Cyclooxygenase-2 Induction by Arsenite Is through a Nuclear Factor of Activated T-cell-dependent Pathway and Plays an Antiapoptotic Role in Beas-2B Cells
2006; Elsevier BV; Volume: 281; Issue: 34 Linguagem: Inglês
10.1074/jbc.m600751200
ISSN1083-351X
AutoresJin Ding, Jingxia Li, Caifang Xue, Kangjian Wu, Weiming Ouyang, Dongyun Zhang, Yan Yan, Chuanshu Huang,
Tópico(s)Nuclear Receptors and Signaling
ResumoArsenite is a well known metalloid human carcinogen, and epidemiological evidence has demonstrated its association with the increased incidence of lung cancer. However, the mechanism involved in its lung carcinogenic effect remains obscure. The current study demonstrated that exposure of human bronchial epithelial cells (Beas-2B) to arsenite resulted in a marked induction of cyclooxygenase (COX)-2, an important mediator for inflammation and tumor promotion. Exposure of the Beas-2B cells to arsenite also led to significant transactivation of nuclear factor of activated T-cells (NFAT), but not activator protein-1 (AP-1) and NFκB, suggesting that NFAT, rather than AP-1 or NFκB, is implicated in the responses of Beas-2B cells to arsenite exposure. Furthermore, we found that inhibition of the NFAT pathway by either chemical inhibitors, dominant negative mutants of NFAT, or NFAT3 small interference RNA resulted in the impairment of COX-2 induction and caused cell apoptosis in Beas-2B cells exposed to arsenite. Site-directed mutation of two putative NFAT binding sites between–111 to +65 in the COX-2 promoter region eliminated the COX-2 transcriptional activity induced by arsenite, confirming that those two NFAT binding sites in the COX-2 promoter region are critical for COX-2 induction by arsenite. Moreover, knockdown of COX-2 expression by COX-2-specific small interference RNA also led to an increased cell apoptosis in Beas-2B cells upon arsenite exposure. Together, our results demonstrate that COX-2 induction by arsenite is through NFAT3-dependent and AP-1- or NFκB-independent pathways and plays a crucial role in antagonizing arsenite-induced cell apoptosis in human bronchial epithelial Beas-2B cells. Arsenite is a well known metalloid human carcinogen, and epidemiological evidence has demonstrated its association with the increased incidence of lung cancer. However, the mechanism involved in its lung carcinogenic effect remains obscure. The current study demonstrated that exposure of human bronchial epithelial cells (Beas-2B) to arsenite resulted in a marked induction of cyclooxygenase (COX)-2, an important mediator for inflammation and tumor promotion. Exposure of the Beas-2B cells to arsenite also led to significant transactivation of nuclear factor of activated T-cells (NFAT), but not activator protein-1 (AP-1) and NFκB, suggesting that NFAT, rather than AP-1 or NFκB, is implicated in the responses of Beas-2B cells to arsenite exposure. Furthermore, we found that inhibition of the NFAT pathway by either chemical inhibitors, dominant negative mutants of NFAT, or NFAT3 small interference RNA resulted in the impairment of COX-2 induction and caused cell apoptosis in Beas-2B cells exposed to arsenite. Site-directed mutation of two putative NFAT binding sites between–111 to +65 in the COX-2 promoter region eliminated the COX-2 transcriptional activity induced by arsenite, confirming that those two NFAT binding sites in the COX-2 promoter region are critical for COX-2 induction by arsenite. Moreover, knockdown of COX-2 expression by COX-2-specific small interference RNA also led to an increased cell apoptosis in Beas-2B cells upon arsenite exposure. Together, our results demonstrate that COX-2 induction by arsenite is through NFAT3-dependent and AP-1- or NFκB-independent pathways and plays a crucial role in antagonizing arsenite-induced cell apoptosis in human bronchial epithelial Beas-2B cells. Arsenic is an environmental toxin widely distributed in water, food, air, and soil (1Chappell W.R. Beck B.D. Brown K.G. Chaney R. Cothern R. Cothern C.R. Irgolic K.J. North D.W. Thornton I. Tsongas T.A. Environ. Health Perspect. 