Induction of orphan nuclear receptor Nur77 gene expression and its role in cadmium-induced apoptosis in lung

Hye-Jin Shin, Byung-Hoon Lee1, Myeong Goo Yeo, Seon-Hee Oh1, Jung-Duck Park2, Kun-Koo Park3, Jin-Ho Chung4, Chang-Kiu Moon4 and Mi-Ock Lee5

Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, 1 College of Pharmacy, Wonkwang University, Jeonbuk 570-749, 2 School of Medicine, Chung-Ang University, Seoul 110-799, 3 PhamacoGenechips Inc., Kangwon 200-160 and 4 College of Pharmacy, Seoul National University, Seoul 110-799, Korea

5 To whom correspondence should be addressed Email: molee{at}sejong.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cadmium is an environmentally widely dispersed and highly toxic heavy metal that has been classified as a human carcinogen. Using the suppression subtractive hybridization technique, we identified previously 29 cadmium-inducible genes, primarily involved in inflammation, cell survival and apoptosis. Among these genes, we are particularly interested in Nor-1, because this gene belongs to the Nur77 family, which plays a key role in the apoptotic processes of a variety of cells and tissues, including the lung. In the present study, we characterized the induction of the Nur77 family genes in the lungs after cadmium exposure. Nur77, Nor-1 and Nurr1 were all induced after cadmium treatment in a dose- and time-dependent manner in WI-38 and A549 lung cell lines. Treatment with inhibitors of signaling pathways, such as PD98059 and H89, almost completely blocked the expression of Nur77, indicating that the extracellular signal-regulated kinase and protein kinase A signaling pathways are important in cadmium-induced Nur77 expression. When a plasmid encoding dominant-negative Nur77 was transfected into A549 cells, cadmium-induced apoptotic changes, such as chromosomal condensation and Bax expression, were significantly reduced, suggesting that the expression of Nur77 plays an important role in cadmium-induced apoptosis. Furthermore, the number of apoptotic cells and the expression of Nur77 was increased in lung tissues collected from cadmium-treated (30 µmol/kg body wt) Wistar rats. Taken together, these results demonstrate that cadmium induces the expression of Nur77 family genes, leading to apoptosis in lung cells, which may cause pulmonary toxicity in response to cadmium exposure.

Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NBRE, Nur77-binding response element; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PKA, protein kinase A; RT–PCT, reverse transcriptase–polymerase chain reaction; TCR, T-cell receptor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cadmium is an environmentally widely dispersed and highly toxic heavy metal that has been classified as a human carcinogen (1). The lung is a primary target organ of systemic exposure to cadmium because cadmium is mainly absorbed through the inhalation of industrial pollution and tobacco smoke, resulting in the accumulation of this metal in the lung. Lung toxicity includes acute inflammation, chronic edema and bronchitis, as well as cancer (24). Recently, an increasing volume of evidence suggests that the induction of apoptosis in target cells is associated with cadmium exposure (5,6). Although the mechanism of cadmium-induced apoptosis has not been clearly elucidated, cadmium may alter the intracellular signaling pathways that regulate apoptosis. Among the three major mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) and p38 cooperatively participate in cadmium-induced apoptosis in non-small-cell lung carcinoma cells, whereas a decrease in extracellular signal-regulated kinase (ERK) induced by low doses of cadmium contributes to growth inhibition or apoptosis (7). Cadmium affects the activities of protein kinase C and the calcium-signaling pathway (8,9). Oxidative stress is implicated in cadmium-induced lung toxicity in that oxidative stress-induced genes are up-regulated and redox-sensitive transcription factors are activated during cadmium-induced apoptosis of rat lung epithelial cells (10). However, at low concentrations, cadmium does not increase the formation of reactive oxygen species in cadmium-induced apoptosis in primary rat lung cells (6). Cadmium also induces apoptosis in normal human lung cells; this is mediated by mitochondria but independent of caspases (11).

