Differential requirement of signal pathways for benzo[a]pyrene (B[a]P)-induced nitric oxide synthase (iNOS) in rat esophageal epithelial cells
Jingyuan Chen 1, 2,
,
Yan Yan 1, 2,
,
Jingxia Li 1,
,
Qian Ma 1,
Gary D. Stoner 3,
Jianping Ye 4 and
Chuanshu Huang 1, *
1 Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA, 2 Department of Occupational and Environmental Health Sciences, Fourth Military Medical University, Xi'an, Shaanxi, 710032, China, 3 Division of Environmental Health Sciences, School of Public Health, The Ohio State University, Columbus, OH 43210, USA and 4 Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA 70808, USA
* To whom correspondence should be addressed. Tel: +1 845 731 3519; Fax: +1 845 351 2118; Email: chuanshu{at}env.med.nyu.edu
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Abstract
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Overexpression of inducible nitric oxide synthase (iNOS) has been reported in several human cancers, including esophageal squamous cell carcinoma (SCC). Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon carcinogen found in tobacco smoke and in the environment, induces cancer in multiple organ sites in animals and may be a causative agent for certain human cancers, such as esophageal cancer. In the present study, the effects of B[a]P on the induction of iNOS and the signaling pathways that lead to the induction were investigated in cultured rat esophageal epithelial (RE-149) cells. Treatment of RE-149 cells with B[a]P led to a marked increase in the expression of iNOS. The induction of iNOS by B[a]P was found to occur through an extracellular signal-regulated protein kinases (ERKs)-dependent pathway, since inhibition of ERKs by either pretreatment of RE-149 cells with PD98059, an inhibitor of ERKs upstream kinase MEK1/2, or overexpression of DN-ERK2, blocked the induction of iNOS by B[a]P. Furthermore, impairing nuclear factor-
B (NF
B) activation by either NEMO-BDBP, an NF
B specific inhibitor, or overexpression of DN-I
B
or IKK-KM markedly inhibited the expression of B[a]P-induced iNOS, suggesting that the NF
B pathway is also required for the induction of iNOS by B[a]P. In addition, treatment of RE-149 cells with either SB202190, a p38 kinase inhibitor, or c-JunN-terminal kinase inhibitor II, resulted in an increased induction of iNOS. Pretreatment of RE-149 cells with wortmannin, a PI-3K inhibitor, or with rapamycin, an mTOR/p70S6K pathway inhibitor, had no effect on the expression of iNOS. These results suggest that B[a]P initiates the signaling pathways leading to the induction of iNOS in cultured rat esophageal epithelial cells. In view of the potential role of iNOS in the development of esophageal SCC in humans, we speculate that the induction of iNOS by B[a]P may be one mechanism by which B[a]P could produce carcinogenic effects in the human esophagus.
Abbreviations: B[a]P, benzo[a]pyrene; B[a]PDE, (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide; CDE, chrysene-1,2-diol-3,4-epoxide; DMSO, dimethyl sulfoxide; ERKs, extracellular signal-regulated protein kinases; FBS, fetal bovine serum; I
B
, inhibitory subunit kappa-B; IKKß, I
B kinase ß; iNOS, inducible nitric oxide synthase; JNKs, c-Jun N-terminal kinases; 5-MCDE, (±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide; MAPKs, mitogen-activated protein kinases; NF
B, nuclear factor-
B; PAHs, polycyclic aromatic hydrocarbons.
