FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080217327> ?p ?o ?g. }

• W2080217327 endingPage "39032" @default.
• W2080217327 startingPage "39022" @default.
• W2080217327 abstract "The carcinogenicity of nickel compounds has been well documented both in vitro and in vivo; however, the molecular mechanisms by which nickel compounds cause cancers are far from understood. Because suppression of apoptosis is thought to contribute to carcinogenesis, we investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in increased cyclooxygenase-2 (COX-2) expression and that small interfering RNA (siCOX-2) knockdown of COX-2 expression resulted in increased cell sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction has an anti-apoptotic effect on Beas-2B cells. Overexpression of IKKβ-KM, a kinase inactive mutant of IKKβ, blocked NF-κB activation and COX-2 induction by nickel compounds, indicating that activated NF-κB may be a mediator for COX-2 induction. To further explore the contribution of the NF-κB pathway in COX-2 induction and in protection from nickel exposure, mouse embryonic fibroblasts deficient in IKKβ, IKKα, p65, and p50 were analyzed. Loss of IKKβ impaired COX-2 induction by nickel exposure, whereas knockout of IKKα had a marginal effect. Moreover, the NF-κB p65, and not the p50 subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKβ or p65 rendered cells more sensitive to nickel-induced apoptosis as compared with those in wild type cells. Finally, it was shown that reactive oxygen species H2O2 were involved in both NF-κB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds occurs via an IKKβ/p65 NF-κB-dependent but IKKα- and p50-independent pathway and plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells. The carcinogenicity of nickel compounds has been well documented both in vitro and in vivo; however, the molecular mechanisms by which nickel compounds cause cancers are far from understood. Because suppression of apoptosis is thought to contribute to carcinogenesis, we investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in increased cyclooxygenase-2 (COX-2) expression and that small interfering RNA (siCOX-2) knockdown of COX-2 expression resulted in increased cell sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction has an anti-apoptotic effect on Beas-2B cells. Overexpression of IKKβ-KM, a kinase inactive mutant of IKKβ, blocked NF-κB activation and COX-2 induction by nickel compounds, indicating that activated NF-κB may be a mediator for COX-2 induction. To further explore the contribution of the NF-κB pathway in COX-2 induction and in protection from nickel exposure, mouse embryonic fibroblasts deficient in IKKβ, IKKα, p65, and p50 were analyzed. Loss of IKKβ impaired COX-2 induction by nickel exposure, whereas knockout of IKKα had a marginal effect. Moreover, the NF-κB p65, and not the p50 subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKβ or p65 rendered cells more sensitive to nickel-induced apoptosis as compared with those in wild type cells. Finally, it was shown that reactive oxygen species H2O2 were involved in both NF-κB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds occurs via an IKKβ/p65 NF-κB-dependent but IKKα- and p50-independent pathway and plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells. Aerosols of nickel salts can be generated in electroplating and electrolysis areas of nickel refineries (1International Agency for Research on CancerIARC Sci. Publ. 1990; 49: 257-445Google Scholar). The release of nickel into the environment represents a potential for nonoccupational exposure (2Denkhaus E. Salnikow K. Crit. Rev. Oncol. Hematol. 2002; 42: 35-36Crossref PubMed Scopus (875) Google Scholar). The routes for nickel uptake include inhalation, ingestion, and dermal penetration (1International Agency for Research on CancerIARC Sci. Publ. 1990; 49: 257-445Google Scholar, 3Sivulka D.J. Regul. Toxicol. Pharmacol. 