Nur77 (also known as NGFI-B, N10, TIS1 and Nak-1) is an orphan member of the steroid/thyroid receptor super-family of transcription factors that positively or negatively regulate gene expression. It is composed of an N-terminal transactivation domain, a DNA-binding domain, and a C-terminal ligand-binding domain (12). Nur77 is involved in the apoptosis of many cell types in response to a variety of stimuli. Nur77 is induced rapidly by T-cell receptor (TCR) signaling in immature thymocytes and T-cell hybridomas, after which apoptotic cell death occurs (1215). A dominant-negative form of Nur77 that lacks the N-terminal transactivation domain blocks TCR-mediated apoptosis, indicating that Nur77 induction plays a crucial role in the TCR-mediated death of T cells (14). Nur77 is involved in activation-induced cell death in macrophages, because Nur77 induction correlates well with cell death and this activation-induced cell death is less likely in Nur77-deficient macrophages (16). We and others have also shown that Nur77 is important in the apoptosis of malignant cells, such as colon and prostate cancer cells, when they are treated with chemotherapeutic agents (17,18). Nur77 binds as a monomer to the Nur77-binding response element (NBRE), which contains the hexanucleotide (5'-AGGTCA-3'), a typical recognition motif of the RAR/RXR family, and two A residues preceding this hexanucleotide (19). Nur77 also binds DNA as a homodimer or as a heterodimer with the retinoid X receptor (20,21). Nur77 is constitutively active when over-expressed, suggesting that the orphan receptor does not require ligand stimulation. Nur77 belongs to the Nur77 protein family (12,22,23), which contains two other nuclear receptors, Nurr1 (Not 1) and Nor-1 (MINOR). The family members share extensive homology in the DNA-binding domain, but diverge outside this domain. They bind to the same response elements in promoters, although differential tissue expression and independent roles have also been described for each member.

Using the suppression subtractive hybridization technique (24), we demonstrated previously that expression of Nor-1 is induced after exposure to cadmium. In this study, we characterized the induction of Nur77 family genes, i.e. Nur77, Nor-1 and Nurr1, in lung cells after cadmium exposure, and we illustrate the possible roles of Nur77 in cadmium-induced apoptosis in lung cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cells and reagents
The human lung fibroblast cell line WI-38 (ATCC CCL-75), the human lung carcinoma cell line A549 (ATCC CCL-185) and green monkey kidney epithelial cell line, CV-1 (ATCC CCL-70) were obtained from the American Type Culture Collection and maintained in RPMI containing 10% fetal bovine serum. For treatment with cadmium, cells were seeded and incubated in media containing 1% fetal bovine serum. Cadmium acetate (CdAc), PD98059, H89, wortmannin and cyclosporine A were purchased from Sigma (St Louis, MO). The other chemicals used were of the purest grade available from Sigma.

Reverse transcriptase–polymerase chain reaction (RT–PCR)
Total RNA was prepared using the Qiagen RNeasy kit and single-stranded DNA was synthesized from RNA in a reaction mixture containing 100 ng of random hexamer and 200 U of murine Moloney leukemia virus reverse transcriptase (GibcoBRL, Grand Island, NY). PCR reaction was performed as described previously with specific primers for Nur77 (forward: 5'-CGACCCCCTGACCCCTGAGTT-3', reverse: 5'-GCCCTCAAGGTGTTGGAGAAGT-3'), Nurr1 (forward: 5'-CGACCCCCTGACCCCTGAGTT-3', reverse: 5'-GCCCTCAAGGTGTTG GAGAAGT-3'), Nor-1 (forward: 5'-CGACCCCCTGACCCCTGAGTT-3', reverse: 5'-GCCCT CAAGGTGTTGGAGAAGT-3'), Bax (forward: 5'-CAGCTCTGAGCAGATCATGAAGAC-A-3', reverse: 5'-GCCCATCTTCTTCCAGATGGTGAGC-3') and ß-actin (forward: 5'-CGTGGGCC GCCCTAGGCACCA-3', reverse: 5'-TTGGCCTTAGGGTTCAGGGGGG-3') (24,25). The genes were analyzed under conditions in which PCR products were exponentially amplified.

Immunoprecipitation/western blot analysis
To detect Nur77 protein, cells were lysed in a lysis buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 10% NP-40, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 10 µg/ml pepstatin. One milligram of whole cell lysate was incubated with 2 µg rabbit polyclonal anti-Nur77 antibodies (Santa Cruz Biotech, Santa Cruz, CA). The resulting immune complex was precipitated and analyzed by western blotting using mouse anti-Nur77 antibody (PharMingen, San Diego, CA) as described previously (26). Fifty micrograms of protein from whole cell lysates was analyzed for {alpha}-tubulin using anti-{alpha}-tubulin antibodies (Oncogene, Boston, MA). The protein concentration was quantified by bicinchoninic acid assay (Pierce, Rockford, IL).

