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Carcinogenesis Advance Access originally published online on October 25, 2006
Carcinogenesis 2007 28(4):883-891; doi:10.1093/carcin/bgl186
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

JNK1, but not JNK2, is required for COX-2 induction by nickel compounds

Dongyun Zhang, Jingxia Li, Kangjian Wu, Weiming Ouyang, Jin Ding, Zheng-gang Liu1, Max Costa and Chuanshu Huang*

Nelson Institute of Environmental Medicine, New York University School of Medicine 57 Old Forge Road, Tuxedo, NY 10987, USA
1 Cell and Cancer Biology Branch, Center for Cancer Research NCI, National Institutes of Health, Bethesda, MD 20892, USA

*To whom correspondence should be addressed. Tel: +1 845 731 3519; Fax: +1 845 351 2320; Email: chuanshu{at}env.med.nyu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activation of the signaling pathways leading to gene expression regulation is critical in the carcinogenic effects of nickel exposure. In the present study, we found nickel exposure could induce cyclooxygenase-2 (COX-2) expression at transcriptional and protein levels in both human bronchoepithelial cells (Beas-2B) and murine embryonic fibroblasts (MEFs). We further provided direct evidence for the specific involvement of the JNK1 signaling pathway in the COX-2 induction using specific gene knockout approaches. Our results demonstrated that COX-2 induction by nickel was impaired in JNK1–/– MEFs, but not in JNK2–/– MEFs. Moreover, re-constitutional expression of JNK1 restored COX-2 induction, confirming the specific requirement of JNK1 in COX-2 induction. Further investigation revealed that JNK1 mediated the nickel-induced COX-2 expression in a c-Jun/AP-1-dependent manner. Ectopic expression of TAM67, a c-Jun dominant negative mutant, also suppressed the COX-2 induction. Our results demonstrate that the JNK1/c-Jun/AP-1 pathway, but not the JNK2 pathway, plays a critical role in nickel-induced COX-2 expression.

Abbreviations: AP-1, activator protein-1; Beas-2B, human bronchoepithelial cells; COX-2, cyclooxygenase-2; JNKs, c-Jun N-terminal kinases; MEFs, mouse embryonic fibroblasts


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The responses of cells to extracellular stimuli are mediated by the activation of protein kinases in the signaling transduction pathways. c-Jun N-terminal kinases (2JNKs) are among the well-characterized mitogen-activating protein kinases (1), and have been shown to be rapidly activated in response to various extracellular stimuli, including growth factors, cytokines and cellular stresses (2). JNKs are encoded by three different genes, jnk1, jnk2 and jnk3 (2,3). The jnk1 and jnk2 genes are ubiquitously expressed. In contrast, the jnk3 gene is selectively expressed in the brain, heart and testis (4). It has been reported that JNK1 and JNK2 play both overlapping and differential roles in certain cellular events, such as proliferation (5), death (6) and tumor promotion (7,8). However, the functional differences between JNK1 and JNK2 still remain largely unknown.

The primary exposure of humans to nickel occurs in occupational settings, such as nickel refineries and stainless steel welding. Also environmental exposure from air pollution is another principal source (9). Epidemiological studies have associated occupational exposure to nickel compounds to elevated incidences of human cancer, such as lung and nasal cancers (10). Nickel has been proposed to contribute to human carcinogenesis by multiple mechanisms (11–13), which include both genetic and epigenetic routes (14–16). A broad spectrum of epigenetic effects of nickel contains the alterations of the related gene expression by DNA hypermethylation, histone hypoacetylation, as well as the activation or silencing of certain genes and transcription factors, especially those involved in tumor initiation and promotion (10,14).

