ToxSci Advance Access originally published online on February 14, 2006
Toxicological Sciences 2006 91(1):299-308; doi:10.1093/toxsci/kfj131
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Cadmium-Induced Apoptosis in Rat Kidney Epithelial Cells Involves Decrease in Nuclear Factor-Kappa B Activity
Department of Biomedical and Pharmaceutical Sciences and Center for Molecular Toxicology, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881
1 Corresponding author: zshaikh{at}uri.edu.
Received November 9, 2005; accepted January 30, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Renal epithelial cells undergo apoptosis upon exposure to cadmium (Cd). Transcription factors, such as nuclear factor-kappa B (NF-
B), mediate the expression of a number of genes involved in apoptosis. The present study was designed to examine the involvement of this transcription factor in Cd-induced apoptosis. Rat kidney proximal tubular epithelial cells, NRK-52E, were incubated with up to 20µM CdCl2 in serum-free medium for 5 h followed by incubation in serum-containing medium (without Cd) for an additional 12 h. The cells accumulated 582 ± 19 ng Cd/mg protein after 5-h exposure to 20µM Cd. As a result of Cd exposure, the DNA-binding activity of the p65 subunit of NF-B was decreased in a concentration- and time-dependent manner. The activity of tumor necrosis factor-
–induced inhibitor of kappa B (IB) kinase was also inhibited by Cd. In addition, the phosphorylation of IB- and NF-B p65, as well as the levels of NF-B target gene products, cIAP-1 and cIAP-2, were reduced. Pretreatment of the cells with the antioxidant U83836E or butylated hydroxytoluene preserved the DNA-binding activity and blocked the Cd-induced decease in IB- phosphorylation. Cd exposure caused the activation of caspase-3, -7, and -9 and DNA fragmentation. By flow cytometry, 14.6 and 30.5% apoptosis was detected at 6 and 12 h after stopping the Cd exposure. Overexpression of NF-B p65 by transient transfection protected the cells from the Cd-induced apoptosis. Conversely, attenuation of NF-B activity by pretreatment with SN50, an NF-B nuclear translocation inhibitor, potentiated apoptosis. These results suggest that Cd-induced apoptosis involves suppression of NF-B activity which may be mediated by oxidative stress.
Key Words: cadmium; nuclear factor-kappa B (NF-B); IKK; IB; IAPs; apoptosis; kidney; epithelial cells; oxidative stress.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cadmium (Cd) is a known occupational hazard and an environmental pollutant. Renal impairment is the main effect observed upon chronic Cd exposure, and the proximal tubules of the kidney are the primary target (Goyer and Clarkson, 2001
). The cellular and molecular mechanisms by which Cd causes nephrotoxicity have been extensively studied. Several investigations report that Cd induces apoptosis in different cell types, including the renal tubular epithelial cells (Hart et al., 1999; Ishido et al., 1995; Thevenod et al., 2000). The oxidative stress elicited by Cd is a likely contributor to this process (Shaikh et al., 1999). Although Cd cannot undergo oxidation/reduction to generate reactive oxidative species (ROS) directly, it can depress the level of endogenous antioxidant glutathione, inhibit the mitochondrial electron transport chain, and thus cause the generation of superoxide radicals (O'Brien and Salacinski, 1998; Robertson and Orrenius, 2000; Stohs et al., 2001; Tang and Shaikh, 2001).