Plasmids, transient transfection and reporter gene assay
The NBRE-tk-Luc reporter, the eukaryotic expression vector for Nur77, pECE-Nur77 and the dominant-negative Nur77 construct, pCDNA3-DN-Nur77 have been described previously (14,25,26). A549 cells were transfected with reporter plasmid (0.1 mg) together with ß-galactosidase (ß-gal) expression vector (0.2 mg) using LipofectaminePlus® (GIBCO BRL) and then incubated for 6 h with 10 mM CdAc. For transient transfection of CV-1 cells, a modified calcium phosphate precipitation procedure was used. Cells (5 x 104 cells/well) were seeded in a 24-well culture plate and transfected with DNA mixture (1 µg/well) containing reporter plasmid, 0.1 µg, ß-gal expression vector, 0.15 µg, and various combinations of receptor expression vectors with carrier DNA (pBluescript) (25). At the end of incubation, luciferase activity was determined using an analytical luminescence luminometer. Luciferase activity was normalized for transfection efficiency by the corresponding ß-galactosidase (ß-gal) activity. For transient expression of DN-Nur77, A549 (2 x 106 cells/dish) were seeded in 60-cm2 dishes and incubated overnight. The cells were transfected with 2-µg expression vectors using LipofectaminPlus® (GIBCO BRL) according to the manufacturer's instructions. Transfection efficiency was ~25% when measured by the plasmid encoding Green fluorescence protein, pEGFP (Clontech, Palo Alto, CA).

Measurement of apoptosis
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays as described previously (25). Cells were seeded at an initial density of 3000 cells/well in 96-well plates, incubated overnight, and treated with various concentrations of CdAc. At the end of treatment, the number of viable cells was determined by measuring their capacity to convert a tetrazolium salt into a blue formazan product. The results obtained from MTT assays reflect both cytotoxic and cytostatic effects of cadmium.

To examine nuclear morphology, cells were collected, washed once and resuspended in phosphate-buffered saline (PBS). Cells were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min and then washed three times with PBS before being mounted onto glass slides with 50% glycerol in PBS. The nuclear morphology of cells was examined with a UV fluorescence microscope and photographed. To assess subdiploid DNA content, total cells were collected and fixed in 70% ethanol in PBS at –20°C. Cells were then washed and stained with 50 µg/ml propidium iodide in the presence of 100 µg/ml RNase A for 30 min at 37°C in the dark. DNA content was analyzed by a FACStar Plus flow cytometer (Becton Dickson, Mountain View, CA). Apoptotic cells with subdiploid DNA staining were found in the ‘sub-G0/G1 peak and the percentage of such cells was calculated.

For DNA fragmentation analysis, cells were exposed to cadmium (10 mM) for various time periods. Attached and floating cells were harvested, washed with PBS. DNA was prepared with Wizard genomic DNA purification Kit (Promega, Madison, WI), and then subjected to electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.

Animals and immunohistochemistry
Four- to five-week-old female Wistar rats (Charles River, Calco, Italy) were used for in vivo experiments. Animals were housed three to four per cage over woodchip bedding and allowed food and water supplied ad libitum and maintained under constant temperature (22 ± 2°C) and humidity (55 ± 2%). The experimental protocol was approved by the Committee for the Care and Use of Laboratory Animals at Chung-Ang University, according to the Guide for Animal Experiments edited by the Korean Academy for Medical Sciences. Cadmium was administered by subcutaneous injection with a dose of 30 µmol/kg body wt prior to 24 or 48 h of killing. Rats were killed under ether anesthesia and blood was collected and the lung was removed and paraffinized. Total RNA was prepared from 1 x 107 peripheral blood mononuclear cells (PBMCs) or from 50 mg lung tissue as described above (24).

Apoptotic cells present in the lung were detected by indirect terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method using VasoTACS In Situ Apoptosis Detection Kits (Trevigen, Gaithersburg, MD) according to the manufacturer's protocol. Briefly, tissue sections were deparaffinized in xylene and re-hydrated in phosphate-buffered saline. After blocking the endogenous peroxidase activity, the sections were treated with proteinase K (20 µg/ml in Tris–HCl) for 10 min at room temperature, and incubated with TdT and fluorescein isothiocyanate-labeled dUTP. The slides were rinsed and incubated with the anti-fluorescein isothiocyanate-peroxidase conjugate. TACS-nucleaseTM was used to generate DNA breaks for positive control. The slides were developed with 3,3'-diaminobenzidine tetrahydrochloride and counterstained with methyl green. To visualize the expression of Nur77 in the lung, an Ultravision Mouse Tissue Detection System (Lab Vision, Fremount, CA) was used. After re-hydration, the tissue sections were digested with pepsin solution for 5 min. To identify Nur77 protein, samples were incubated with anti-Nur77 antibody (1:100 dilution, Santa Cruz Biotech) at 4°C overnight and treated with secondary antibody. Peroxidase activity was detected with 3-amino-9-ethylcarbazole.

