). For example, cobalt is thought to stabilize HIF1
by inhibiting the prolyl hydroxylase domain-containing enzymes (PHDs), a family of enzymes that play a key role in oxygen dependent degradation of the transcription factor (Epstein et al., 2001
). The characterization of the PHD family of enzymes offers a direct link between metal exposure and HIF mediated signaling. This direct link and the overlap between gene expression patterns between hypoxia and cobalt exposure have led us to hypothesize that HIFs may be necessary to the toxic effects of cobalt (Vengellur et al., 2003
). This hypothesis was tested using HIF1
/ cells and several markers of toxicity. Our results demonstrate that HIF1
plays an important role in metal-induced toxicity. Among the HIF1
dependent genes whose expression was altered by cobalt treatment are the pro-apoptotic factors, BNIP3 and NIX. Overall, these results suggest that cobalt-induced toxicity is dependent upon the HIF1
protein and its ability to induce the expression of cell death promoting genes.
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MATERIALS AND METHODS |
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Cell culture and toxicity assay. Mouse embryonic fibroblast cell lines were maintained in modified DMEM media (10% heat inactivated FBS, penicillin-streptomycin [10 U/ml], non-essential amino acid [10 µg/ml], L-glutamine [2 mM]). The cells were treated with 0 and 100 µM of CoCl2 for 48 h. Cells were trypsinized, counted with a hemacytometer and 10,000 cells were plated in 6 cm cell culture dishes. Cells were counted with a hemacytometer on one, two, three, four, and five days after plating.
MTT assay. Cells were grown in 96 well plates and treated with 0, 50, 100, 150, and 200 µM of CoCl2 for 72 h. The MTT assay was performed by replacing the cobalt containing media with 100 µl of media containing 0.5 mg/ml of MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide). The plates were then incubated for 4 h (37°C). Media was then removed by aspiration and 200 µl of solvent (1:1 DMSO:ethanol) was added and the formazan crystals solubilized with continuous agitation. Optical density (OD) measurements were taken at 550 and 630 nm and the difference in OD relative to untreated controls was taken as a measure of cell viability (van de Loosdrecht et al., 1991). For the CoCl2 time course experiment cells were treated with 150 µM CoCl2 and MTT assay was performed on four consecutive days and values are represented as a percent of time matched controls within cell types.
Protein extraction and Western blotting. Wild type and HIF1/ cells were grown under normoxic (20% O2), or hypoxic (1% O2) conditions (NAPCO 7000 incubator, NAPCO, Winchester, VA) or in the presence of 100 µM or 150 µM CoCl2 and protein extracts were prepared as described previously (LaPres et al., 2000
). Briefly, cells were washed with cold PBS (4°C) and removed from surface by scraping on cold PBS and collected by centrifugation. Soluble proteins were extracted with cell lysis buffer (25 mM HEPES, pH = 7.6, 2 mM EDTA, 10% glycerol, 1 µg/ml of aprotinin, leupeptin, pepstatin A, and 1 mM PMSF) and three rounds of sonication (5 s, 4°C). Insoluble material was removed by centrifugation (16,000 x g, 1 h). Protein concentrations were determined using Bio-Rad Bradford assay kit and BSA standards (Lowry et al., 1951
). An equal amount of protein was separated by SDS-PAGE, and Western blotting was performed with BNIP3 and NIX specific antibody (Sigma, St. Louis, MO and Exalpha Biologicals, Maynard, MA respectively) using ECL chemiluminescent detection kit (Amersham Pharmacia). A ß-actin specific antibody (a generous gift of Dr. John Wang, MSU) was used to verify equal loading.
RNA extraction and reverse transcription. RNA extraction was performed using TriZol reagent (Invitrogen) via manufacturer's instructions. Briefly, cells were treated for the specific duration and washed in 1X PBS (4°C). Cells were removed by scraping in the presence of 1 ml of TriZol reagent. Phase separation was accomplished by addition of chloroform and centrifugation (16,000 x g, 15 min). RNA was precipitated using isopropanol and was quantitated spectrophotometrically. One µg total RNA was used in subsequent reverse transcription reaction using Superscript II RNase H-Reverse Transcriptase (Invitrogen) via manufacturer's instructions.