Cd may induce apoptosis by altering the activity of various oxidative stress–sensitive transcription factors that are responsible for regulating apoptotic gene expression (Watkin et al., 2003). One of these factors, nuclear factor-kappa B (NF-B), is a prominent factor in the cell death/survival balance. In the cytoplasm, NF-B exists as both a homodimer and a heterodimer of Rel-related proteins. The most common form of NF-B is composed of p50 and p65 subunits associated with inhibitor of kappa B (IB). Diverse cellular stimuli lead to phosphorylation of IB by IB kinase (IKK) and release of NF-B that translocates to the nucleus, binds to its consensus sequence, and initiates the transcription of target genes (Schoonbroodt and Piette, 2000). Optimal activation of NF-B and induction of NF-B target genes also requires phosphorylation of NF-B p65 protein by IKK, along with the activation of a variety of other kinases (Viatour et al., 2005). Among the target genes, the inhibitors of apoptosis proteins (IAPs) are considered critical cell survival signals. The IAPs bind to caspases and inhibit apoptosis (Lee and Collins, 2001). Downregulation of these proteins and subsequent activation of caspases is one of the mechanisms of apoptosis (Bergmann et al., 2004; Lee and Collins, 2001; Yang and Li, 2000). Although NF-B can be either pro- or anti-apoptotic, under most circumstances, it acts as a stimulus for cell survival (Bours et al., 2000) and reduction in its activity leads to apoptosis (Feinman et al., 2002; Jones et al., 2000; Liu et al., 2002; Mathas et al., 2003; Piccioli et al., 2001).
Divalent metals, such as mercury, arsenic, zinc, and Cd, that have high affinities for sulfhydryl groups have been shown to inhibit NF-B DNA binding to DNA in vitro (Shumilla et al., 1998). In addition, in different cell types, mercury, arsenic, zinc, and copper have been shown to induce apoptosis by suppressing NF-B binding to DNA (Dieguez-Acuna et al., 2004; Mathas et al., 2003; Uzzo et al., 2002; Zhai et al., 2000). Since little is known about the involvement of NF-B in Cd-induced apoptosis, the present study was designed to explore this in a rat kidney epithelial cell line.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
Rat kidney epithelial cells, NRK-52E, were obtained from American Type Culture Collection (ATCC, Manassas, VA). Tissue culture reagents, including Dulbecco's modified Eagle's medium (DMEM), calf serum, trypsin, penicillin/streptomycin, protease inhibitor cocktail, CdCl2, and all other chemicals and biological reagents were obtained from Sigma (St. Louis, MO). T4 polynucleotide kinase and NF-
B consensus oligonucleotide were purchased from Promega (Madison, WI). 
-32P-ATP was obtained from PerkinElmer (Boston, MA). Colorimetric substrates for caspases were from Biomol (Plymouth Meeting, PA). SN50 was from Calbiochem (La Jolla, CA). IB- (1–317), phospho-IB- (Ser 32), actin, IKK, cIAP-1 and cIAP-2, and agarose-conjugated anti-IKK antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal antibody against phospho-NF-B p65 (Ser 276) was purchased from Cell Signaling (Beverly, MA). Monoclonal antibody against the p65 subunit of NF-B was obtained from Geneka (Montreal, Canada). LipofectAMINE and Plus reagent were purchased from Invitrogen (Carlsbad, CA). NF-B p65-enhanced green fluorescence protein (EGFP) expression plasmid was kindly provided by Dr. J. Schmid (Vienna, Austria), and pEGFP-N3 vector (Clontech Laboratories, Heidelberg, Germany) was a gift from Dr. S. Mathas (Berlin, Germany). Construction of NF-B p65-EGFP has been described by Schmid et al. (2000).
Cell culture and treatment.
The cells were cultured according to the instructions provided by ATCC for culturing the NRK-52E cells. The cells were grown in 75-cm2 culture flasks at 37°C in ATCC-modified DMEM that contained 4.5 g/l glucose and 1.5 g/l sodium bicarbonate. It was supplemented with 5% calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The medium was formulated for use with an atmosphere of 5% CO2/95% air. The cells used for the experiments were approximately 80% confluent at the time of Cd exposure.