Statistical analysis
Experimental values were expressed as mean ± SD. The significance of differences was determined by Student's t-test and expressed as a probability value.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Cadmium induces expression of orphan nuclear receptors, Nur77, Nor-1 and Nurr1 in lung cells
We reported previously that the expression of Nor-1 is induced after cadmium exposure (24). Nor-1 is a member of the steroid/thyroid hormone receptor super-family, which includes various ligand-dependent transcription factors. Nor-1 exhibits close sequence homology to Nur77, and therefore belongs to the Nur77 gene family together with Nur77 and Nurr1 (12). In the present investigation, we first examined the induction of Nur77, Nor-1 and Nurr1 in WI-38 normal human lung fibroblast and A549 human lung carcinoma cell lines by RT–PCR. Consistent with our previous report (24), Nor-1 was markedly induced after cadmium exposure (Figure 1). Similarly, Nur77 and Nurr1 were induced by cadmium treatment in a dose- and time-dependent manner in both WI-38 and A549 cell lines (Figure 1).



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Fig. 1. Induction of Nur77, Nor-1 and Nurr1 transcripts after cadmium exposure. (A) WI-38 cells were plated at a density of 2 x 106 cells in 100-cm2 dishes and incubated overnight. Cells were treated with the indicated concentration of CdAc for 6 h (upper panel) or with 10 µM CdAc for the indicated time periods (lower panel). (B) A549 cells were plated at a density of 2 x 106 cells in 100-cm2 dishes and incubated overnight. Cells were treated with the indicated concentration of CdAc for 6 h. The expression of Nur77, Nor-1 and Nurr1 transcripts was analyzed by RT–PCR with primers that are specific for each gene as described in the Materials and methods. ß-Actin is shown to indicate that equal amounts of RNA were analyzed. A representative result obtained from at least three independent experiments is shown.

 
Next, we confirmed the induction of Nur77 at the protein level by western blot analysis. As shown in Figure 2A, Nur77 protein was induced by cadmium treatment in a dose-dependent manner. The induction was transient: maximal induction was achieved after 6 h exposure to cadmium, and the protein returned to baseline levels after 24 h. We also checked whether the increased Nur77 protein was functional as a transcription factor, using the reporter construct, NBRE-tk-Luc, which encodes the specific target sequences of Nur77 (25,26). Reporter gene activity was induced dose-dependently after cadmium treatment (Figure 2B). This result, together with that of RT–PCR analysis, demonstrates that cadmium induces Nur77 at the transcription level to generate functionally active Nur77 protein.



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Fig. 2. Induction of Nur77 at protein- and function-level by cadmium in lung cells. (A) A549 cells (2 x 106 cells/dish) were seeded in 100-cm2 dishes and incubated overnight. The cells were treated with the indicated concentration of CdAc for 6 h (upper panel) or with 20 µM CdAc for the indicated time periods (lower panel). After cells were lysed, 1 mg whole cell lysate was immunoprecipitated by rabbit anti-Nur77 antibody and Nur77 protein was identified by western blotting using mouse anti-Nur77 antibody. {alpha}-Tubulin is shown to indicate that equal amounts of protein were analyzed. A representative result obtained from at least three independent experiments is shown. (B) The NBRE-tk-Luc reporter (0.2 µg) together with ß-gal expression vector was transiently transfected into A549 cells as described in the Materials and methods. Transfected cells were treated with the indicated concentration of CdAc for 6 h, and then cell lysates were assayed for luciferase activity. Corresponding ß-gal activity was used to normalize luciferase activity. Data shown are the mean ± SD of three independent experiments.

 
Multiple signaling pathways are involved in cadmium-induced Nur77 gene expression
Protein kinase A (PKA) and MAPK calcium-signaling pathways are associated with the induction and activation of Nur77 by stimuli such as corticotrophs and luteinizing hormone (27,28). To determine the signaling pathway involved in cadmium-triggered Nur77 gene expression, we used the specific inhibitors PD98059, H89, wortmannin and cyclosporine A, which block the ERK, PKA, phosphatidylinositol-3 kinase (PI3K), and calcineurin signaling pathways, respectively. As shown in Figure 3, western blot analysis indicated that the induction of Nur77 was almost completely abolished by 40 µM PD98059 or 10 µM H89. In contrast, 2 µg/ml cyclosporine A decreased Nur77 expression by ~25%, whereas 10 µM wortmannin had no effect. The concentrations of the inhibitors used are known to be relatively specific for each signaling pathway (2931). These results suggest that both ERK and PKA are largely involved in the cadmium-mediated induction of the Nur77 gene.