1997; 105: 1060-1067Crossref PubMed Scopus (82) Google Scholar). Arsenic, combined with oxygen, chlorine, and sulfur, is called inorganic arsenic, which represents the most common forms of either arsenite or arsenate in the environment (2Knowles F.C. Benson A.A. Z. Gesamte Hyg. 1984; 30: 625-626PubMed Google Scholar). Humans are exposed to arsenic mainly by inhalation, ingestion, and skin contact (3Bettley F.R. O'Shea J.A. Br. J. Dermatol. 1975; 92: 563-568Crossref PubMed Scopus (70) Google Scholar, 4Landolph J.R. Environ. Health Perspect. 1994; 102: 119-125Crossref PubMed Scopus (68) Google Scholar). The inhalation route is mainly associated with occupational exposure of ore smelters, insecticide manufacturers, and sheep dip workers. Previous studies have shown that environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin and lung cancers. Although arsenic itself is not a mutagen of DNA, it has some deleterious effects, such as the potential for DNA damage by other agents and inhibition of DNA repair (5Tsuda T. Babazono A. Yamamoto E. Kurumatani N. Mino Y. Ogawa T. Kishi Y. Aoyama H. Am. J. Epidemiol. 1995; 141: 198-209Crossref PubMed Scopus (275) Google Scholar, 6Bates M.N. Smith A.H. Cantor K.P. Am. J. Epidemiol. 1995; 141: 523-530Crossref PubMed Scopus (213) Google Scholar, 7Chiou H.Y. Hsueh Y.M. Liaw K.F. Horng S.F. Chiang M.H. Pu Y.S. Lin J.S. Huang C.H. Chen C.J. Cancer Res. 1995; 55: 1296-1300PubMed Google Scholar, 8Ishinishi N. Kodama Y. Nobutomo K. Hisanaga A. Environ. Health Perspect. 1977; 19: 191-196Crossref PubMed Scopus (46) Google Scholar). More importantly, arsenite resembles many other classic carcinogens in inducing cell tumorigenesis by activating certain genes, especially those involved in tumor promotion. Nonetheless, the mechanism by which arsenite causes human lung cancers remains to be intensively elucidated (9Lee T.C. Tanaka N. Lamb P.W. Gilmer T.M. Barrett J.C. Science. 1988; 241: 79-81Crossref PubMed Scopus (184) Google Scholar, 10Li J.H. Rossman T.G. Mol. Toxicol. 1989; 2: 1-9PubMed Google Scholar, 11Li J.H. Rossman T.G. Biol. Trace Elem. Res. 1989; 21: 373-381Crossref PubMed Scopus (111) Google Scholar). Cyclooxygenase (COX) 3The abbreviations used are: COX, cyclooxygenase; AP-1, activator protein-1; FBS, fetal bovine serum; NFAT, nuclear factor of activated T-cells; DN-NFAT, dominant negative mutant of NFAT; NFκB, nuclear factor-κB; siRNA, small interfering RNA; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethy)ester; CsA, cyclosporin A; siNFAT3, small interference RNA specific for NFAT3; siCOX-2, COX-2 small interference RNA; DMEM, Dulbecco's modified Eagle's medium; PARP, poly(ADP-ribose) polymerase. 3The abbreviations used are: COX, cyclooxygenase; AP-1, activator protein-1; FBS, fetal bovine serum; NFAT, nuclear factor of activated T-cells; DN-NFAT, dominant negative mutant of NFAT; NFκB, nuclear factor-κB; siRNA, small interfering RNA; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethy)ester; CsA, cyclosporin A; siNFAT3, small interference RNA specific for NFAT3; siCOX-2, COX-2 small interference RNA; DMEM, Dulbecco's modified Eagle's medium; PARP, poly(ADP-ribose) polymerase.-2, also named prostaglandin endoperoxide synthase 2, is an essential enzyme involved in the inflammation processes and other pathogenesis (12Kuwano T. Nakao S. Yamamoto H. Tsuneyoshi M. Yamamoto T. Kuwano M. Ono M. FASEB J. 2004; 18: 300-310Crossref PubMed Scopus (242) Google Scholar). Previous studies have demonstrated that COX-2 is constitutively overexpressed in a variety of human malignancies, especially in primary lung adenocarcinoma (13Dannenberg A.J. Lippman S.M. Mann J.R. Subbaramaiah K. DuBois R.N. J. Clin. Oncol. 2005; 23: 254-266Crossref PubMed Scopus (338) Google Scholar, 14Dempke W. Rie C. Grothey A. Schmoll H.J. J. Cancer Res. Clin. Oncol. 2001; 127: 411-417Crossref PubMed Scopus (378) Google Scholar). There is a growing amount of evidence both in vitro and in vivo indicating that chronic inflammation and its mediator COX-2 play an important role in cancer development (15Pikarsky E. Porat R.M. Stein I. Abramovitch R. Amit S. Kasem S. Gutkovich-Pyest E. Urieli-Shoval S. Galun E. Ben-Neriah Y. Nature. 