2005; 43: 117-133Crossref PubMed Scopus (58) Google Scholar). Epidemiological studies have associated occupational exposure to nickel compounds to elevated incidences of human cancer such as lung and nasal cancers (4Kasprzak K.S. Sunderman J.F.W. Salnikow K. Mutat. Res. 2003; 533: 67-97Crossref PubMed Scopus (616) Google Scholar). Numerous studies from cell culture models and experimental animal models have also confirmed the carcinogenic potency of nickel compounds (4Kasprzak K.S. Sunderman J.F.W. Salnikow K. Mutat. Res. 2003; 533: 67-97Crossref PubMed Scopus (616) Google Scholar, 5Cangul H. Broday L. Salnikow K. Sutherland J. Peng W. Zhang Q. Poltaratsky V. Yee H. Zoroddu M.A. Costa M. Toxicol. Lett. (Amst.). 2002; 127: 69-75Crossref PubMed Scopus (87) Google Scholar). Although the mechanisms implicated in the carcinogenic effect of nickel compounds are not well understood, it is accepted that the carcinogenic effects of nickel occur through alterations in cancer development-related gene expression (6Li J. Davidson G. Huang Y. Jiang B.-H. Shi X. Costa M. Huang C. Cancer Res. 2004; 64: 94-101Crossref PubMed Scopus (70) Google Scholar). On the other hand, it has been reported that nickel compounds can promote the generation of reactive oxygen species (ROS), 2The abbreviations used are: ROS, reactive oxygen species; COX-2, cyclooxygenase-2; AP, activator protein; siRNA, small interfering RNA; IKK, I-κB kinase; NF-κB, nuclear factor κB; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; HA, hemagglutinin; RT, reverse transcription; WT, wild type; MEF, mouse embryonic fibroblast; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase; mCat, mitochondrial catalase. which can regulate the expression of specific genes related to tumor development (7Salnikow K. Costa M. J. Environ. Pathol. Toxicol. Oncol. 2000; 19: 307-318PubMed Google Scholar). Apoptosis plays an essential role as a protective mechanism against neoplastic development in the organism by eliminating genetically damaged cells (8Wyllie A.H. Bellamy C.O. Bubb V.J. Clarke A.R. Corbet S. Curtis L. Harrison D.J. Hooper M.L. Toft N. Webb S. Bird C.C. Br. J. Cancer. 1999; 80: 34-37PubMed Google Scholar, 9Liu Z.G. Lewis J. Wang T.H. Cook A. Methods Cell Biol. 2001; 66: 187-195Crossref PubMed Google Scholar). Cyclooxygenase-2 (COX-2) is implicated in the suppression of apoptosis, in some experimental systems, leading to the development of cancer (10Subbaramaiah K. Dannenberg A.J. Trends Pharmacol. Sci. 2003; 24: 96-102Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 11Gasparini G. Longo R. Sarmiento R. Morabito A. Lancet Oncol. 2003; 4: 605-615Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Previous studies have documented that COX-2 is constitutively overexpressed in a variety of human malignancies, especially in primary lung carcinoma (12Kuwano T. Nakao S. Yamamoto H. Tsuneyoshi M. Yamamoto T. Kuwano M. Ono M. FASEB J. 2004; 18: 300-310Crossref PubMed Scopus (250) Google Scholar, 13Dempke W. Rie C. Grothey A. Schmoll H.J. J. Cancer Res. Clin. Oncol. 2001; 127: 411-417Crossref PubMed Scopus (379) Google Scholar, 14Dannenberg A.J. Lippman S.M. Mann J.R. Subbaramaiah K. DuBois R.N. J. Clin. Oncol. 2005; 23: 254-266Crossref PubMed Scopus (348) Google Scholar). In the current study we utilized human bronchial epithelial Beas-2B cells to define whether nickel compounds are able to promote survival by inducing COX-2 expression and to define the signals regulating nickel-induced COX-2 expression. Plasmids, Antibodies, and Other Reagents—HA-tagged full-length IKKβ (HA-IKKβ) were provided by Dr. Zheng-Gang Liu (NCI/National Institutes of Health, Bethesda, MD) (15Tang G. Minemoto Y. Dibling B. Purcell N.H. Li Z. Karin M. Lin A. Nature. 2001; 414: 313-317Crossref PubMed Scopus (666) Google Scholar). Mitochondrial catalase expression vector pZeoSV/mCAT was described previously by Rodríguez et al. (16Rodríguez A.M. Carrico P.M. Mazurkiewicz J.E. Meléndez J.A. Free Radic. Biol. Med. 2000; 29: 801-813Crossref PubMed Scopus (115) Google Scholar). The antibodies against phospho-IKKα/β, IKKα, IKKβ, phospho-IκBα,IκBα, caspase-3, and PARP were purchased from Cell Signaling Technology (Beverly, MA). Anti-HA antibody was purchased from Upstate Biotechnology (Lake Placid, NY); anti-FLAG and anti-β-actin antibodies were obtained from Sigma; anti-COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI). Anti-catalase antibody was purchased from Calbiochem (EMD Biosciences, Inc., La Jolla, CA). Nickel compounds were purchased from Aldrich; and