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Fig. 3. MAPK and PKA signaling pathways are mainly involved in cadmium-induced Nur77 expression. A549 cells (2 x 106 cells/dish) were seeded in 100-cm2 dishes and incubated overnight. The cells were pre-treated with PD98059 (PD; 40 µM), H89 (10 µM), wortmannin (WM; 10 nM) or cyclosporine A (CsA; 2 µg/ml) for 1 h, and subsequently treated with 20 µM CdAc for 6 h. After cells were lysed, 1 mg whole cell lysate was immunoprecipitated and probed with specific anti-Nur77 antibodies. {alpha}-Tubulin is shown to indicate that equal amounts of protein were analyzed. A representative result obtained from at least three independent experiments is shown. See online supplementary material for colour version of this figure.

 
Cadmium induces apoptosis in lung cells
A role for Nur77 in apoptotic cell death has been proposed because constitutive expression of Nur77 leads to massive apoptosis, with high-level expression of the Fas ligand, in the thymocytes of transgenic mice (32). Therefore, we expected that high-level expression of Nur77 after cadmium exposure might cause the induction of apoptosis in lung cells. To check whether cadmium induces apoptotic death in lung cells, we first measured the viability of WI-38 and A549 cells after cadmium treatment using MTT assays. As shown in Figure 4A, cadmium decreased cell viability in both cell lines in a dose- and time-dependent manner. The concentrations of CdAc required for LD50 (lethal dose 50%) values after treatment for 24 h were ~10 and 40 µM for WI-38 cells and A549 cells, respectively. When cells were treated with cadmium, they became shrunken and rounded, eventually detaching and floating in the medium. This is typical of cells undergoing apoptosis (Figure 4B). When chromosomes were visualized with DAPI staining, a proportion of cells contained condensed and fragmented chromatin, which was highly fluorescent (Figure 4C). Analysis of cellular DNA content by flow cytometry showed profiles typical of cells undergoing apoptosis. We determined the percentage of cells in a distinct, easily quantifiable region below the G1 flow cytometry profile; these formed a subpopulation of apoptotic cells with condensed chromatin and degraded DNA, which reduced their stainability with propidium iodide. When A549 cells were treated with cadmium, the percentage of subdiploid cells increased in a dose-dependent manner (Figure 4D). In WI-38 cells, cadmium induced the DNA laddering pattern typical of apoptosis when the cells were analyzed by agarose gel electrophoresis (Figure 4E). Finally, the expression of Bax, a proapoptotic gene, was remarkably increased by cadmium as early as 1 h after treatment (Figure 4F). Together, these results indicate that the decrease in cell viability after cadmium treatment is due to lung cells undergoing apoptosis.



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Fig. 4. Cadmium induced apoptosis in lung cells. (A) WI-38 or A549 cells were plated at a density of 3000 cells/well in a 96-well plate and incubated overnight. Cells were treated with the indicated concentration of CdAc for 3 h (filled circle), 10 h (clear circle), 18 h (inverted filled triangle) and 24 h (clear square). Cytotoxicity was determined by MTT assays as described in the Materials and methods. Cell viability represents [(OD at 570 nm measured with CdAc-treated cells/OD at 570 nm measured with vehicle-treated control cells) x 100 (%)]. Data shown are the mean ± SD of three independent determinations. (B) WI-38 or A549 cells were treated with 10 or 20 µM CdAc, respectively, for 16 h and photographed by phase-contrast microscope. (C) WI-38 or A549 cells were treated with 10 or 20 µM CdAc, respectively, for 16 h and then cells were detached, fixed and stained with DAPI as described in the Materials and methods. Stained cells were examined by a fluorescence microscope. (D) A549 cells were treated with the indicated concentrations of CdAc for 24 h and stained with propidium iodide as described in Materials and methods. The percenatge of apoptotic cells with subdiploid DNA staining was determined by flow cytometry. Data shown are the mean ± SD of three independent experiments. (E) WI-38 cells were treated with 10 µM CdAc for the indicated time periods. After cells were harvested, DNA was extracted, subjected to agarose gel electrophoresis, and stained with ethidium bromide. M indicates the migration pattern of 100 bp ladder. (F) A549 cells (2 x 106 cells) were treated with of 10 µM CdAc for the indicated time periods. After cells were harvested, expression of Bax and Nur77 transcripts was analyzed by RT–PCR with specific primers as describe in the Materials and methods. ß-Actin is shown to indicate that equal amounts of RNA were analyzed. A representative result obtained from at least three independent experiments is shown. See online supplementary material for colour version of this figure.