Real-time quantitative PCR analysis. The measurement of BNip3 and NIX mRNA levels were performed using real-time PCR technology and SYBR Green as a detector. Primers were designed using the web based application, Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) biasing towards the 3' end of the transcript giving a gene-specific product (Table 1). The primers were characterized by BLAST, and the amplicon size was verified by gel electrophoresis. The primer and Mg2+ concentrations were optimized and the PCR was performed using 5% of the reverse transcription product described above. All standards, unknowns, and no template controls assays were performed with at least three biological replicates. All of the assays were performed on an ABI 7700 under standard thermal cycling parameters: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 60 s. The mRNA expression for each gene was determined by comparing it with a standard curve of known quantities of the specific target. This measurement was controlled for RNA quality, quantity, and RT efficiency by normalizing it to the expression level of the murine hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. HPRT was used as a control gene because it was shown to be unaffected by any treatment used.
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Caspase assays were performed using EnZChek caspase-3 assay kit #2 (Molecular Probes) via the manufacturer's instructions. Briefly, cells were left untreated or exposed to CoCl2 (150 µM, 48 h) or staurosporine (1 µM, 4 h). Cell extracts were obtained by scraping the cells in lysis buffer and cleared by centrifugation. The caspase-3 activity in the supernatant was analyzed spectrophotometrically (caspase activity in the cell lysate leads to the cleavage of the non-florescent substrate into a florescent product). The specificity of the caspase 3 activity was determined by the addition of a caspase-3 inhibitor (Ac-DEVD-CHO Inhibitor).
Statistics. Statistical analysis was performed between treated and untreated or vehicle controls using t-test (two tailed, unequal variance, p 0.05 cut-off).
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RESULTS |
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Western Blot Analysis of BNIP3 and NIX Levels
Western blot analysis was performed to determine the levels of BNIP3 and NIX proteins in CoCl2 treated cells. Wild type and HIF1/ cells were treated with cobalt (100 and 150 µM) or hypoxia (1% O2) for 48 h and total protein was extracted and separated by SDS-PAGE. There was a dramatic increase in BNIP3 levels following cobalt and hypoxia exposure and this increase was dependent upon the presence of HIF1
(Fig. 5, upper panel). The multiple bands represented in the BNIP3 Western blot are due to progressive proteolysis and has been previously reported to be indicative of cellular stress (Chen et al., 1997
). NIX protein levels also showed a marginal increase in CoCl2 and hypoxia treated wild type cells (Fig. 5, middle panel). Both BNIP3 and NIX levels were unchanged in CoCl2 treated HIF1
/ cells. ß-actin was used to verify equal protein loading (Fig. 5, lower panel).
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Cells were left untreated or exposed to cobalt (150 µM), or staurosporine (0.01 µM) for 48 h and stained with Hoechst33342 nuclear dye. Staurosporine is a known inducer of apoptosis and was used as a positive control (Tamaoki et al., 1986). Cobalt induced a moderate level of chromatin condensation in the WT cells, while little, if any, was observed in the HIF1
/ cells. In contrast, staurosporine treated WT and HIF1
/ cells showed marked chromatin condensation (Fig. 6A). The percentage of condensed nuclei in each treatment was also determined. Cobalt treated wild type cells induced a three-fold increase in the percentage of condensed nuclei (Fig. 6B). These experiments, taken together with the previous gene expression and Western blot results, suggest that HIF1
-mediated gene activation may be involved in promoting cobalt-induced cell death.
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DISCUSSION |
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Cell viability and proliferation studies show that wild type cells are more susceptible to cobalt-induced toxicity when compared to HIF1/ cells. Given that HIF1
is a transcription factor, it seemed likely that this toxicity is dependent upon gene activation. Previous genomic screens and other reports have identified BNip3 and NIX as target genes of hypoxia signaling (Bruick, 2000
; Guo et al., 2001
; Sowter et al., 2001
; Vengellur et al., 2003
). Expression of these pro-apoptotic factors was shown to be HIF1
dependent and to occur at a dose and in a time frame similar to that of cobalt-induced cell damage (Figs. 2 and 4). These results offer a direct link between the cobalt exposure, hypoxia signaling, and activation of genes involved in cell injury. BNIP3 and NIX are BH3 domain-containing proteins belonging to the pro-apoptotic family of genes and their increased expression is correlated with increased cell death (Chen et al., 1999
). Here we show that these mitochondrial proteins are not mediating cell death through the classical caspase-3 activation pathway. There have been earlier reports that increases in BNIP3 lead to caspase activation in primary cardiac myocytes undergoing hypoxia (Regula et al., 2002
). However, in other cell types, BNIP3 induces a cell death similar to necrosis, which doesn't involve caspase activation (Kubasiak et al., 2002
). In addition, it has been shown that BNIP3 cause a necrosis-like cell death in cells through the mitochondrial permeability transition pore which involves the loss of mitochondrial potential but without caspase activation and cytochrome C release (Vande Velde et al., 2000
). This is not surprising, as there are alternate pathways of apoptosis that do not require caspase activation such as the AIF pathway (Lorenzo et al., 1999
). One important point is that caspase-dependent apoptosis is an energy requiring process. At least during hypoxia, ATP levels in the cell are low due to the inhibition of oxidative phosphorylation. Therefore, it is possible that the cells initiate an apoptotic process but resort to a necrotic pathway due to reduced ATP levels. At present it is not clear if ATP levels are reduced under cobalt treatment; however, the morphological study of CoCl2 treated cells show moderate chromatin condensation in the absence of caspase 3 activation. Taken together, it seems likely that the HIF1
-dependent increase in BNIP3 and NIX leads to caspase-independent, necrotic-like cell death similar to what has been demonstrated in 293T, MCF7, other MEFs and various tumors (Sowter et al., 2001
, 2003
; Vande Velde et al., 2000
).