2004; 431: 461-466Crossref PubMed Scopus (1947) Google Scholar, 16Clevers H. Cell. 2004; 118: 671-674Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). It has also been reported that arsenite exposure can stimulate COX-2 expression through activating the nuclear factor κB (NFκB) pathway in endothelial cells (17Tsai S.-H. Liang Y.-C. Chen L. Ho F.-M. Hsieh M.-S. Lin J.-K. J. Cell. Biochem. 2002; 84: 750-758Crossref PubMed Scopus (69) Google Scholar). Whether arsenite is able to induce COX-2 expression in human bronchial epithelial cells and, if it does, which signaling pathway mediates its induction, as well as what the role of COX-2 is in cell responses to arsenite exposure, have not yet been investigated. The present study documents that arsenite can markedly induce COX-2 expression through the calcineurin/NFAT-dependent pathway in human bronchial epithelial Beas-2B cells, and we also demonstrate that elevated COX-2 protein expression mediates the protection of Beas-2B cells from apoptosis caused by arsenite. Cell Culture and Reagents—Beas-2B cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Calbiochem) supplemented with 10% fetal bovine serum (FBS), 5% penicillin/streptomycin, and 2 mml-glutamine (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO2 in air. Sodium metavanadate (vanadate), and sodium arsenite were purchased from Aldrich. Benzo[a]pyrene was from Eagle-Picher Industries, Inc. The substrate for the luciferase assay was purchased from Promega (Madison, WI). NFAT inhibitor peptide was from Calbiochem. Antibodies against COX-2, NFAT3, and hemagglutinin tag were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-β-actin antibody was from Sigma. Antibodies for caspase-3 and PARP were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Plasmids and Construction of siRNA Vectors—The COX-2-luciferase reporter plasmid containing the upstream 5′-flanking region of human COX-2 gene promoter linked to a luciferase reporter was previously described (18Subbaramaiah K. Bulic P. Lin Y. Dannenberg A.J. Pasco D.S. J. Biomol. Screen. 2001; 6: 101-110PubMed Google Scholar). The p-AP-1-Luc reporter plasmid was purchased from Stratagene (La Jolla, CA). The NFAT-luciferase reporter plasmid and NFκB-luciferase reporter plasmid were constructed as described previously (19Li J. Chen H. Ke Q. Feng Z. Tang M.S. Liu B. Amin S. Costa M. Huang C. Mol. Carcinog. 2004; 40: 104-115Crossref PubMed Scopus (35) Google Scholar, 20Rincon M. Flavell R.A. Mol. Cell. Biol. 1997; 17: 1522-1534Crossref PubMed Scopus (108) Google Scholar). The dominant negative mutant of NFAT (DN-NFAT) eukaryotic expression construct, kindly provided by Dr. Chi-Wing Chow (Albert Einstein College of Medicine), contains a deletion mutant of NFAT that can inhibit the transcription activity of all NFAT isoforms (21Chow C.W. Rincon M. Davis R.J. Mol. Cell. Biol. 1999; 19: 2300-2307Crossref PubMed Scopus (155) Google Scholar). The specific small interference RNAs targeted human NFAT3 or COX-2, were designed with siRNA converter on the Web site of Ambion Inc. (Austin, TX) according to the gene sequences and the siRNA design guidelines, and synthesized by Invitrogen. The target sequences were 5′-gaactggactcagaggatg-3′ (human NFAT3 mRNA) and 5′-agacagatcataagcgagg-3′ (human COX-2 mRNA). The siRNA sequences were controlled via BLAST search and did not show any homology to other known human genes. The siRNAs were inserted into pSuppressor vector and verified by DNA sequencing. The siRNA constructs for NFAT3 and COX-2 were named as siNFAT3 and siCOX-2, respectively. Point Mutation of NFAT Binding Site in COX-2 Promoter-Luciferase Reporter—To generate a site-directed mutant of the NFAT binding sequence in the COX-2 promoter region of the COX-2-luciferase reporter plasmid, the QuikChange mutagenesis kit (Stratagene) was used according to the manufacturer's instruction. The sense primer was 5′-gaggaggtaaaaatttgtggggggtacgaaaaggctgaaagaaacag-3′, and the antisense primer was 5′-ctgtttctttcagccttttcgtaccccccacaaatttttacctcctc-3′. (The underlined bold characters indicate the mutated