 
Expression of a dominant-negative Nur77 mutant represses apoptotic death of lung cells
To test whether Nur77 plays an active role in cadmium-induced apoptosis, we transfected a eukaryotic expression vector encoding a dominant-negative Nur77 mutant, pCDNA3-DN–Nur77, into A549 cells. When the repressive function of the dominant-negative Nur77 was tested using an NBRE-Luc reporter construct, it efficiently inhibited the transcriptional activity of Nur77, which is consistent with previous results (Figure 5A) (14,26). As shown in Figure 5B, the number of apoptotic nuclei apparent after cadmium treatment was significantly lower when the function of Nur77 was blocked by the dominant-negative Nur77. The number of fragmented nuclei was reduced by ~70%. The percentage of subdiploid cells was significantly lower when dominant-negative Nur77 was transfected (Figure 5C). The apoptotic process in lung cells after cadmium exposure included a dramatic induction of the apoptotic gene, Bax, as shown in Figure 4C. Therefore, we asked whether dominant-negative Nur77 blocks the induction of Bax. As expected, the expression of Bax decreased by ~55% when dominant-negative Nur77 was transfected (Figure 5D). These results provide evidence that Nur77 is actively involved in the cadmium-induced apoptosis of lung cells.



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Fig. 5. Cadmium-induced apoptosis was repressed by the DN-Nur77. (A) Repressive function of the DN-Nur77 was tested by reporter gene analysis. NBRE-Luc (0.1 µg) was co-transfected with 10 ng pECE-Nur77 and the indicated amount of pCDNA3-DN-Nur77 into CV-1 cells. After incubation overnight, cell lysates were obtained and analyzed. Luciferase activity was normalized for transfection efficiency by corresponding ß-gal activity. Data shown are the mean ± SD of three independent determinations. (B) A549 cells were transfected with pCDNA3-DN-Nur77 (2 µg) or empty vector (pCDNA3, 2 µg) as indicated. Transfected cells were treated with vehicle or 20 µM CdAc for 6 h. Cells were fixed, stained with DAPI and examined by fluorescence microscope (upper panel). Apoptotic nuclei were quantified by counting 100 cells for each experiment (lower panel). Data shown are the mean ± SD of at least five independent determinations. *P < 0.01 compared with empty vector transfected and CdAc treated control. (C) A549 cells were transfected as described above and treated with 100 µM CdAc for 20 h. At the end of treatment, cells were stained with propidium iodide and the percentage of apoptotic cells with subdiploid DNA staining was determined by flow cytometry. Data shown are the mean ± SD of three independent experiments. *P < 0.01 compared with empty vector transfected and CdAc-treated control. (D) A549 cells were transfected and treated with 20 µM CdAc for 6 h. Total RNA was obtained and analyzed for the expression of Bax transcripts by RT–PCR. The expression of ß-actin was monitored as control (upper panel). The density of electrophoretically separated PCR products was determined using an image analysis system. The values were normalized to that of ß-actin and expressed as fold inductive to untreated control value (lower panel). Data shown are the mean ± SD of three independent experiments. *P < 0.01 compared with empty vector transfected and CdAc treated control.

 
Cadmium induces the expression of Nur77 in vivo
To investigate whether the results obtained in vitro were reproducible in vivo, we administereyyd a single dose of cadmium (30 µmol/kg body wt) or vehicle to Wistar rats by subcutaneous injection. First, TUNEL staining was performed to analyze apoptotic cell death in the lung. Stained cells were not detected in vehicle-treated controls, whereas strong positive staining was observed in positive controls. In cadmium-treated rats, positive staining was detected in alveolar epithelial cells after treatment for 24 h. The number of TUNEL-positive cells was increased after treatment for 48 h (Figure 6). Next, we tested the induction of Nur77 family genes in PBMCs and lung tissue collected from cadmium-treated rats. Consistent with the results of in vitro experiments, transcripts of Nur77, Nor-1 and Nurr1 were induced strongly in both PBMCs and lungs of cadmium-exposed animals (Figure 7A). Finally, the induction of Nur77 protein was analyzed in lung tissue using immunohistochemistry. The expression of Nur77 protein increased substantially following cadmium treatment. Strong positive staining was detected in the cytoplasm of bronchiolar and alveolar epithelial cells at 48 h after treatment, whereas the bronchiolar epithelial cells of control rats showed faint staining (Figure 7B). These results show that cadmium induces apoptosis and Nur77 expression in vivo, and confirms the in vivo relevance of the in vitro data.