Cobalt and nickel are known to activate hypoxic signaling and nickel-induced transformation of fibroblasts requires a functional hypoxia signaling pathway (Salnikow et al., 1997, 2003
). The mechanism of action of CoCl2 mediated stabilization of HIF1
under normoxia is not completely elucidated. It is thought to inhibit the iron containing HIF prolyl hydroxylase enzyme, which plays a critical role in mediating the normal hypoxic signaling by modifying HIF1
protein and targeting it for degradation. The chemical characteristics of cobalt also allow it to compete for iron at reactive sites of various enzymes, rendering these enzymes inactive. The first published reports of the PHD family of enzymes characterized this inhibition and helped explain the ability of cobalt to act as a hypoxic mimic (Epstein et al., 2001
). Recent reports also suggest that cobalt exposure may not displace iron at the catalytic site within the hydroxylase but may sequester the available ascorbate in the cell. Since ascorbate is necessary for the transition of iron between oxidation states, this would effectively inhibit PHD activity (Salnikow et al., in press).
The hypoxic signaling pathway is known to activate cell survival genes involved in glycolysis, angiogenesis, and erythropoiesis (Levy et al., 1995, Semenza et al., 1994
; Wang et al., 1995
). In addition, in some cell types, cobalt and hypoxia exposure has been shown to inhibit apoptotic pathways (Piret et al., 2004
). Under chronic hypoxia, however, this pathway also activates genes involved in cell cycle arrest and death including pro-apoptotic genes (Bruick, 2000
). These reports and our current results suggest a complex and at times, contradictory picture for cobalt induced damage. The protective effects of cobalt were shown using a very different experimental paradigm and this may explain the differences in results. Piret et al. exposed HepG2 cells to tert-butyl hydroperoxide (t-BHP) under serum-free conditions to characterize cobalt's inhibitory effects (Piret et al., 2004
). In contrast, we utilized MEFs in the presence of serum and absence of outside apoptotic stimuli. Treatment time may have also been a factor since the HepG2 cell's cobalt exposure was limited to 8 h in the serum containing controls. These differences highlight the complexity in metal-induced toxicity and suggest multiple pathways may be involved. For example, the observation that cobalt toxicity was only partially attenuated in the HIF1
/ cells suggests that cobalt-induced cell death involves HIF1
dependent and independent mechanisms (Fig. 1). HIF1
independent mechanisms may be dependent upon other functioning HIFs in the MEFs or the possible disruption of one or more of the essential enzymes that require iron as a cofactor, which may ultimately affect cell viability. Also, the effect of oxidative stress due to the production of reactive oxygen species on various cell processes and integrity of cellular components such as proteins, DNA and lipid bi-layer cannot be underestimated. Consistent with this hypothesis, cobalt chloride treatment is known to induce stress responsive proteins such as metallothionein in wild type and HIF1
/ cells (Murphy et al., 1999
; Vengellur et al., 2003
).
In summary, WT and HIF1/ cells were treated with cobalt chloride and toxicity was studied using cell count and MTT assays. Wild type cells showed a marked decrease in cell viability as well as cell proliferation compared to HIF1
/ cells. Wild type cells showed an increase in the expression of the cell death gene, BNip3 mRNA and protein upon CoCl2 treatment. The cell death and expression of BNip3 mRNA overlapped in both the time course and dose response studies. This study shows that cobalt chloride exposure in mouse embryonic fibroblast leads to a necrosis-like cell death, which is dependent on the presence of functional HIF1
protein. This indicates that the pathology of cobalt-induced toxicity might be due to the activation of aberrant hypoxic signaling leading to the increase in the cell death promoting genes such as BNIP3 and NIX levels and subsequent necrosis.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at 402 Biochemistry Building, Michigan State University, East Lansing, MI 48824-1319. Fax: (517) 353-9334. E-mail: lapres{at}msu.edu.
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