nucleotides.) The COX-2 promoter-luciferase reporter plasmid with mutation at both NFAT binding sites was identified and designated as COX-2-luc NFATmut. Stable Transfection—Beas-2B cells were cultured in a 6-well plate until 90% confluence. The COX-2 reporter construct in combination with either mock control, siNFAT3, or DN-NFAT, as well as the siCOX-2 construct, were co-transfected with a hygromycin-resistant plasmid into Beas-2B cells by the Lipofectamine transfection kit (Invitrogen) according to the manufacturer's instructions. The stable transfectants, including Beas-2B-COX-2-luc mass1, Beas-2B-COX2-luc NFATmut mass1, Beas-2B/siNFAT3 COX-2-luc mass1, Beas-2B/DN-NFAT COX-2-luc mass1, and Beas-2B-siCOX-2 mass1, were established by selection with 400 μg/ml hygromycin. The established stable transfectants were cultured in hygromycin-free 10% FBS DMEM for at least two passages before each experiment. The stable transfectants for the luciferase reporters were identified by measuring the basal level of luciferase activity, and the stable transfectants of siNFAT3 or siCOX-2 were verified by analyzing their specific gene expression with Western blot. Beas-2B/DN-NFAT COX-2-luc mass1 was identified by analyzing the ectopic expression of Flag tag. 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 in air. After being cultured at 37 °C overnight, the cells were treated with a different concentration of arsenite for various time points as indicated. Cells were then lysed with 50 μl of lysis buffer, and the luciferase activity was finally measured using a Promega luciferase assay reagent with a luminometer (Wallac 1420 Victor2 multipliable counter system). The results were expressed as transcription factor activation relative to control medium (relative NFAT, NFκB, or AP-1 activation) 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 of Beas-2B transfectants were cultured in each well of 6-well plates until 70–80% confluence; the culture medium was replaced with 0.1% FBS DMEM. After being cultured for 24 h, the cells were exposed to the indicated amount of arsenite for 12 or 24 h. The cells were then washed once with ice-cold phosphate-buffered saline and extracted with a SDS-sample buffer. The cell extracts were separated on SDS-polyacrylamide gels, transferred, and probed with a rabbit-specific antibody against COX-2. The protein band, specifically bound to the primary antibody, was detected using an anti-rabbit IgG-AP-linked and an ECF Western blotting system (Amersham Biosciences). Cell Death Assay—The arsenite-treated Beas-2B cells were collected by pooling cells from the culture medium as well as the trypsinized adherent cells. The trypan blue exclusion method was used to determine the dead cells. Induction of COX-2 by Arsenite in Beas-2B Cells—Previous studies have shown that environmental and occupational exposure to arsenite is associated with an increase in the risk of lung cancer (22Pold M. Krysan K. Pold A. Dohadwala M. Heuze-Vourc'h N. Mao J.T. Riedl K.L. Sharma S. Dubinett S.M. Cancer Res. 2004; 64: 6549-6555Crossref PubMed Scopus (32) Google Scholar), and the carcinogenic effect of arsenite has also been verified in various experimental models (6Bates M.N. Smith A.H. Cantor K.P. Am. J. Epidemiol. 1995; 141: 523-530Crossref PubMed Scopus (213) Google Scholar, 7Chiou H.Y. Hsueh Y.M. Liaw K.F. Horng S.F. Chiang M.H. Pu Y.S. Lin J.S. Huang C.H. Chen C.J. Cancer Res. 1995; 55: 1296-1300PubMed Google Scholar). It has also been documented that COX-2 is constitutively overexpressed in the human lung cancers (23Riedl 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. Update. 