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Fig. 6. Induction of apoptotic cell death after cadmium exposure in lung in vivo. Paraffin sections of lung were prepared from Wistar rats treated with either vehicle (a) or cadmium (30 µmol/kg body wt) for 24 h (b) or 48 h (c). TACS-nucleaseTM was used to generate DNA breaks for positive control (d). The cells undergoing apoptosis were identified using an in situ apoptosis detection kit as described in the Materials and methods. Examples of apoptotic cells are indicated by arrows. Data are representative of at least three independent experiments.

 


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Fig. 7. Expression of Nur77, Nor-1, and Nurr1 after exposure to cadmium in vivo. Rats were treated with vehicle or with cadmium (30 µmol/kg body wt) for 24 or 48 h. C represents vehicle-treated control. (A) PBMCs and lung tissues were obtained from the experimental rats and analyzed for expression of the indicated transcripts using RT–PCR with specific primers. ß-actin is shown to indicate that equal amounts of RNA were analyzed. (B) Paraffin sections of lung were prepared and analyzed for expression of Nur77 by immunohistochemistry using specific antibodies for Nur77 as described in the Materials and methods. The expression of Nur77 was detected in alveolar epithelial cells (arrow), and bronchiolar epithelial cells (arrow head). Representative results obtained from at least three independent experiments is shown.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
We identified previously 29 different cadmium-inducible genes primarily involved in inflammation/immunity and cell survival/apoptosis (24). Of these genes, we are particularly interested in Nor-1 because this gene belongs to the Nur77 family, which is important in the apoptotic processes of a variety of cells and tissues, including the lung. In this study, we further characterized the induction of the Nur77 family genes in lung cells in vitro and in vivo after exposure to cadmium, and illustrated the possible roles of these proteins in cadmium-induced apoptosis in the lung.

We confirmed that cadmium induces apoptosis in lung cells in vitro and in vivo (Figures 4 and 6). Lag et al. consistently showed that apoptosis is induced by cadmium in alveolar type 2 cells and Clara cells isolated from rat lung. In these lung cells, increased levels of p53 and Bax accompany cadmium-induced apoptosis (6). Shih et al. reported that cadmium induces apoptosis in normal human fetal lung fibroblast cells, and that this apoptosis is mitochondria-mediated but caspase-independent (11). Similarly, cadmium induces apoptosis in a rat lung epithelial cell line, and reactive oxygen species play a role in the process (10). Interestingly, however, repeated low-dose exposure to cadmium leads to the development of adaptive survival responses that significantly attenuate oxidant-induced apoptosis in alveolar epithelial cells (33,34). The mechanism by which cadmium induces apoptosis is not clearly understood; nevertheless, apoptosis may be an important cellular event causing acute and chronic lung injury after cadmium exposure.

In this study, we demonstrated that Nur77 has a critical role in inducing apoptosis in lung cells. Although the molecular mechanism by which Nur77 induces apoptosis is largely unknown, possible roles for Nur77 include regulating the expression of modulators of apoptosis. In this respect, the expression of the proapoptotic gene Bax might be a target of Nur77, because this gene was significantly repressed when dominant-negative Nur77 was expressed in cells (Figure 5). However, the induction of Bax preceded the expression of Nur77, which implies that factors other than Nur77 are responsible for the induction of Bax in response to cadmium in the initial phase of apoptosis (Figure 4F). During apoptotic processes, cytosolic Bax is translocated into the outer membrane of the mitochondria and cytochrome c is subsequently released into the cytosol, after which caspase-3 is activated (35). Similarly, in response to apoptotic stimuli, Nur77 is translocated from the nucleus to the cytoplasm, where it targets mitochondria to induce cytochrome c release (36). Whether Nur77 affects cadmium-induced apoptosis by repressing Bax at the level of transcription or by cooperating in the mitochondrial translocation of Bax is an interesting question for further investigation.