A filter-sterilized 20mM CdCl2 stock solution in water was used to administer Cd to the cell culture medium just before use. Since serum proteins can form complexes with Cd, thus reducing its uptake and toxicity (Gennari et al., 2003), our laboratory routinely treats the cells with Cd in DMEM without serum. After Cd exposure for 5 h, the cells were washed and incubated in DMEM containing 5% calf serum for an additional 6 or 12 h. To examine the protective effect of antioxidants, the cells were treated for 30 min with 25µM butylated hydroxytoluene (BHT) or 10µM U83836E prior to exposure to Cd. The effect of Cd on IKK was studied in cells in which the activity of this enzyme was stimulated with 20 ng/ml tumor necrosis factor (TNF)-, 10 min prior to cell harvest. Nuclear NF-B activity was decreased by pretreatment of cells with 20µM SN50 for 30 min prior to coincubation with 10µM Cd.
Determination of cellular Cd concentration.
The cells were incubated with 20µM Cd in serum-free DMEM for 5 h. At the end of Cd treatment, the cells were quickly washed twice with phosphate-buffered saline (PBS) containing 2mM EGTA and once with PBS without ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). The washed cells were digested for 10 min in 1 ml of 50% nitric acid using a MARS 5 microwave-accelerated reaction system (CEM Corporation, Matthews, NC). The Cd concentration was determined by measuring 111Cd using an XSeries ICP-MS system (Thermo Electron Corporation, Madison, WI).
Cytoplasmic and nuclear protein preparations.
The cytoplasmic and nuclear proteins were prepared according to the standard method (Active Motif, Carlsbad, CA). After the Cd treatment, the cells were washed twice with ice-cold PBS, pH 7.4, and collected in cold PBS by gentle scraping. The cell suspension was centrifuged at 500 x g for 10 min. The pellet was lysed with buffer A (10mM HEPES [pH 7.9], 10mM KCl, 1mM ethylenediaminetetraacetic acid (EDTA), 0.2% NP40, 1mM dithiothreitol [DTT], and 0.5mM phenylmethylsulfonyl fluoride [PMSF]) containing 1% (vol/vol) protease inhibitor cocktail. After 10-min incubation on ice, the cell lysate was centrifuged at 14,000 x g for 15 s. The supernatant containing cytoplasmic proteins was aliquoted and stored at –80°C for further analysis. The pellet containing the nuclei was washed twice with buffer A and then resuspended in buffer B (20mM HEPES [pH 7.9], 420mM NaCl, 1mM EDTA, 1mM DTT, and 1mM PMSF) containing 1% (vol/vol) protease inhibitor cocktail. The suspension was incubated on ice for 30 min, and centrifuged for 5 min at 15,000 x g. The supernatant containing the nuclear proteins was aliquoted, and stored at –80°C.
Protein assay.
The protein concentration was determined using the Micro BCA reagent kit from Pierce (Rockford, IL) according to the manufacturer's instructions.
Electrophoretic mobility shift assay.
The electrophoretic mobility shift assay (EMSA) was performed as described by Zhang et al. (1991). Double stranded oligonucleotide containing a consensus NF-B–binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3') was labeled with T4 polynucleotide kinase and -32P-ATP. Ten micrograms of nuclear protein was mixed with 0.5 ng of radiolabeled oligonucleotide in the binding buffer [10mM Tris-HCl (pH 7.5), 1mM MgCl2, 0.5mM EDTA, 0.5mM DTT, 50mM NaCl, 10% glycerol, 0.05 µg/µl poly(dIdC)·poly(dIdC)]. The final volume of the reaction mixture was brought up to 20 µl with this buffer. After 20 min incubation at room temperature, the samples were resolved on a nondenaturing 6% polyacrylamide gel. The gel was subsequently dried and autoradiographed by a Typhoon Variable Mode Imager (Amersham Biosciences, Piscataway, NJ). The optical density of each DNA-binding band was quantified using ImageQuant version 5.2 software (Amersham Biosciences). The quantification method involved drawing a rectangular box around each band of interest. The background was corrected using the local average method, which determined the average of all the pixel values in the object outline and used this value for the background. The background-corrected intensity of pixels in the box area was combined to represent the DNA-protein binding activity in the band.