It is widely accepted that chronic inflammation is one of the causes for tumor promotion (16,17). Cyclooxygenase-2 (COX-2), an essential enzyme in inflammatory reactions, has been reported to be constitutively overexpressed in a variety of malignancies and is frequently elevated in human cancers (18–22). However, it remains to be elucidated how external stimuli, such as environmental carcinogens, induce COX-2 expression. In the present study, we demonstrate that nickel exposure induces COX-2 expression at both transcriptional and protein levels. Moreover, the COX-2 induction requires JNK1-, but not JNK2-, associated signaling activation in a c-Jun/AP-1-dependent manner.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture
The human bronchoepithelial cells (Beas-2B) (23), wild-type (WT), JNK1 deficient (JNK1–/–) (24) and JNK2 deficient (JNK2–/–) (25) mouse embryonic fibroblasts (MEFs), as well as their stable transfectants were cultured at 37°C in 5% CO2 incubator with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 25 µg/ml of gentamicin. The cultures were dissociated with trypsin and transferred to new 75 cm2 culture flasks (Fisher) twice a week. DMEM, L-glutamine and gentamicin were from Sigma; and FBS was from Nova-Tech.

Plasmid constructs and transfection
The p-AP-1 Luc plasmid was purchased from Stratagene. The COX-2-luciferase reporter plasmid containing the upstream 5'-flanking region of the COX-2 gene promoter linked to the luciferase reporter was described in the previous study (26). The HA-tagged murine JNK1 and JNK2 full-length cDNAs were constructed into a pcDNA3 expression vector and named as HA-JNK1/pcDNA3 and HA-JNK2/pcDNA3, respectively (27). An expression construct for the c-Jun dominant negative mutant (pcDNA3.1/TAM67) was kindly provided by Dr Tim G.Bowden (College of Pharmacy, University of Arizona, Tucson, AZ) and Dr Matthew Young (The Center for Cancer Research, National Cancer Institute, Frederick, MD) (28). Transfection experiments were performed with Lipofectamine2000 reagent (Invitrogen), following the manufacturer's instructions. Briefly, cells were cultured in 6-well plates until 80% confluence. Five micrograms of the indicated plasmids were mixed with 10 µl of Lipofectamine2000 reagent and then added into cells cultured in serum-free medium. After 6–8 h, the medium was replaced with 10% serum medium. Approximately 30–36 h post-transfection, the cells were digested, re-plated into 75 m2 culture flasks and cultured for 24–28 days for selection. The stable transfectants were established and cultured in antibiotics-free DMEM for at least two passages before performing experiments.

Luciferase reporter assays
A confluent monolayer of cells with AP-1-luciferase reporter or COX-2-luciferase reporter were trypsinized, and 8 x 103 viable cells suspended in 100 µl of 10% serum medium were added to each well of 96-well plates and incubated until 80% confluence. The cells were treated with nickel compounds at the indicated concentrations and for various time periods as described in the figure legends. After nickel treatment, the cells were lysed with 50 µl lysis buffer, and collected to analyze luciferase activity by using a luminometer (Wallac 1420 Victor2 multipliable counter system). The results were expressed as AP-1 activation relative to the medium control (relative AP-1 activation) or COX-2 induction relative to the medium control (relative COX-2 induction) (23,29–31).

RT–PCR
Six or twelve hours after exposed to nickel compounds, the cells were collected and total RNA was extracted by using Trizol reagent (Gibco). The cDNAs were synthesized from 1 µg total RNA by a ThermoScriptTM RT–PCR system (Invitrogen) and then used as templates for the subsequent PCR assay. The PCR primer sequences for mouse COX-2 were 5'-TCC TCC TGG AAC ATG GAC TC-3' (sense) and 5'-GCT CGG CTT CCA GTA TTG AG-3' (antisense) and for human COX-2 were 5'-TGA AAC CCA CTC CAA ACA CA-3' (sense) and 5'-AAC TGA TGC GTG AAG TGC TG-3' (antisense). The internal control mouse ß-actin PCR primers were 5'-GAC GAT GAT ATT GCC GCA CT-3' (sense) and 5'-GAT ACC ACG CT T GCT CTG AG-3' (antisense), and human ß-actin primers were 5'-GCG AGA AGA TGA CCC AGA TCA T-3' (sense) and 5-GCT CAG GAG GAG CAA TGA TCT T-3' (antisense).