Cadmium interferes with cellular signaling and gene regulation at multiple levels. It enters cells through calcium ion channels in the plasma membrane (37). It also causes an increase in the intracellular calcium concentration by inhibiting the Ca2+ pump in the endoplasmic reticulum and activates or inhibits some calcium-related enzymes (9,38). Among the three major MAPKs, JNK and p38 cooperatively participate in cadmium-induced apoptosis in non-small-cell lung carcinoma cells, whereas a decrease in ERK induced by low doses of cadmium contributes to growth inhibition or apoptosis (7). Cadmium induces the activation of metal-regulatory transcription factor 1 through protein kinases such as protein kinase C, JNK and PI3K (39). On the other hand, many physiological and pharmacological stimuli induce Nur77 gene expression. Nur77 is an immediate-early-response gene that is induced by growth factors and mitogens (40,41). Nur77 is also rapidly induced by TCR signaling in immature thymocytes and T-cell hybridomas, and this induction is followed by apoptotic cell death (1215). Nur77 and Nurr1 are induced by hormones such as corticotrophs and luteinizing hormone (27,28). Calcium, PKA, protein kinase C and MAPK pathways are involved in the process of Nur77 induction (27,28). These signaling pathways may trigger transcriptional factors, such as myocyte enhancer factor 2, AP-1 and cyclic-AMP-response-element-binding protein, that bind to and regulate the promoter of the Nur77 gene (4244). In our study, inhibitors of the PKA and ERK pathways almost completely blocked Nur77 expression in the presence of cadmium, whereas inhibitors of the calcineurin or PI3K pathways did not. These results indicate that cadmium induces Nur77 gene expression predominantly through the activation of the ERK and PKA signaling pathways (Figure 3).

In addition to cadmium, other environmental toxicants have been reported to induce Nur77 expression. Di-n-butyltin dichloride (DBTC) is one of the organotin compounds that have a wide array of industrial applications, for example, as stabilizers for polyvinylchloride plastics and as biocides used in the preservation of paper and wood. DBTC induces thymotoxicity and Nur77 expression in thymocytes in vitro and in vivo (45). Inhibition of Nur77 expression with antisense oligonucleotides prevents the DBTC-induced apoptosis, suggesting Nur77 has a role in organotin-induced apoptotic cell death. Furthermore, one of the well-known endocrine-disrupting chemicals, bisphenol A, induces Nur77 gene expression in testicular Leydig cells and mouse testes, where Nur77 has an important role in steroidogenesis (46). The induction of Nur77 gene expression that affects steroidogenesis may be the mechanism by which bisphenol A acts as an endocrine disruptor. These previous findings, together with our results, indicate that Nur77 rapidly and drastically responds to certain environmental toxicants. This in turn suggests the induction of the Nur77 gene family could be used as a biomarker for exposure to these toxicants.

Recently, a large body of data has indicated the physiological importance of the Nur77 gene family. High-level expression of Nur77 and Nurr1 in the rat brain suggests their involvement in the development and maturation of a specific set of central nervous system neurons and a possible association between the deregulation of Nur77 and neuron degeneration in Parkinson's disease (47,48). Corticotrophin-releasing hormone induces the expression of Nur77 in synovial tissue (49). Luteinizing hormone induces the Nur77 that regulates steroidogenesis in testicular Leydig cells and testes (28). Similarly, parathyroid hormone increases Nur77 expression in osteoblasts, indicating that Nur77 may regulate bone metabolism (50). Nor-1 is over-expressed in human coronary atherosclerotic lesions, and balloon angioplasty transiently induced Nor-1 in porcine coronary arteries (51). Nur77 is also highly expressed and co-localizes with plasminogen activator inhibitor 1, the main fibrinolysis inhibitor, in atherosclerotic tissues, implicating the Nur77 gene family in vascular disease (52). We and others have reported that viral transactivators, such as hepatitis B virus X protein and TAX of human T lymphotropic virus type 1, trigger the induction of Nur77 (26,53). Given the importance of Nur77 in such fundamental physiology and in human disease, the induction of functionally active Nur77 by cadmium is likely to be one critical mechanism by which cadmium exerts its adverse effects on human health.


    Supplementary material
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material can be found at: http://www.carcin.oupjournals.org/.


    Acknowledgments
 
This study was supported by grants from the Ministry of Environment as ‘The Eco-technopia 21 project’, and the Ministry of Science and Technology (M1-0312-04-0002 and M1-0311-00-0089).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received September 22, 2003; revised February 23, 2004; accepted February 27, 2004.