The identity of the DNA-NF-B complex was confirmed both by a competition test and by a supershift test using anti-p65 antibody. In the competition experiment, 100-fold excess unlabeled NF-B oligonucleotide was added to the reaction mixture before the addition of 32P-labeled oligonucleotide. For the supershift test, 1 µg monoclonal antibody against p65 subunit of NF-B was added to the reaction mixture and incubated for 20 min prior to the addition of 32P-labeled NF-B oligonucleotide.
Western blotting.
A total of 20 µg cytoplasmic protein was applied to a sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) for electrophoresis. Upon completion of the electrophoresis, the protein bands were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA), blotted in Li-Cor blotting buffer, incubated with the appropriate antibodies labeled with infrared dyes and detected by a Li-Cor infrared imager (Li-Cor, Lincoln, NE).
DNA fragmentation.
The procedure used for extraction of DNA was that described by Luo et al. (1998). Cells (
1 x 106) were harvested by gentle scraping and centrifuged at 500 x g for 5 min at 4°C. The pellet was resuspended in 5mM Tris-HCl buffer (pH 8.0) containing 20mM EDTA and 0.5% (vol/vol) Triton X-100 at 4°C and maintained for 20 min. To remove high molecular weight DNA, the sample was centrifuged at 14,000 x g for 30 min in the presence of 0.1% SDS. The sample was then sequentially extracted from the supernatant with equal volumes of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform. DNA was precipitated overnight after adding 0.1 volume of 5M NaCl and 2 volumes of ethanol at –70°C. The precipitate was resuspended in 30 µl Tris-EDTA buffer, and the RNA was removed by digestion with 0.1 mg/ml RNase for 3 h at 30°C. The resulting extract was electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, visualized using a UV light box, and photographed.
IKK assay.
To determine the IKK activity, 200 µg of cytoplasmic extract was incubated overnight with 5 µl agarose-conjugated IKK antibody at 4°C. The immunoprecipitate was washed twice with buffer A (see above) and once with kinase buffer (20mM HEPES [pH 7.4], 10mM MgCl2, 20mM ß-glycerophosphate, 1mM DTT, 1mM Na3VO4). The washed agarose beads were incubated at 30°C for 30 min with 20 µl kinase buffer containing 100µM ATP, 5 µCi -32P-ATP, and 1 µg IB- (1–317) as substrate. The reaction mixture was separated by SDS-PAGE. The gel was dried and autoradiographed by the Typhoon Variable Mode Imager. The cytoplasmic protein extract was analyzed by Western blotting with IKK antibody as a reference.
Transient transfection with NF-B plasmid.
The cells were plated in six-well plates in DMEM supplemented with 5% calf serum at a density of 1.5 x 105 cells per well. After reaching 80% confluence, the cells were transfected using LipofectAMINE and Plus reagent. Briefly, 800 ng of NF-B p65-EGFP or pEGFP-N3 vector was mixed with 16 µl of Plus reagent diluted in 100 µl serum-free DMEM and incubated at room temperature for 15 min. To the mixture, 4 µl LipofectAMINE reagent diluted in 100 µl serum-free DMEM was added and incubated at room temperature for another 15 min. The final transfection complex (800 ng/well) was added to a monolayer of NRK-52E cells in 0.8 ml serum-free DMEM and incubated at 37°C. After 3-h incubation, more serum-containing DMEM was added to bring the total volume to 5 ml normal growth medium. After 24 h, the cells were exposed to Cd as described above.
Flow cytometry.