Western blotting analysis
Beas-2B, MEFs or their transfectants were cultured in 6-well plates until 80% confluence. The cells were then treated with nickel compounds for various time points as indicated in the figure legends. The treated cells were washed once with ice-cold PBS and extracted with SDS-sample buffer. Whole-cell extracts were sonicated and denatured by heating at 100°C for 5 min. The cell extracts were quantified with a Dc protein assay kit (Bio-Rad), separated on polyacrylamide–SDS gels, transferred and probed with one of the antibodies against phosphor-specific c-Jun, phosphor-specific JNK1/2, non-phosphorylated c-Jun, JNK1/2 (Cell Signaling Technology), COX-2 (Cayman Chemical Co.) and ß-actin (Sigma). Primary antibody-bound proteins were detected by using an alkaline phosphatase-linked secondary antibody and an ECF Western blotting system (Amersham).

Statistical analysis
The Student's t-test was used to determine the significance of differences of COX-2 induction and AP-1 activation between nickel-treated cells and medium control cells or among various cell lines. The differences were considered significant at a P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Nickel exposure induced COX-2 expression in both Beas-2B cells and MEFs
Occupational exposure to nickel via inhalation has been shown to increase the risk of lung and other respiratory cancers (32), and also there is concern that nickel may contribute to the environmental risk of lung cancer as a component of air pollution (33). Recent increasing evidence indicates that constitutive COX-2 overexpression plays an important role in cancer development (34). In order to test whether COX-2 induction is associated with nickel exposure, we treated Beas-2B cells and MEFs with nickel chloride (NiCl2) and nickel sulfide (NiS). As shown in Figure 1A, treatment with either NiCl2 or NiS for 24 or 48 h dramatically induced COX-2 protein expression in both types of cells, and this induction appeared to be in dose- and time-dependent features. Moreover, the mRNA level of COX-2 was also elevated following nickel exposure in Beas-2B cells (Figure 1B) and MEFs (Figure 3A). To investigate whether transcriptional regulation was operative in this COX-2 upregulation, we established the stable transfectants with COX-2-luciferase reporter and found that exposure to nickel compounds led to marked increases in COX-2 promoter-driven luciferase activity (Figure 1C). Moreover, treating MEFs with actinomycin D (Act D) in combination with nickel compounds caused obvious blockage of nickel-induced COX-2 protein expression (Figure 1D). These results indicated that nickel was able to induce COX-2 expression which was largely due to upregulation of transcription in both Beas-2B cells and MEFs.


Figure 1
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  		Fig. 1 Induction of COX-2 expression by nickel exposure in Beas-2B cells and MEFs. (A) Beas-2B cells and MEFs (2 x 105) were seeded into each well of 6-well plates, and exposed to various doses of nickel chloride (NiCl2) or nickel sulfide (NiS) as indicated for 24 or 48 h. The cells were extracted and analyzed by western blotting as described in the Materials and Methods. (B) Beas-2B cells were exposed to NiCl2 (0.5 mM) and NiS (2 µg/cm2) for 12 h and extracted with Trizol for the analysis of COX-2 mRNA by RT–PCR. (C) MEFs COX-2 Luc stable transfectants (8 x 103) were seeded into each well of 96-well plates, and treated with NiCl2 and NiS at the indicated doses for 24 h. The cells were then harvested for analysis of luciferase reporter gene activities as described in the Materials and Methods. The results were expressed as COX-2 induction relative to the medium control (relative COX-2 induction). Each bar indicated the mean and standard deviation of triplicate tests. *P < 0.05, significant increase from medium control. (D) MEFs were pretreated with gene transcription inhibitor Act D for 30 min and then exposed to nickel compounds as indicated for 12 h. The cells were extracted and analyzed by western blotting.