substrate for the luciferase assay was purchased from Promega (Madison, WI). Cell Culture and Transfection—Beas-2B cells, wild type (WT) mouse embryonic fibroblasts (MEFs), and their stable transfectants were cultured in Dulbecco's modified Eagle's medium (DMEM, Calbiochem) supplemented with 10% fetal bovine serum (FBS), 5% penicillin/streptomycin, and 2 mm l-glutamine (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO2. To block IKK/NF-κB pathway activation, IKKβ-KM expressing plasmid was used to transfect Beas2B cells and WT cells together with NF-κB gene reporter plasmid or COX-2 gene reporter plasmid. Stable transfection was performed with Lipofectamine reagent according to the manufacturer's instruction. After co-transfection with hygromycin B-resistant plasmid, cells were subjected to hygromycin B drug selection to generate stable transfectants. The stable transfectants were identified by analyzing basal luciferase activity or FLAG tag overexpression. IKKβ–/– MEFs and IKKα–/– MEFs were kind gifts from Dr. Zheng-Gang Liu (NCI, Bethesda, MD) and Dr. Michael Karin (University of California at San Diego). p50–/– MEFs and p65–/– MEFs were isolated from p50–/– or p65–/– mice and provided by Dr. Jianping Ye (Pennington Biomedical Research Center, Louisiana State University, Baton Rouge). All MEFs and their transfectants were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mm l-glutamine. Gene Reporter Assays—Confluent monolayers of stable luciferase reporter transfectants were trypsinized, and 8×103 viable cells suspended in 100 μl of 10% FBS/DMEM were added to each well of 96-well plates. Plates were incubated at 37 °C in a humidified atmosphere of 5% CO2. After cell density reached 80–90%, the cells were treated with nickel compounds for different time periods as indicated. Cells were then lysed with 50 μl of lysis buffer, and luciferase activity was measured using a Promega assay reagent with a luminometer (Wallac 1420 Victor2 multiple counter system). The results are expressed as NF-κB activation relative to the control medium (NF-κB activity) or COX-2 induction relative to control medium (relative COX-2 induction). Student's t test was used to determine the significance of the differences, and the differences were considered significant at p ≤ 0.05. Western Blot Assay—2×105 cells were cultured in each well of 6-well plates to 70–80% confluence. The culture medium was replaced with 0.1% FBS/DMEM. After being cultured for 24 h, the cells were exposed to nickel compounds. The cells were then washed once with ice-cold PBS and extracted with SDS-sample buffer. The cell extracts were separated on polyacrylamide-SDS gels, transferred, and probed with a rabbit-specific antibody against target protein. The protein band, specifically bound to the primary antibody, was detected using an anti-rabbit IgG-AP-linked antibody and an ECF Western blotting system (Amersham Biosciences). Reverse Transcription Polymerase Chain Reaction (RT-PCR)—Total RNA was extracted with Trizol reagent (Invitrogen), and cDNAs were synthesized with ThermoScript™ RT-PCR system (Invitrogen). For detection of COX-2 expression, a pair of oligonucleotides (5′-tgaaacccactccaaacaca-3′ and 5′-aactgatgcgtgaagtgctg-3′) were designed according to human COX-2 gene sequence. The human β-actin cDNA was amplified at the same time by the primers 5′-gcgagaagatgacccagatcat-3′ and 5′-gctcaggaggagcaatgatctt-3′. Cell Death Assay—Beas-2B cells treated with nickel compounds were collected by pooling the cells from the culture medium as well as the trypsinized adherent cells. Dead cells were counted by the trypan blue exclusion method and flow cytometric analysis following propidium iodide staining of the nuclei. Briefly, the cells were fixed in ice-cold 80% ethanol at –20 °C overnight. The fixed cells were permeabilized in buffer containing 100 mm sodium citrate/0.1% Triton X-100 at room temperature for 15 min as well as RNase A (0.2 mg/ml) (Sigma) for 10 min, stained with propidium iodide (50 μg/ml) at 4 °C for at least 1 h, and then analyzed using the Epics XL FACS (Beckman-Coulter, Miami, FL) as described in our previous publication (3Sivulka D.J. Regul. Toxicol. Pharmacol. 2005; 43: 117-133Crossref PubMed Scopus (58) Google Scholar, 17Ouyang W. Ma Q. Li J. Zhang D. Liu Z.