The cells were washed twice with ice-cold PBS, dislodged by trypsin digestion, centrifuged at 500 x g for 10 min, and resuspended in PBS. The density of the cells was adjusted with PBS to 1 x 106 to 3 x 106 cells/ml, and 1 ml cell suspension was centrifuged at 500 x g for 10 min. Approximately 0.1 ml supernatant was retained, and the cell pellet was mixed on a vortex mixer for 10 s. In a dropwise manner, 1 ml ice-cold ethanol (75%, vol/vol) was added to the cells. The cell suspension was mixed for 10 s in between drops. Tubes were capped and the cells were fixed in ethanol overnight at 4°C. After fixation, the cells were mixed again and centrifuged at 1000 x g for 5 min to remove the ethanol. The cells were stained with propidium iodide (50 µg/ml) containing RNase A (100 U/ml) at room temperature for at least 30 min and analyzed within 24 h by flow cytometry using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). Analyses were performed at a setting of 20,000 events per sample. Apoptotic cells were quantified by analysis of the sub-G1 (subdiploid) peak using the ModFit LT version 2 for Macintosh data acquisition software package.
Caspase assays.
The caspase activity was determined by the colorimetric method described by Biomol. Ac-DEVD-pNA was used as a substrate for caspase-3 and -7, and Ac-LEHD-pNA for caspase-9. Cell extract (40 µg protein/20 µl) was incubated with 178 µl reaction buffer (100mM HEPES [pH 7.5], 20% vol/vol glycerol, 5mM DTT, 0.5mM EDTA) and 2 µl of 10mM substrate in dimethylsulfoxide. The samples were incubated at 37°C for 30 min, and the release of p-nitroanilide was measured at 410 nm in a microtiter plate reader.
Statistics.
Statistical analyses were performed by one-way analysis of variance, followed by Newman-Keuls test at p < 0.05 (n = 3).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhibition of NF-
B DNA-Binding Activity by CdAfter 5-h exposure to 20µM Cd, the cells accumulated 582 ± 19 ng Cd/mg protein. The effect of Cd exposure on NF-
B DNA binding was evaluated at the completion of Cd exposure (0 h) and 6 and 12 h later. The identity of the NF-B p65 band was verified by the supershift assay (data not shown). Cd exposure decreased the DNA binding of NF-B p65. While 5 and 10µM Cd had no effect, 15 and 20µM Cd decreased DNA binding by 36 and 53%, respectively (Fig. 1A). Persistence of the effect was evident from further decrease in NF-B p65 DNA binding in cells exposed to 20µM Cd and cultured for an additional 12-h period. These cells displayed a 65% decrease in DNA binding (Fig. 1B).
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Protection against the Effect of Cd on NF-
B DNA BindingSince Cd causes oxidative stress that is known to influence NF-
B activity, the effect of antioxidants on preserving the NF-B DNA-binding activity in Cd-treated cells was evaluated. As shown in Figure 2, pretreatment of cells with 10µM U83836E or 25µM BHT, 30 min prior to Cd exposure, limited the decrease in DNA binding to only 5 and 18%, respectively, as compared to the 54% decrease observed in the absence of the antioxidants.
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Inhibition of IKK Activity and I
B PhosphorylationOne of the possible mechanisms of Cd-mediated decrease in NF-
B activity could be the blockage of phosphorylation and subsequent degradation of IB by IKK. To investigate this possibility, an agarose-conjugated monoclonal IKK antibody was used to immunoprecipitate this enzyme from cells exposed to Cd. The cells were treated with 20 ng/ml TNF- for 10 min before the harvest in order to induce both IKK and ß. As depicted in Figure 3A, incubation of cells with 20µM Cd for 5 h reduced IKK activity. Even though Cd was removed from the medium, the IKK activity remained inhibited for at least an additional 12-h period. Lack of associated decrease in IKK protein suggested that Cd inhibited the enzyme activity but not its protein level. Further proof of the inhibition of IKK activity was obtained by examining the level of phosphorylated IB- using anti-phospho-IB- antibody. The decrease in phosphorylated IB- was detected as early as 6 h after stopping the Cd exposure (Fig. 3B), which was consistent with the inhibition of IKK activity. Pretreatment of cells with 10µM U83836E or 25µM BHT, 30 min prior to Cd exposure, blocked the Cd-induced decrease in IB- phosphorylation (Fig. 3C).