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Introduction
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Esophageal cancer is a significant health problem throughout the world, and is the seventh leading cause of cancer death in males in USA (1). Epidemiologic studies indicate that cigarette smoke and alcohol are important etiologic agents for esophageal squamous cell carcinoma (SCC) (2,3). Cigarette smoke contains polycyclic aromatic hydrocarbons (PAHs), which may contribute to the etiology of the disease. Moreover, analysis of raw and cooked food obtained from Linxian, China, a high risk area for esophageal SCC, revealed the presence of high levels of the PAHs, benzo[a]pyrene (B[a]P) (4). Samples of urine obtained from Linxian residents contained high levels of 1-hydroxypyrene, a urinary metabolite of PAHs (5). In addition, tissue samples of human esophagus obtained from the Linxian residents were stained positively for nuclear PAHsDNA adducts compared with esophageal tissues from USA (6). Results from animal bioassays also suggest that exposures to B[a]P produce carcinogenic effects in the esophagus (7,8). Dunham et al. (8) reported that the instillation of B[a]P into the diet of hamsters twice a week for 5 weeks produced cellular atypia, papillomatosis and papillary carcinomas in the esophagus of hamsters. Repeated intracolonic instillation of Swiss mice with B[a]P for up to 10 weeks produced neoplasms in the esophagus (7). B[a]P administered to female B6C3F1 mice in the diet also showed an induction of esophageal papillomas and/or carcinomas (911). Therefore, B[a]P may be an important carcinogen in the development of esophageal tumors.
Inducible nitric oxide synthase (iNOS) is one of the three isoforms of nitric oxide synthase, which catalyze the oxidative deamination of L-arginine to produce citrulline and nitric oxide. Nitric oxide reacts with superoxide anion to form the highly reactive oxidant, peroxynitrite (1216). Several studies have found that iNOS is overly expressed in esophageal SCC of the human esophagus, as well as in adenocarcinoma and its precursor lesion, Barrett's esophagus (1719). The overexpression in Barrett's esophagus, and in adenocarcinomas arising from Barrett's, suggests that iNOS plays a role in the multistage development of esophageal cancer.
In the present study, we determined whether treatment of cultured rat esophageal epithelial cells with PAHs would lead to an increase in the induction of iNOS and, if so, the signaling pathways that might be involved in this induction. Our results demonstrated that the exposure to PAHs could result in an induction of iNOS in esophageal epithelial cells and that this induction occurs through extracellular signal-regulated protein kinases (ERKs)- and nuclear factor-
B (NF
B)-dependent pathways.
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Materials and methods
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Cell culture
The rat esophageal epithelial cell line, RE-149, has the potential for prolonged subculture in vitro (>150 population doublings); however, they are non-tumorigenic following xenotransplantation to either syngeneic 1-day-old rats or athymic, nude mice (20). Since RE-149 cells produce high molecular weight keratins typical of keratins seen in human esophagus (G.Stoner, unpublished data), they are considered to exhibit the differentiative properties of squamous epithelial cells of the human esophagus. The cells were cultured routinely in complete PFMR-4 medium, which consists of Ham's F-12 medium (Calbiochem, San Diego, CA) supplemented with 2% fetal bovine serum (FBS), 2% MEM amino acid solution (GIBCO BRL, Rockville, MD), 1% MEM non-essential amino acid solution (GIBCO BRL), 0.1% trace elements A (Cellgro, Herndon, VA), 0.1% trace elements B (Cellgro), 5 ng/ml epithelial growth factor (EGF, Sigma, St Louis, MO), 2 µg/ml insulin (Sigma), 5 µM hydrocortisone (Sigma), 144 ng/ml phosphoethanolamine, 10 mM HEPES, 100 µM CaCl2, 2 mM L-glutamine, and 1% penicillin/streptomycin (GIBCO BRL). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in the air.