 
JNK1, but not JNK2, was specifically required for the COX-2 induction by nickel exposure
JNKs are encoded by three different genes, namely jnk1, jnk2 and jnk3, and each of them has multiple isoforms generated from their mRNA alternative splicing processes (3). Recent findings have strongly suggested that JNK1 and JNK2 have not only overlapping but also distinct functions under various stresses (5–7,25). To test the involvement of JNKs in the nickel-associated COX-2 induction, we employed specific JNK1 and JNK2 knockout cells to address the question directly. We first exposed WT MEFs to nickel compounds, and found that exposure to nickel led to a marked increase in JNK1/2 phosphorylations, while the total amount of JNK1 and JNK2 proteins were not affected by nickel exposure (Figure 2A), indicating that nickel did induce JNK1 and JNK2 activation. To figure out whether activation of JNK1 and/or JNK2 was necessary for the COX-2 induction, we next compared the COX-2 induction among WT, JNK1–/– and JNK2–/– MEFs. Initially, the JNK1 or JNK2 deficiencies were confirmed by western blotting. As shown in Figure 2B, the basal JNK1 protein expression was deficient in JNK1–/– MEFs, and JNK2 protein was deficient in JNK2–/– MEFs, as compared with those in WT MEFs. It may be noted that in JNK1–/– MEFs there was a weak band expressed at the 46 KD position, which was likely the p46 isoform of JNK2 as previously reported (2). Interestingly, the nickel-induced COX-2 protein expression was totally impaired in JNK1–/– MEFs as compared with that in WT and JNK2–/– MEFs (Figure 2C). So it was suggested that JNK1, but not JNK2, was selectively required for COX-2 induction by nickel compounds. Consistently, the nickel-induced COX-2 promoter-driven luciferase activity was also blocked in JNK1–/– MEFs (Figure 2D), indicating that the reduction of COX-2 induction in JNK1–/– MEFs was due to the impairment of COX-2 gene transcription, which was further confirmed by the data from RT–PCR (Figure 3A). However, in JNK2–/– cells the induction of COX-2 gene transcription (Figure 2D) as well as mRNA expression (Figure 3A) was not obviously affected compared with that in WT cells.


Figure 2
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  		Fig. 2 JNK1, but not JNK2, was specifically required for the COX-2 induction. (A) WT MEFs were seeded into each well of 6-well plates (2 x 105), and exposed to nickel compounds as indicated for 6 h. The cells were extracted and subjected to western blotting analysis for detection of JNK1/2 status. (B) The gene deficiency of JNK1–/– and JNK2–/– MEFs was identified by western blotting analysis. (C) WT, JNK1–/– and JNK2–/– MEFs were exposed to nickel compounds for 12 h, induction of COX-2 and phosphorylation of JNK1/2 were detected by western blotting. (D) WT, JNK1–/– and JNK2–/– MEFs COX-2 Luc stable transfectants were seeded into each well of 96-well plates and treated with nickel compounds. The cells were then harvested for analysis of luciferase reporter gene activities. The results were expressed as COX-2 induction relative to the medium control (relative COX-2 induction). Each bar indicated the mean and standard deviation of triplicate tests. *P < 0.05, significant increase from medium control. {clubsuit}P < 0.05, significant decrease from the induction of WT cells.

 


Figure 3
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  		Fig. 3 JNK1 reconstitution rescued COX-2 induction in JNK1–/– MEFs. (A) WT, JNK1–/–, JNK2–/– MEFs as well as their transfectants (2 x 105) were seeded into each well of 6-well plates, and exposed to 0.5 mM of NiCl2 as indicated for 6 h (left panel) or indicated time (right panel). The cells were extracted and subjected to RT–PCR to determine the mRNA level of COX-2. (B and C) The indicated cells were treated with nickel compounds for 24 h and extracted for western blotting analysis.