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Inhibition of NF-
B p65 PhosphorylationBecause phosphorylation of the p65 subunit of NF-
B by IKK and other protein kinases is required for optimal activation and translocation of NF-B to the nucleus, the effect of Cd on the phosphorylation was also evaluated. As shown in Figure 4, both cytoplasmic and nuclear phosphorylated NF-B p65 levels were decreased at 6 and 12 h following the Cd exposure.
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Suppression of IAP Expression
Among its various functions, NF-
B is responsible for the transcriptional regulation of IAP family proteins. To investigate the possibility that Cd exposure affected the expression of these proteins, the levels of cIAP-1 and cIAP-2 were analyzed. The results showed that Cd decreased the levels of both proteins at 6 and 12 h after stopping the Cd exposure (Fig. 5).
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Cd-Induced Apoptosis
Apoptosis was assessed by DNA fragmentation, caspase activation, and flow cytometry. Consistent with the inhibition of IKK activity and decline in NF-
B DNA binding, apoptotic DNA fragmentation was observed 6 h following the termination of exposure to 20µM Cd (Fig. 6). Similarly, significant elevation in the activities of caspase-3, -7, and -9 was evident 6 h after the Cd exposure and continued to progress further during the experimental period (Fig. 7). As observed by flow cytometry, the cells exhibited 14.6 and 30.5% apoptosis, respectively, at 6 and 12 h after the Cd exposure (Fig. 8).
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Protection from Cd-Induced Apoptosis by NF-
B p65 OverexpressionTo investigate the relationship between NF-
B and Cd-induced apoptosis, NF-B protein expression was increased in the cells by transient transfection with the NF-B p65-EGFP expression plasmid. As compared to the cells transfected with the vector alone, the overexpression of NF-B p65-GFP was evident in the p65-EGFP transfected cells (Fig. 9A), as was the increase in their DNA-binding activity (Fig. 9B). Overexpression of NF-B resulted in 47 and 48% reduction in Cd-induced cell death at 6 and 12 h after the Cd exposure, respectively (Fig. 10).
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Enhancement of Cd-Induced Apoptosis by SN50
To further delineate the role of NF-
B in Cd-induced apoptosis, a noncytotoxic concentration of a peptide (SN50) that lowers the nuclear translocation of NF-B/Rel complexes (Lin et al., 1995) was used to impair NF-B DNA-binding activity. The cells were exposed to a lower concentration of Cd (10µM) than that used in the other experiments. At this concentration, Cd had no significant effect on NF-B DNA binding (Fig. 11). In comparison, cells pretreated with 20µM SN50 significantly reduced the NF-B binding activity in both control and Cd-exposed cells. As expected, cells incubated with 10µM Cd alone exhibited relatively mild (7.8%) apoptosis (Fig. 12). In comparison, the cells pretreated with SN50 enhanced the cytotoxicity of Cd and the fraction of cells undergoing apoptosis increased 2.3-fold. These results indicated that attenuation of NF-B activity by SN50 rendered the NRK-52E cells more prone to Cd-induced apoptosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, after 5-h exposure to 20µM Cd in serum-free DMEM, the cells accumulated 582 ± 19 ng Cd/mg protein. Tohyama and Shaikh (1981)
studied renal toxicity in rats exposed to 5 µmol Cd/kg/day, 5 days a week. After 9 weeks of treatment, the Cd concentration in the renal cortex reached 240 µg/g wet weight and produced renal dysfunction. Assuming that the protein content of the rat kidney cortex is about 9.5% of the wet weight (Xie and Shaikh, unpublished data), the Cd concentration associated with renal damage is estimated to be about 2.5 µg/mg protein. In comparison, the cellular Cd burden in the present study was about one fourth of this concentration. Thus, the in vitro Cd exposure model employed in the present study is relevant to understanding the mechanism of in vivo toxicity.