-G. Rustgi A.K. Huang C. Cancer Res. 2005; 65: 9287-9293Crossref PubMed Scopus (75) Google Scholar). H2O2 Staining Assay—Beas-2B transfectants were seeded (2 × 104) into each well of a 96-well plate. After cell density reached 80–90%, the cells were washed thoroughly with PBS and incubated with dichlorofluorescein diacetate (DCFH-DA) at 10 μm in PBS (stock concentration is 10 mm in Me2SO) for 20 min. Cells were then washed with PBS to remove the dye completely and exposed to UVC radiation (60 J/m2). The cells were incubated at 37 °C for another 10 min, and the oxidative product was detected by using an HTS7000 Bio-Assay reader (PerkinElmer Life Sciences) with excitation 488 nm and emission 530 nm. The results were expressed as relative H2O2 production compared with the cells without exposure to UVC radiation. Electrophoretic Mobility Shift Assays—Nuclear proteins were prepared with a Cellytic™ NuCLEAR™ extraction kit (Sigma) following the manufacturer's protocols. 5 μg of nuclear protein was subjected to a gel shift assay by incubating it with 1 μg of poly(dI-dC) DNA carrier in DNA-binding buffer (10 mm Tris, pH 8.0, 150 mm KCl, 2 mm EDTA, 10 mm MgCl2, 10 mm dithiothreitol, 0.1% bovine serum albumin, 20% glycerol) in a final volume of 10 μl on ice for 10 min. Then, 105 cpm (108 cpm/μg) of the 32P-labeled double-stranded oligonucleotide (2 μl) was added, and the reaction was incubated at room temperature for 30 min. For competition experiments, a 20-fold molar excess amount of the unlabeled oligonucleotide was added before the addition of the probe. For the super-gel shift assay, nuclear extracts were incubated with 2 μg of either rabbit IgG or the rabbit anti-p65 antibodies for 30 min at 4 °C before addition of the probe. DNA-protein complexes were resolved by electrophoresis in 5% nondenaturing glycerol/polyacrylamide gels. The synthetic oligonucleotides (5′ to 3′) used as probe binding to NF-κB was GAGTTGAGGGGACTTTCCCAGGC. Nickel Exposure Rendered Beas-2B Cells Resistant to the Pro-apoptotic Effects by Induction of COX-2—Previous studies have demonstrated that environmental and occupational exposure to nickel compounds is associated with an increased risk of lung cancer (3Sivulka D.J. Regul. Toxicol. Pharmacol. 2005; 43: 117-133Crossref PubMed Scopus (58) Google Scholar). Epidemiological evidence also indicates that COX-2 is constitutively overexpressed in most human lung cancers (18Riedl K. Krysan K. Pold M. Dalwadi H. Heuze-Vourc'h N. Dohadwala M. Liu M. Cui X. Figlin R. Mao J.T. Strieter R. Sharma S. Dubinett S.M. Drug Resist. Updat. 2004; 7: 169-184Crossref PubMed Scopus (82) Google Scholar, 19Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1572) Google Scholar, 20Gorgoni B. Caivano M. Arizmendi C. Poli V. J. Biol. Chem. 2001; 276: 40769-40777Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Thus, it was of interest to investigate the potential contributions of elevated COX-2 protein in response to nickel-caused biological effects and their mechanisms. We first sought to determine whether nickel compounds induce COX-2 expression in human bronchial epithelial cells. As shown in Fig. 1, a and b, treatment of Beas-2B cells with water-soluble NiCl2 or water-insoluble NiS led to marked increases of COX-2 transcription as monitored using a COX-2-luciferase reporter. The nickel-dependent induction of COX-2 was both dose- and time-dependent. RT-PCR and Western blotting confirmed that endogenous COX-2 levels are also nickel-responsive (Fig. 1, c and d). These results verified that nickel exposure led to an increase in the COX-2 mRNA and protein expression in Beas-2B cells. To evaluate the role of nickel-dependent COX-2 production in cell survival, specific human COX-2 siRNA (siCOX-2) was used decrease COX-2 level. Stable transfection of siCOX-2 led to an almost complete block of COX-2 expression in response to nickel exposure, whereas control siRNA had no effect (Fig. 1e). Knockdown of COX-2 protein expression by siCOX-2 resulted in significant increases in the sensitivity of Beas-2B cells to nickel-triggered cell death (Fig. 1f). Analysis of apoptosis using either propidium iodide staining (to evaluate hypodiploid DNA content) or caspase-3 or PARP cleavage demonstrated loss of COX-2 nickel-induced cell death (Fig. 1, g and h). These results show that COX-2 is responsible for preventing cell death in the presence of nickel. Activation of IKK/NF-κB Pathway Is Required for COX-2 Induction by Nickel Compounds in Beas-2B Cells—The COX-2 promoter region contains several NF-κB binding sites thought to be involved in the regulation of its expression (21Yoon J.M. Lim J.