 
To extensively confirm the role of JNK1 in nickel-induced COX-2 expression, and to rule out the possibility of other gene changes rather than JNK1 deficiency during the establishment of the cells, a plasmid encoding the HA-tagged full-length JNK1 was stably transfected into JNK1–/– MEFs and selected as a mass culture pool (35). As expected, the reconstitution of JNK1 expression restored the nickel-induced COX-2 expression, at both the mRNA and protein levels (Figures 3A and B). To exclude the non-specific effect of ectopic expression of JNK protein in their deficient MEFs, JNK2 was also reconstituted into JNK2–/– MEFs. However, the reconstituted JNK2 did not cause any observed differences on COX-2 induction compared with that in the JNK2–/– parental cells (Figure 3C). These data provided strong evidence that impairment of JNK1 blocked nickel-induced COX-2 expression, suggesting that JNK1, but not JNK2, was specifically required for COX-2 induction by nickel compounds. Therefore, in the following studies, we focused on the investigations of the relevance of JNK1 to COX-2 induction.

c-Jun/AP-1 was the JNK1 downstream mediator for nickel-associated COX-2 induction
To determine whether the JNK1 downstream pathway was responsible for the COX-2 induction by nickel compounds, the major downstream target, c-Jun, was then tested for its activation. As shown in Figure 4A, exposure of WT MEFs to nickel compounds led to marked increases in c-Jun phosphorylation, suggesting that c-Jun was activated by nickel treatment. c-Jun was a key component of AP-1, which acted as a ‘master switch’ for a variety of stimuli by the regulation of target gene transcription (36). The transcription activity of AP-1 was also evaluated in WT MEFs. As shown in Figure 4B and C, AP-1 was activated significantly after nickel exposure in both dose- and time-dependent manners. Then, the status of c-Jun phosphorylation and AP-1 transactivation were determined in nickel-treated JNK1–/– MEFs. As shown in Figure 5A and B, both c-Jun phosphorylation and AP-1 transactivation were evidently blocked in JNK1–/– MEFs as compared with those in WT MEFs. So the data suggest that the abrogation of c-Jun/AP-1 transcription activity due to JNK1 knockout might account for the absence of COX-2 induction in nickel response. To this end, TAM67, a well-characterized c-Jun dominant negative mutant, was transfected into WT cells, and the stable transfectants were established and identified by pan-c-Jun antibody (Figure 6A). As shown in Figure 6B and D, ectopic expression of TAM67 obviously blocked c-Jun phosphorylation and AP-1 activity induced by nickel compounds. More importantly, it also resulted in a remarkable reduction of COX-2 promoter transcription activity as well as protein expression (Figure 6E and C). All the results strongly demonstrate that c-Jun/AP-1 is a downstream mediator for nickel-associated COX-2 induction in the JNK1-dependent pathway.


Figure 4
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  		Fig. 4 c-Jun and AP-1 were activated in response to nickel compounds. (A) WT MEFs (2 x 105) were seeded into each well of 6-well plates, and exposed to nickel compounds as indicated for 12 h. The cells were then extracted, and the phosphor-specific and non-phosphorylated c-Jun protein levels were detected by western blotting. (B and C) 8 x 103 cells of WT MEFs AP-1 Luc mass1 were seeded into each well of 96-well plates, and treated with NiCl2 and NiS at the indicated doses or time periods. The cells were then harvested for analysis of luciferase reporter gene activities. The results were expressed as AP-1 activation relative to the medium control (relative AP-1 activation). Each bar indicated the mean and standard deviation of triplicate tests. *P < 0.05, significant increase from medium control.