Exposure to Cd causes apoptotic cell death in different cell types (Hart et al., 1999; Ishido et al., 1995; Thevenod et al., 2000). Positive apoptotic signals such as pro-apoptotic Bcl-2 proteins, mitochondrial permeability transition, cytochrome C release, ROS, and protein kinase modulation have all been implicated in Cd-induced apoptosis (Li et al., 2003; Oh et al., 2004; Pulido and Parrish, 2003; Shih et al., 2004). However, the negative apoptotic signaling pathways that involve anti-apoptotic transcription factors, repair and detoxifying enzymes, and caspase inhibitors have not been well studied. It has been suggested that susceptibility to Cd-induced apoptosis is dependent on the level of metallothionein, a potential negative regulator of apoptosis (Chabicovsky et al., 2004; Pulido and Parrish, 2003). Similarly, glutathione is identified as another protector against cell death (Jimi et al., 2004). In the present study, the modulation of an anti-apoptotic cell signaling pathway by Cd was investigated in rat kidney epithelial cells. The results showed that at concentrations of Cd that produced apoptosis, the activity of transcription factor NF-B, a key protective molecule in cell survival/death processes, was markedly reduced. Overexpression of NF-B p65 protected the cells from undergoing apoptosis. Conversely, suppression of nuclear translocation of NF-B by SN50 enhanced the Cd-induced apoptosis. Together, these observations suggest that NF-B indeed plays an important role in the cytotoxicity of Cd. The downregulation of the NF-B–dependent anti-apoptotic gene products IAPs appears to be a link between the effect of Cd on NF-B activity and resultant apoptosis.
As a ubiquitous multifunctional signaling system, members of the NF-B family play a prominent role in the cell death/survival balance. Under many circumstances, activation of NF-B turns on the powerful protective genes for cell survival (Bours et al., 2000). In comparison, suppression of NF-B activity sensitizes a variety of rodent and human cell lines to apoptotic stimuli or even directly induces apoptosis (Cahir-McFarland et al., 2000; Choi et al., 2000; Izban et al., 2001; Millet et al., 2000). Dieguez-Acuna et al. (2004) reported that mercury suppressed TNF-–induced NF-B activation and increased the sensitivity of NRK-52E cells to apoptosis. Other divalent metals like arsenic, zinc, cisplatin, and copper also produce similar effects. Mathas et al. (2003) found that arsenic rapidly inhibited IKK and NF-B activities and induced apoptosis in L540Cy HRS cells. The NF-B target genes, including cIAP-2, were downregulated. Furthermore, overexpression of NF-B p65 in the HRS cells protected the cells from arsenic-induced apoptosis. Similarly, physiological levels of zinc reduced NF-B activation and cIAP-2 expression in PC-3 and DU-145 human prostate cancer cells, and the cells became sensitized to cytotoxic agents like TNF- and paclitaxel (Uzzo et al., 2002). Suppression of NF-B and its target genes such as xIAP and TRAF2 was reported in cisplatin-induced apoptosis in Hep3B cells (Kim et al., 2004). Similarly, copper-induced apoptosis in a pro-B cell line, BA/F3beta, was achieved via inactivation of NF-B (Zhai et al., 2000). The present study demonstrated a link between Cd-induced apoptosis and NF-B DNA-binding activity. Thus, it appears that suppression of NF-B and its downstream pathway is a common underlying mechanism of apoptosis by a number of metals. The binding of metals to the sulfhydryl groups of target protein molecules and the oxidative stress caused by these metals are two of the plausible explanations for their effects on the NF-B activity.