-J. Yoo C.-G. Lee C.-T. Bang Y.-J. Han S.K. Shim Y.-S. Kim Y.W. Lung Cancer. 2005; 48: 201-209Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 22Weng M.-W. Hsiao Y.-M. Chen C.-J. Wang J.-P. Chen W.-C. Ko J.-L. Toxicol. Lett. (Amst.). 2004; 151: 345-355Crossref PubMed Scopus (31) Google Scholar). Thus, we next evaluated the role of NF-κB in nickel-induced COX-2 expression in Beas-2B cells. NF-κB is normally sequestered in the cytoplasm as an inactive form bound to the I-κBα inhibitor. NF-κB activation requires the phosphorylation I-κBα by I-κB kinase (IKK) leading to I-κBα degradation and subsequent release and nuclear translocation of NF-κB, where it regulates the transcription of its target genes (23Karin M. Greten F.R. Nat. Rev. Immunol. 2005; 5: 749-759Crossref PubMed Scopus (2546) Google Scholar, 24Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar). Our findings indicate that exposure to nickel leads to an increase in IKK phosphorylation, IκBα phosphorylation, and degradation (Fig. 2, a and b). In turn, this results in NF-κB transactivation (Fig. 2, c and d) in Beas-2B cells, consistent with our previous finding that nickel exposure may lead to NF-κB activation (25Huang Y. Davidson G. Li J. Yan Y. Chen F. Costa M. Chen L. Huang C. Environ. Health Perspec. 2002; 110: 835-839Crossref PubMed Scopus (44) Google Scholar). Furthermore, analysis of NF-κB binding activity using an electrophoretic mobility shift assay indicated (Fig. 2e) that NiCl2 treatment enhanced binding in a dose-dependent manner that was abolished in the presence of a 20-fold excess of unlabeled probe. The specificity of this binding was further confirmed in the supergel shift assay with antibody specific for the p65 subunit of NF-κB (Fig. 2e). NF-κB activation in the time course studies reached a peak at 24 h after exposure to nickel, which occurred earlier than the COX-2 induction, suggesting that NF-κB may play a role in COX-2 induction in response to nickel. To determine the role of IKK/NF-κB pathway in COX-2 induction, an inactive mutant of IKKβ, IKKβ-KM, was utilized to establish stable Beas-2B transfectants. Fig. 3a confirmed the overexpession IKKβ-KM using FLAG-tagged analysis. Overexpression of IKKβ-KM in Beas-2B cells significantly blocked IκBα phosphorylation and degradation in response to nickel compounds (Fig. 3, b and c). Furthermore, overexpression of IKKβ-KM impaired NF-κB transactivation (Fig. 3, d and e). Consistent with the blockage of IKK/NF-κB pathway activation, nickel-induced COX-2 expression was also blocked (Fig. 3f). Collectively, these data indicate that the IKK/NF-κB pathway plays an important role in COX-2 induction by nickel compounds. IKKβ, but Not IKKα, Plays a Major Role in COX-2 Induction by Nickel Compounds—To provide direct evidence for the requirement for the IKK/NF-κB pathway in nickel-induced COX-2 expression, we used several IKK/NF-κB gene knockout cell lines (MEFs). Similar to the results obtained with Beas-2B cells, COX-2 transcriptional induction by nickel exposure was significantly increased in a time- and dose-dependent manner in the WT MEFs (Fig. 4, a–c). Activation of the IKK/NF-κB pathway in WT MEFs was also demonstrated (Fig. 4, d–g). Furthermore, NF-κB activation preceded COX-2 induction. Overexpression of IKKβ-KM in WT MEFs (Fig. 4h) not only impaired I-κBα phosphorylation and degradation (Fig. 4i) but also blocked COX-2 protein expression induced by nickel compounds (Fig. 4j). The IKK complex consists of two highly homologous kinase subunits, IKKα and IKKβ, and a nonenzymatic regulatory component, IKKγ/NEMO (26Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). It has been established that two NF-κB activation pathways exist (27Luo J.-L. Kamata H. Karin M. J. Clin. Investig. 