 


Figure 5
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  		Fig. 5 Activation of c-Jun/AP-1 in response to nickel compounds was impaired by JNK1 deficiency. (A) WT and JNK1–/– MEFs (2 x 105) were seeded into each well of 6-well plates, and treated with NiCl2 and NiS for different time periods and c-Jun basal and phosphorylation levels were detected by western blotting. (B) WT MEFs AP-1 Luc mass1 and JNK1–/– MEFs AP-1 Luc mass1 (8 x 103) were seeded into each well of 96-well plates, and were treated with 0.25 mM of NiCl2 and 2 µg/cm2 of NiS for 24 h. The cells were then harvested, extracted with lysis buffer and the luciferase activity was measured. The results were expressed as AP-1 activation relative to the medium control (relative AP-1 activation). Each bar indicated the mean and standard deviation of triplicate tests. *P < 0.05, significant increase from medium control. {clubsuit}P < 0.05, significant decrease from the activation of WT cells.

 


Figure 6
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  		Fig. 6 Blockage of nickel-induced COX-2 induction by overexpression of TAM67. (A) Expression of TAM67 was identified by western blotting assay. (B, C) WT and WT/TAM67 MEFs were cultured into each well of 6-well plates and treated with nickel compounds as indicated for 12 h (B) or 24 h (C). The cells were extracted, and western blotting was carried out as described in Materials and Methods. (D and E) WT AP-1 Luc mass1, WT/TAM67 AP-1 Luc mass1 (D) and WT COX-2 Luc mass1, WT/TAM67 COX-2 Luc mass1 (E) were seeded into each well of 96-well plates, and treated with 0.25 mM of NiCl2 and 2 µg/cm2 of NiS for 24 h. The cells were then extracted with lysis buffer, and the luciferase activity was measured using the Promega luciferase assay kit. The results were expressed as relative AP-1 activation or relative COX-2 induction. Each bar indicated the mean and standard deviation of triplicate tests. *P < 0.05, significant increase from the medium control. {clubsuit}P < 0.05, significant decrease from the activation of parental cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Occupational exposure to nickel has been associated with an increased risk of lung and other respiratory cancers (10), and there is also concern that environmental exposure to nickel via air pollution may also contribute to an increased cancer risk (37). Overexpression of COX-2 is observed in a variety of human malignancies, especially in primary lung carcinoma (19). It is of interest and importance to determine whether nickel compounds are able to induce COX-2 expression. If it does, which signaling pathway is involved. This study demonstrated that nickel exposure led to a marked increase of COX-2 expression at both the transcriptional and protein levels in Beas-2B cells and MEFs. The induction of COX-2 by nickel was largely due to upregulation of COX-2 transcription, which was specifically dependent on the JNK1-signaling pathway because JNK1 deficiency impaired this induction, but JNK2 deficiency did not show any reductive effects. Furthermore, re-constitutional expression of JNK1 in its deficient MEFs restored the nickel-induced COX-2 transcription and protein expression. Finally, c-Jun/AP-1 was identified as the JNK1 downstream mediator responsible for nickel-induced COX-2 induction. We demonstrated that JNK-, but not JNK2-, mediated signaling pathway specifically participated in the COX-2 induction in a c-Jun/AP-1-dependent manner.

Multiple lines of evidence suggest that COX-2 plays an important role in carcinogenesis (21). Overexpression of COX-2 was detected in both transformed cells (38) and various cancer tissues, including lung cancer (22), colorectal adenomas (39) and adenocarcinoma (40). The COX-2 specific inhibitors, such as celecoxib and rofecoxib, have proven to be beneficial in both the prevention and therapy of certain cancers (18–22,41–43). Therefore, investigating nickel-associated COX-2 induction might help us to clearly understand the mechanisms of nickel in carcinogenesis. To our knowledge, this is the first report to demonstrate that nickel compounds lead to the substantial increase of COX-2 transcription and protein expression.