2004; 7: 169-184Crossref PubMed Scopus (80) Google Scholar, 24Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1516) Google Scholar, 25Gorgoni B. Caivano M. Arizmendi C. Poli V. J. Biol. Chem. 2001; 276: 40769-40777Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). It is, therefore, interesting to determine whether COX-2 is inducible in human bronchial epithelial cells by arsenite. To address this question, Beas-2B cells were exposed to arsenite. As shown in Fig. 1, a and b, treatment of Beas-2B cells with arsenite resulted in marked COX-2 induction in COX-2-luciferase reporter assay. This induction appeared to be in both dose- and time-dependent manners (Fig. 1). To further confirm this finding, the levels of COX-2 protein expression induced by arsenite were also determined by Western blot. The results showed that arsenite exposure led to an increase in the COX-2 protein level in Beas-2B cells (Fig. 1c), suggesting that arsenite is a potent carcinogen for COX-2 expression in human bronchial epithelial cells. Arsenite Exposure Led to Activation of NFAT but Not AP-1 and NFκB in Beas-2B Cells—The COX-2 gene promoter region contains NFκB, AP-1, and NFAT binding sites, which can be recognized by these transcription factors and in turn lead to COX-2 transcription (26Hao C.M. Yull F. Blackwell T. Komhoff M. Davis L.S. Breyer M.D. J. Clin. Invest. 2000; 106: 973-982Crossref PubMed Scopus (140) Google Scholar, 27Chen J. Zhao M. Rao R. Inoue H. Hao C.-M. J. Biol. Chem. 2005; 280: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 28Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Previous studies have indicated that COX-2 regulation appears to involve diversified mechanisms based on cell types and stimuli (26Hao C.M. Yull F. Blackwell T. Komhoff M. Davis L.S. Breyer M.D. J. Clin. Invest. 2000; 106: 973-982Crossref PubMed Scopus (140) Google Scholar, 27Chen J. Zhao M. Rao R. Inoue H. Hao C.-M. J. Biol. Chem. 2005; 280: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 28Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 29Hernandez G.L. Volpert O.V. Iniguez M.A. Lorenzo E. Martinez-Martinez S. Grau R. Fresno M. Redondo J.M. J. Exp. Med. 2001; 193: 607-620Crossref PubMed Scopus (383) Google Scholar, 30Sugimoto T. Haneda M. Sawano H. Isshiki K. Maeda S. Koya D. Inoki K. Yasuda H. Kashiwagi A. Kikkawa R. J. Am. Soc. Nephrol. 2001; 12: 1359-1368Crossref PubMed Google Scholar, 31Duque J. Fresno M. Iniguez M.A. J. Biol. Chem. 2005; 280: 8686-8693Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 32de Gregorio R. Iniguez M.A. Fresno M. Alemany S. J. Biol. Chem. 2001; 276: 27003-27009Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Breyer and co-workers (26Hao C.M. Yull F. Blackwell T. Komhoff M. Davis L.S. Breyer M.D. J. Clin. Invest. 2000; 106: 973-982Crossref PubMed Scopus (140) Google Scholar) reported that NFκB activation was responsible for COX-2 induction following dehydration or hypertonic stress in renal medullary interstitial cells (26Hao C.M. Yull F. Blackwell T. Komhoff M. Davis L.S. Breyer M.D. J. Clin. Invest. 2000; 106: 973-982Crossref PubMed Scopus (140) Google Scholar, 27Chen J. Zhao M. Rao R. Inoue H. Hao C.-M. J. Biol. Chem. 2005; 280: 16354-16359Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Whereas in the Jurkat human leukemic T cells, the COX-2 induction was mediated by AP-1 and NFAT (28Iniguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). It has also been reported that COX-2 can be regulated by NFAT in nonlymphoid tissues (29Hernandez G.L. Volpert O.V. Iniguez M.A. Lorenzo E. Martinez-Martinez S. Grau R. Fresno M. Redondo J.M. J. Exp. Med. 2001; 193: 607-620Crossref PubMed Scopus (383) Google Scholar, 30Sugimoto T. Haneda M. Sawano H. Isshiki K. Maeda S. Koya D. Inoki K. Yasuda H. Kashiwagi A. Kikkawa R. J. Am. Soc. Nephrol. 2001; 12: 1359-1368Crossref PubMed Google Scholar). Therefore, it is important to know which transcription factors mediate COX-2 induction by arsenite in Beas-2B cells. To test this, stable Beas-2B transfectants with NFκB-, AP-1-, and NFAT-luciferase reporters were exposed to arsenite. As shown in Fig. 2, a–d, arsenite exposure did not show significant effects on activation of AP-1 (Fig. 2, a and b) or NFκB (Fig. 2, c and d). To rule out the deficiency of activation of AP-1 and NFκB in arsenite treatment due to any reason from either luciferase reporter or a defect of signaling pathway leading to AP-1 and NFκB activation in Beas-2B cells, vanadate and benzo[a]pyrene were used as positive controls for activation of AP-1 and NFκB, respectively. The results showed that vanadate