IKK is a serine-specific kinase that is responsive to a number of potent NF-B activators like TNF-. Two members of the mitogen-activated protein kinase family, NF-B–inducing kinase and mitogen-activated protein/extracellular signal–regulated kinase kinase 1, have been shown to directly interact with IKK (Schoonbroodt and Piette, 2000). IKK activity is redox sensitive and is regulated by ROS (Bowie and O'Neill, 2000; Schoonbroodt and Piette, 2000). Phosphorylation of IB by IKK is the key step in NF-B activation, translocation, and induction of its target genes (Viatour et al., 2005). In the present study, a marked decrease in IKK activity and phosphorylation of IB was observed after the Cd exposure. The decrease in IB phosphorylation by Cd was prevented by pretreatment with antioxidants. Thus, Cd appears to decrease NF-B activity by decreasing its phosphorylation steps and it is reasonable to suggest that Cd-induced oxidative stress is involved in this enzymatic regulation.
A number of studies have reported that ROS serve as common intracellular mediators of IB degradation and subsequent NF-B activation in the cytoplasm (Flohe et al., 1997). In addition, a variety of antioxidants are capable of preventing NF-B activation (Asehnoune et al., 2004; Brennan and O'Neill, 1996; Park et al., 2004). There are other reports that suggest that the DNA-binding activity of NF-B is reduced by ROS (Mihm et al., 1995; Shumilla et al., 1998). Furthermore, Fernandez et al. (1999) reported that the decline in NF-B activation by dithiocarbamates was blocked by N-acetyl-L-cysteine in primary and transformed T cells. In contrast, Woods et al. (1999) found that in NRK-52E cells, the lipopolysaccharide-induced NF-B activation was not altered by either pretreatment with N-acetylcysteine or depletion of glutathione. They concluded that cellular redox status was not involved in the regulation of NF-B in the kidney cells, and instead a redox-insensitive calcium-dependent pathway regulated NF-B. N-acetylcysteine and glutathione form complexes with thiol-reactive metals like mercury and Cd. The formation of a complex may alter metal uptake, distribution, availability for interaction with protein thiols, and resultant toxicity. To avoid this complication, in the present study, two non-thiol antioxidants, U83836E and BHT, were used. Both antioxidants protected against the suppression of NF-B activity by Cd, indicating that under our experimental conditions, redox status appears to play a role in maintaining the activity of this transcription factor in the NRK-52E cells.
Shumilla et al. (1998) have demonstrated that preincubation of A549 cell nuclear extract with up to 500µM CdCl2 for 15 min caused a concentration-dependent decrease in NF-B DNA binding. We observed similar results in the NRK-52E nuclear extract at Cd concentrations equal to or greater than 200µM (Xie and Shaikh, unpublished data). The molecular mechanism of Cd toxicity is believed to be largely due to its high affinity for sulfhydryl groups (Zalups and Ahmad, 2003). A direct interaction between Cd and critical sulfhydryl groups of NF-B could take place if the nuclear Cd concentration could reach such high levels. The possibility of such occurrence, however, is highly unlikely.
In conclusion, the results of the present study indicate that reduction of NF-B activity plays an important role in Cd-induced apoptosis and that its transcriptional target gene products, IAPs, are a possible regulatory step in this process. The effect of Cd on NF-B activity appears to be mediated through its actions on IKK activity and phosphorylation of IB and NF-B proteins. Furthermore, Cd-induced ROS appears to be involved in NF-B–mediated apoptosis in NRK-52E cells.
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ACKNOWLEDGMENTS |
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J.X. received a fellowship through grant # P20RR016457 from the RI-BRIN/INBRE program supported by the National Center for Research Resources, National Institutes of Health. We are grateful for the generous donation of NF-
B p65-EGFP expression plasmid by Dr. Johannes Schmid of the University of Vienna, Vienna, Austria, and pEGFP-N3 vector by Dr. Stephan Mathas of the Max-Delbruck-Center for Molecular Medicine, Berlin, Germany. We thank Dr. Bingfang Yan for helpful suggestions, Dr. Xiulong Song for assistance in performing the transfection, Dr. M. Riaz Basha for help in establishing EMSA, and Dr. Aftab Ahmed for flow cytometry performed in the RI-INBRE Centralized Research Core Facility supported by the above grant. |
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