Mammalian cells respond to extracellular stimuli by activating signaling cascades that are mediated by the MAP kinase family, which include ERKs, JNKs and p38 kinase (44). MAP kinases act by phosphorylating various substrates including transcription factors, which in turn, regulate the expression of a specific set of genes, and, thus, can mediate a specific genetic response to the stimulus (45). JNK1 and JNK2 are shown to have similar roles in regulating T cell apoptosis and proliferation (25). However, in response to UV irradiation, JNK1 and JNK2 display quite distinct effects on cell survival (6,46). Similarly, in the aspect of COX-2 regulation, JNK1 and JNK2 seem to play different roles according to extracellular stimuli (47,48). For example, COX-2 induction by hypertonicity has been shown to selectively require JNK2 activation, but not JNK1 activation (47,48). Therefore, it is necessary to investigate the potential involvement of different JNKs isoforms in COX-2 regulation by nickel exposure. Our results indicated that the nickel-associated COX-2 induction was specifically blocked in JNK1–/– MEFs due to blockage of COX-2 transcription, while JNK2 knockout did not show any inhibitory effect on COX-2 transcription or protein expression. The specific role of JNK1 in COX-2 induction was confirmed by re-constitutional JNK1 expression in its deficient MEFs. Collectively, our findings demonstrated that JNK1, but not JNK2, was critical for the nickel-associated COX-2 induction. Boislève et al. (49) demonstrated that in the dendritic cell (DC) derived from CD34+ cells of human cord blood, JNK inhibition by its chemical inhibitor SP600125 strongly affected the expression of mature molecules (CD83, CD86 and CCR7) induced by NiSO4, suggesting that JNKs might be essential for the nickel-induced maturation process of DC, which is an important event involved in inflammation responses upon nickel exposure. Since chronic inflammation responses require COX-2 expression and DC maturation, which are associated with cancer development (49,50), we presumably anticipate that JNKs might participate in the carcinogenic effect of nickel compounds.

It has been well documented that COX-2 transcription is dependent on the activity of various transcription factors including AP-1 (22). Our data showed that the c-Jun/AP-1 pathway was activated when WT MEFs were exposed to nickel compounds, but was impaired in JNK1–/– MEFs, suggesting that c-Jun/AP-1 was the downstream target of JNK1 in nickel response. In additional, abrogation of c-Jun/AP-1 activation by ectopic expression of TAM67 resulted in the obvious impairment of COX-2 induction by nickel, further demonstrating that the critical role of the JNK1/c-Jun/AP-1 pathway in nickel-associated COX-2 induction. It needs to be mentioned that besides the inducible COX-2 protein level discussed above, the basal COX-2 protein expression level in JNK1–/– cells was also much lower than that in the WT and JNK2–/– cells, which was consistent with the weaker c-Jun basal expression level in JNK1–/– cells. Therefore, it further broadened our conclusion that JNK1/c-Jun was not only essential for COX-2 induction but also important for the basic COX-2 expression in cells in the physiological conditions.

In conclusion, our results strongly showed that COX-2 induction was implicated in the cellular response to nickel exposure, and it was specifically dependent on JNK1, but not JNK2 activation, even though nickel was able to activate both JNK1 and JNK2. The c-Jun/AP-1 was at least one of the major JNK1 downstream pathways responsible for COX-2 induction. Understanding the association of COX-2 in nickel response may not only be helpful for the elucidation of the mechanisms involved in nickel carcinogenesis, but may also provide some information for using JNK1 and COX-2 (51,52) as targets for the prevention, as well as therapy, against the carcinogenic effects of nickel compounds in the nickel pollution affected population.


   Acknowledgments
 
We thank Dr Tim G.Bowden from the College of Pharmacy, University of Arizona, Tucson, AZ, and Dr Matthew Young from the Center for Cancer Research, National Cancer Institute, Frederick, MD, for their generous gift of pcDNA3.1/TAM67 expression construct. We thank Juliana Powell for her editorial assistance. This work was supported in part by grants from NIH/NCI (R01 CA094964, R01 CA112557 and R01 CA103180) and NIH/NIEHS (R01 ES012451).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 28, 2006; revised September 18, 2006; accepted September 27, 2006.


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