and benzo[a]pyrene were able to markedly activate AP-1 (Fig. 2, a and b) and NFκB (Fig. 2, c and d), suggesting that AP-1 and NFκB-Luc reporters and signaling pathways leading to their activation are normal. Interestingly, arsenite exposure specifically resulted in NFAT transcriptional activation in both time- and dose-dependent manners (Fig. 2, e and f). It may be noted that NFAT activation by arsenite in the time course studies reached a peak earlier, as compared with that of COX-2 induction (Fig. 1c versus Fig. 2f), and given the factor that there are two NFAT binding sites in the COX-2 promoter region, we anticipate that NFAT may be involved in COX-2 induction by arsenite in Beas-2B cells. NFAT Activation Is Required for COX-2 Induction by Arsenite—To unravel the role of NFAT in the COX-2 induction in Beas-2B cells by arsenite, NFAT-specific inhibitor, which is a highly selective NFAT peptide inhibitor and is able to inhibit NFAT activation and NFAT-dependent gene expression in T cells, was first used. As shown in Fig. 3a, pretreatment of the cells with NFAT inhibitor impaired the arsenite-associated COX-2 induction, suggesting that NFAT might play a role in the arsenite-induced COX-2 expression in Beas-2B cells. To further address this notion, hemagglutinin-tagged dominant DN-NFAT was stably transfected, and the Beas-2B DN-NFAT COX-2 mass1 was established (Fig. 3c). Overexpression of DN-NFAT resulted in a dramatic inhibition of arsenite-induced COX-2 expression in both the COX-2-luciferase reporter assay and Western blot (Fig. 3, d and g). There are two putative NFAT binding sites in the human COX-2 promoter that were reported to be critical for the COX-2 promoter activity (31Duque J. Fresno M. Iniguez M.A. J. Biol. Chem. 2005; 280: 8686-8693Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). To determine whether NFAT regulated COX-2 expression through direct binding to the COX-2 promoter region, point mutation of the two NFAT binding sites in the promoter region of COX-2-Luc reporter was carried out. The results demonstrated that this mutation resulted in impairment of the COX-2 transcription induced by arsenite exposure (Fig. 3f). All of these data strongly demonstrate that NFAT activation by arsenite is responsible for its COX-2 induction in Beas-2B cells. Previous studies have shown that at least five members of the NFAT family have been identified (32de Gregorio R. Iniguez M.A. Fresno M. Alemany S. J. Biol. Chem. 2001; 276: 27003-27009Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 33Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2195) Google Scholar). NFAT1 and NFAT2 are mainly involved in T-cell activation. NFAT4 is mainly expressed in thymus, whereas NFAT5 is crucially involved in cellular response to hypertonic stress, and NFAT3 is primarily expressed in nonlymphoid tissues (33Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2195) Google Scholar). Since our most recent studies have demonstrated that NFAT3 is a mediator for TNF-α-induced COX-2 expression in mouse Cl41 cells (34Yan Y. Li J. Ouyang W. Ma Q. Hu Y. Zhang D. Ding J. Qu Q. Subbaramaiah K. Huang C. J. Cell Sci. 2006; (in press)Google Scholar), we anticipated that NFAT3 might be a major NFAT isoform involved in COX-2 induction by arsenite. To evaluate the potential role of NFAT3 in COX-2 expression by arsenite, human siNFAT3 was constructed and used. Stable transfection of siNFAT3 in Beas-2B cells resulted in a dramatic reduction of NFAT3 protein expression in Beas-2B cells (Fig. 3b). Specific knockdown of NFAT3 expression by siNFAT3 blocked arsenite-induced COX-2 transcription and protein expression (Fig. 3, e and h). These results distinctly demonstrate that NFAT3 is a major mediator for COX-2 induction by arsenite in Beas-2B cells. Involvement of Ca2+/Calcineurin in COX-2 Induction by Arsenite—Calcineurin signaling has been implicated in a broad spectrum of physiological or pathological conditions in a variety of organ systems (35Aramburu J. Heitman J. Crabtree G.R. EMBO Rep. 2004; 5: 343-348Crossref PubMed Scopus (131) Google Scholar). There is evidence showing