2005; 115: 2625-2632Crossref PubMed Scopus (708) Google Scholar). The first, the classical pathway, is normally triggered in response to microbial and viral infections or by exposure to proinflammatory cytokines that activate the tripartite IKK complex, leading to phosphorylation-mediated I-κBα degradation. This pathway, which mostly targets p50·RelA and p50·c-Rel dimers, depends mainly on IKKβ activity (28Li L. Steinauer K.K. Dirks A.J. Husbeck B. Gibbs I. Knox S.J. Radiat. Res. 2003; 160: 617-621Crossref PubMed Scopus (23) Google Scholar). The second alternative pathway leads to selective activation of p52·RelB dimers by inducing the processing of the NF-κB2/p100 precursor protein, which heterodimerizes with Rel B in the cytoplasm (29Li Z.-W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Crossref PubMed Scopus (824) Google Scholar). This pathway is triggered by certain members of the tumor necrosis factor (TNF) cytokine family through selective activation of IKKα homodimers by the upstream kinase NIK (30Viatour P. Merville M.-P. Bours V. Chariot A. Trends Biochem. Sci. 2005; 30: 43-52Abstract Full Text Full Text PDF PubMed Scopus (1197) Google Scholar). We next addressed the role of these two pathways in NF-κB-mediated COX-2 induction by nickel exposure, by transfecting NF-κB- and COX-2-luciferase reporter transfectants into WT, IKKβ–/–, and IKKα–/– MEFs. As shown in Fig. 5, a–e, compared with WT MEFs, both NF-κB activity and COX-2 transcription was dramatically impaired in IKKβ–/– MEFs in response to nickel compounds. However, only a marginally inhibitory effect was observed in IKKα–/– MEFs, suggesting that IKKβ is a major mediator for COX-2 induction by nickel compounds. To further confirm this finding, IKKβ expression plasmid was used to transfect IKKβ–/– MEFs (Fig. 5f). As shown in Fig. 5g, reconstituted expression of IKKβ in IKKβ–/– MEFs restored the COX-2 induction by nickel compounds, demonstrating that IKKβ is required for nickel-induced COX-2 expression. These findings indicate that nickel-induced NF-κB activation is via the classical IKKβ/NF-κB pathway, which, in turn, mediates COX-2 expression.FIGURE 5IKKβ, but not IKKα, was the major mediator for COX-2 induction by nickel compounds. IKKα–/– and IKKβ–/– were identified as compared with WT MEFs using Western blot with specific antibodies as indicated (a) or an NF-κB-luciferase reporter stable transfection assay (b). Western blot was carried out as described under “Materials and Methods.” An NF-κB activity assay was performed 24 h after cells were exposed to nickel compounds. For COX-2 reporter assay (c and d), 8 × 103 WT COX-2 mass1, IKKα–/– COX-2 mass1, and IKKβ–/– COX-2 mass1 cells were seeded separately into each well of a 96-well plate. After being cultured at 37 °C overnight, the cells were treated with NiCl2 or NiS at the indicated dosage for 48 h. The cells were then extracted with lysis buffer, and luciferase activity was measured (c and d). For detection COX-2 protein expression, 2 × 105 WT, IKKα–/–, IKKβ–/–, or IKKβ–/– (HA-IKKβ) cells were seeded into each well of 6-well plates and cultured in 10% FBS/DMEM at 37 °C. When cell density reached 70–80%, the culture medium was replaced with 0.1% FBS/DMEM. After being cultured for 24 h, cells were exposed to nickel compounds for 48 h. The cells were then extracted with sample buffer, and Western blot was carried out (e–g).View Large Image Figure ViewerDownload Hi-res image Download (PPT) p65NF-κB, but Not p50NF-κB, Is Critical for COX-2 Induction by Nickel Compounds—NF-κB is a homo- or heterodimer formed from a multigene family that encodes five structurally related proteins: p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), c-Rel (Rel), and RelB. p50 and p65 are the two predominant NF-κB components expressed in a variety of cell types (23Karin M. Greten F.R. Nat. Rev. Immunol. 2005; 5: 749-759Crossref PubMed Scopus (2546) Google Scholar, 24Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3380) Google Scholar). It has been documented that p50 and p65 have different roles in regulating NF-κB transcriptional activities and mediates different biological effects of the IKKβ/NF-κB signaling pathway under certain stimulatory conditions (31Beg A.A. Sha W.C. Bronson R.T. Ghosh S. Baltimore D. Nature. 