that NFAT activation was mediated by a Ca2+/calcineurin-dependent pathway (33Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2195) Google Scholar). To evaluate the contribution of cacinuerin in arsenite-induced COX-2 expression, cyclosporin A (CsA), a specific inhibitor of calcineurin, was employed. As shown in Fig. 4, a and b, pretreatment of cells with CsA markedly inhibited NFAT activation. Consistent with inhibition of NFAT activation, COX-2 induction by arsenite was also impaired (Fig. 4, c and d). To test the role of Ca2+ in COX-2 induction by arsenite, 2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethy)ester (BAPTA-AM), a specific Ca2+ chelator (36Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2978) Google Scholar), was used. Pretreatment of cells with BAPTA-AM resulted in a dramatic inhibition of NFAT activation and COX-2 induction (Fig. 4, e and f), whereas it did not show any inhibitory effect on vanadate-induced COX-2 expression (Fig. 4f). These data suggest that arsenite-induced COX-2 transcription may involve calcium signaling and calcineurin activation in Beas-2B cells. COX-2 Induction by Arsenite Provides Antiapoptotic Signaling in Arsenite-treated Beas-2B Cells—It has been reported that COX-2 may play a role in the regulation of cell proliferation, cell survival, and tumorigenesis (37Ali-Fehmi R. Morris R.T. Bandyopadhyay S. Che M. Schimp V. Malone J.J.M. Munkarah A.R. Am. J. Obstet. Gynecol. 2005; 192: 819-825Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Thus, it is of interest to investigate the potential contribution of activated NFAT and elevated COX-2 protein in the arsenite-induced biological effects on Beas-2B cells. As shown in Fig. 5a, inhibition of the calcineurin/NFAT pathway by either pretreatment of cells with CsA or specific knockdown of NFAT3 significantly led to arsenite-associated Beas-2B cell death, whereas either arsenite treatment or CsA pretreatment alone caused only a marginal effect on the cell viabilities (Fig. 5a), suggesting that activation of the calcineurin/NFAT pathway by arsenite provides an inhibitory effect on cell death caused by arsenite. To determine whether calcineurin/NFAT-mediated inhibition of arsenite-induced cell death was due to its antiapoptotic effect, we compared the levels of the arsenite-induced caspase-3 and PARP cleavages among cells from three groups as shown in Fig. 5b. Inhibition of calcineurin by CsA or specific knockdown of NFAT3 significantly increased the levels of cleaved caspase-3 and PARP, suggesting that calcineurin/NFAT provide an inhibitory effect on cell apoptosis caused by arsenite. To further confirm that COX-2 is an NFAT downstream mediator for its antiapoptotic effect, we made a siCOX-2 construct and established a stable Beas-2B-siCOX-2 transfectant. As shown in Fig. 6a, introduction of siCOX-2 was able to knock down COX-2 protein expression in Beas-2B cells and increased the cell death (Fig. 6, b–D) in Beas-2B cells upon arsenite exposure. These data demonstrated that COX-2 appeared to be an NFAT downstream gene product responsible for inhibitory effects on cell death caused by arsenite. Detection of the apoptotic effector by Western blot revealed that the levels of the cleaved caspase-3 and PARP were markedly higher in the siCOX-2 stably transfected cells than those in Beas-2B control cells. Therefore, the data strongly demonstrated that elevated COX-2 protein via a calcineurin/NFAT3-dependent pathway by arsenite treatment rendered Beas-2B cells resistant to the arsenite-induced apoptosis.FIGURE 6COX-2 was an executor in calcineurin/NFAT downstream for antiapoptotic effect in cell response to arsenite exposure. 2 × 105 Beas-2B/control cells and Beas-2B/siCOX-2 mass1 cells were seeded into each well of 6-well plates and cultured in 10% FBS DMEM at 37 °C. When the 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 20 μm arsenite for 12 h. The cell morphological changes were observed, and the photos were taken under the microscope (b); the percentage of cell death was counted and calculated using try
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