1995; 376: 167-170Crossref PubMed Scopus (1640) Google Scholar, 32Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar). To further clarify the roles of p50 and p65 in nickel-induced COX-2 expression, MEFs from p50 and p65 gene knock-out mice were utilized. Genetic ablation of the expression of these two proteins was confirmed by immunoblot analysis (Fig. 6a). We found that genetic ablation of p50 did not affect the nickel-dependent COX-2 induction (Fig. 6b), whereas COX-2 induction was impaired in p65–/– MEFs (Fig. 6, c and d). Furthermore, reconstituted expression of p65 in p65–/– MEFs increased the basal level of COX-2 protein expression and restored COX-2 induction in response to nickel exposure, demonstrating the critical role of p65NF-κB in nickel-induced COX-2 expression. Taken together, these findings show that nickel induction of COX-2 expression occurs through a specific IKKβ/p65 NF-κB-dependent pathway. IKKβ and p65NF-κB, and Not IKKα or p50, Mediate the Anti-apoptotic Effects of Nickel Exposure—Analysis of cell death in the IKKβ or p65-deficient MEF demonstrated an increased sensitivity to nickel-induced cell death when compared with the WT control cells. Furthermore, neither the IKKα–/–-nor p50–/–-deficient MEFs showed cell death under the same experimental conditions (Fig. 7, a, b, d, and e). These data are consistent with siCOX-2 studies in Beas-2B cells showing increased sensitivity to nickel-induced cell death. Furthermore, the results from measurement of cleaved caspase-3 and PARP demonstrated that nickel-induced cell death in IKKβ–/– or p65–/– MEFs is likely attributable to enhanced apoptotic cell death (Figs. 7c and 1f). Taken together, our results demonstrate that the induction of COX-2 via the IKKβ/p65/NF-κB-dependent pathway renders cells resistant to nickel-induced apoptosis. Activation of NF-κB upon Nickel Exposure Is Mediated through Hydrogen Peroxide—Because nickel exposure can activate the IKKβ/p65NF-κB pathway and elevate COX-2 protein expression, we chose to evaluate the mechanism linking nickel stimulation and NF-κB activation. Our previous studies demonstrated that nickel could induce H2O2 generation, which is required for the activation of NFAT (nuclear factor of activated T cell) in PW cells (33Huang C. Li J. Costa M. Zhang Z. Leonard S.S. Castranova V. Vallyathan V. Ju G. Shi X. Cancer Res. 2001; 61: 8051-8057PubMed Google Scholar). Therefore, we evaluated the contribution of H2O2 to the regulation of nickel-dependent NF-κB activation and COX-2 expression. We used a mitochondrial targeted catalase expression (pZeoSV/mCAT) to directly detoxify H2O2 at its primary site of production. As shown in Fig. 8a, the expression of catalase in Beas-2B/mCAT mass2 was increased when compared with Beas-2B/vector control cells. Catalase overexpression efficiently abrogated the generation of H2O2 when compared with that of Beas-2B/vector control cells (Fig. 8b). Furthermore, both the phosphorylation of IκBα and the expression of COX-2 (Fig. 8, c and d) was diminished in response to nickel exposure in the Beas-2B/mCAT mass2 cells when compared with the control cells. These results indicate that H2O2 mediates the activation NF-κB and the subsequent induction of COX-2 expression in response to nickel. Although epidemiological studies have demonstrated that exposure to nickel is associated with increased risk of human lung cancer, the mechanism involved in the carcinogenic effects of nic" @default.
• W2080217327 created "2016-06-24" @default.
• W2080217327 creator A5001171800 @default.
• W2080217327 creator A5025524154 @default.
• W2080217327 creator A5035325283 @default.
• W2080217327 creator A5042809761 @default.
• W2080217327 creator A5067471566 @default.
• W2080217327 creator A5069299119 @default.
• W2080217327 creator A5072795022 @default.
• W2080217327 creator A5072882134 @default.
• W2080217327 creator A5074463821 @default.
• W2080217327 creator A5077713155 @default.
• W2080217327 date "2006-12-01" @default.
• W2080217327 modified "2023-09-29" @default.
• W2080217327 title "Nickel Compounds Render Anti-apoptotic Effect to Human Bronchial Epithelial Beas-2B Cells by Induction of Cyclooxygenase-2 through an IKKβ/p65-dependent and IKKα- and p50-independent Pathway" @default.
• W2080217327 cites W1555487519 @default.
• W2080217327 cites W1585093254 @default.
• W2080217327 cites W1585759889 @default.
• W2080217327 cites W1966473790 @default.
• W2080217327 cites W1975560535 @default.
• W2080217327 cites W1982062664 @default.
• W2080217327 cites W1988924112 @default.
• W2080217327 cites W1989227773 @default.
• W2080217327 cites W1991868611 @default.
• W2080217327 cites W2000821034 @default.
• W2080217327 cites W2025838546 @default.
• W2080217327 cites W2030110600 @default.
• W2080217327 cites W2033069208 @default.
• W2080217327 cites W2035616122 @default.
• W2080217327 cites W2040470926 @default.
• W2080217327 cites W2045182776 @default.
• W2080217327 cites W2046056065 @default.
• W2080217327 cites W2046293893 @default.
• W2080217327 cites W2048133075 @default.
• W2080217327 cites W2049433363 @default.
• W2080217327 cites W2054158147 @default.
• W2080217327 cites W2060280757 @default.
• W2080217327 cites W2060736868 @default.
• W2080217327 cites W2061098044 @default.
• W2080217327 cites W2062026458 @default.
• W2080217327 cites W2066789432 @default.
• W2080217327 cites W2069495273 @default.
• W2080217327 cites W2070539196 @default.
• W2080217327 cites W2080127319 @default.
• W2080217327 cites W2119543039 @default.
• W2080217327 cites W2121431202 @default.
• W2080217327 cites W2123978826 @default.
• W2080217327 cites W2133984207 @default.
• W2080217327 cites W2139853624 @default.
• W2080217327 cites W2145028653 @default.
• W2080217327 cites W2147921405 @default.
• W2080217327 cites W2148119611 @default.
• W2080217327 cites W2148674446 @default.
• W2080217327 cites W2148700883 @default.
• W2080217327 cites W2157942810 @default.
• W2080217327 cites W2157973267 @default.
• W2080217327 cites W2159899427 @default.
• W2080217327 cites W2164053161 @default.
• W2080217327 cites W2164394800 @default.
• W2080217327 cites W2170457651 @default.
• W2080217327 cites W2747063936 @default.
• W2080217327 cites W3205358246 @default.
• W2080217327 cites W4234885891 @default.
• W2080217327 cites W4313335852 @default.
• W2080217327 doi "https://doi.org/10.1074/jbc.m604798200" @default.
• W2080217327 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16982623" @default.
• W2080217327 hasPublicationYear "2006" @default.
• W2080217327 type Work @default.
• W2080217327 sameAs 2080217327 @default.
• W2080217327 citedByCount "46" @default.
• W2080217327 countsByYear W20802173272012 @default.
• W2080217327 countsByYear W20802173272013 @default.
• W2080217327 countsByYear W20802173272014 @default.
• W2080217327 countsByYear W20802173272015 @default.
• W2080217327 countsByYear W20802173272016 @default.
• W2080217327 countsByYear W20802173272017 @default.
• W2080217327 countsByYear W20802173272018 @default.
• W2080217327 countsByYear W20802173272019 @default.
• W2080217327 countsByYear W20802173272020 @default.
• W2080217327 crossrefType "journal-article" @default.
• W2080217327 hasAuthorship W2080217327A5001171800 @default.
• W2080217327 hasAuthorship W2080217327A5025524154 @default.
• W2080217327 hasAuthorship W2080217327A5035325283 @default.
• W2080217327 hasAuthorship W2080217327A5042809761 @default.
• W2080217327 hasAuthorship W2080217327A5067471566 @default.
• W2080217327 hasAuthorship W2080217327A5069299119 @default.
• W2080217327 hasAuthorship W2080217327A5072795022 @default.
• W2080217327 hasAuthorship W2080217327A5072882134 @default.
• W2080217327 hasAuthorship W2080217327A5074463821 @default.
• W2080217327 hasAuthorship W2080217327A5077713155 @default.
• W2080217327 hasBestOaLocation W20802173271 @default.
• W2080217327 hasConcept C100175707 @default.
• W2080217327 hasConcept C104317684 @default.
• W2080217327 hasConcept C181199279 @default.
• W2080217327 hasConcept C185592680 @default.
• W2080217327 hasConcept C190283241 @default.
• W2080217327 hasConcept C2777730290 @default.
• W2080217327 hasConcept C2779689624 @default.

• next