Reagents and plasmids
B[a]P, (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE), chrysene-1,2-diol-3,4-epoxide (CDE) and (±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) were synthesized by Dr Shantu Amin of the Institute for Cancer Prevention (Valhalla, NY) as described (2123). All these compounds were dissolved in dimethyl sulfoxide (DMSO) to make 2 mM stock concentration. The PI-3K inhibitors (wortmannin and Ly294002), MEK1/2 inhibitor (PD98059), p38 kinase inhibitor (SB202190) and c-Jun N-terminal kinases (JNKs) inhibitor (JNK inhibitor II) were purchased from Calbiochem (La Jolla, CA). Phospho-specific antibodies against various phosphorylated sites of ERKs, p38 kinase and inhibitory subunit kappa-B (I
B
), were from Cell Signaling (Beverly, MA). A dominant negative mutant I
B
was purchased from Upstate Biotechnology (Lake Placid, NY). Dominant negative mutant I
B
is a S32A/S36A mutation that renders I
B
non-phosphorylatable, and consequently not degradable. It therefore stays constitutively in a complex with NF
B and completely ablates the NF
B signaling in cells (24,25). A dominant negative mutant I
B kinase ß (IKKß) (pCR-Flag-IKKß-KM) was a gift from Dr Hiroyasu Nakano, Juntendo University, Tokyo, Japan. IKK-KM is a kinase inactive mutant of murine IKKß (IKK-KM), in which a lysine at the ATP binding site in the kinase domain was substituted by an alanine (26,27). Cotransfection of IKK-KM also inhibited the NF
B-dependent reporter gene activity (26). Dominant negative mutant of ERK2 was a generous gift from Dr Melanie H.Cobb, which are K52R mutations and block ERKs activation as shown in previous studies (28,29).
Generation of RE-149 cell lines stably transfected with the iNOSluciferase reporter gene
The iNOSluciferase reporter plasmid containing a 7.2 kb fragment of the upstream 5'-flanking region of the human iNOS gene linked to a luciferase reporter gene was kindly provided by Dr D.A.Geller (30). RE-149 cells were cultured in PFMR-4 medium in 6-well plates until they reached 8590% confluence, after which the medium was replaced with serum-free PFMR-4. Five microgram of iNOSluciferase reporter plasmid DNA, 1 µg of CMV-neo vector and 20 µl of LipofectAMINE reagent (GIBCO BRL) was added to each well to initiate transfection. After 1012 h, the serum-free PFMR-4 was replaced with PFMR-4 containing 2% FBS. Approximately 3036 h after the beginning of the transfection, the cells were digested with 0.033% trypsin and the cell suspensions were plated onto 25 ml culture flasks. The cells were cultured for 2845 days in a medium containing 600 µg/ml of G418 (Gibco BRL) to select the transfectants. Stable transfectants were identified by measuring the basal level of luciferase activity. Stably transfected cells, designated as RE-149 iNOS mass1, were cultured in G418-free PFMR-4 medium for at least two passages before each experiment.
Assays for iNOS induction
Confluent monolayer of RE-149 iNOS mass1 was trypsinized and 8 x 103 viable cells, suspended in 100 µl of PFMR-4 medium supplemented with 1% FBS, were added to each well of 96-well plates. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2 in the air. After the cell density reached 8090%, the medium was replaced with 100 µl of Ham's F-12 medium supplemented with only 0.1% FBS and 2 mM L-glutamine. Twelve hours later, the cells were exposed to either B[a]P, B[a]PDE, CDE or 5-MCDE at concentrations necessary for the induction of iNOS. After different periods of incubation with the individual PAHs, 50 µl of lysis buffer was added to each well for 30 min at 4°C and the luciferase activity was determined by a luciferase assay (Promega, Madison, WI) using a luminometer (Wallac 1420 Victor 2 multilable counter system). Results were expressed as the induction of iNOS in cells cultured in medium containing PAH relative to the control medium containing DMSO only (Relative iNOS induction).
Western blot
RE-149 stable transfectants were cultured in monolayers in 6-well plates. After the cell density reached 7080%, the medium was replaced with 0.1% FBS MEM and cells were incubated for 45 h. Cells were then incubated in MEM containing 0.1% FBS for 34 h at 37°C. Cells were then incubated with various doses of B[a]P for 6 h and extracted with TrisGlycine SDS sample buffer (Invitrogen, Carlsbad, CA). Western blots were performed with either phospho-specific antibodies or non-phosphorylated antibodies against various kinases, including ERKs, p38 kinase and I
B
. 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, Piscataway, NJ).
Statistical analysis
The induction of iNOS in RE-149 mass1 cells treated with PAHs was compared with that in cells treated with DMSO only by the Student's t-test. Differences in the induction of iNOS were considered significant at a P
0.05.
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Results
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Induction of iNOS in RE-149 mass1 cells by PAHs
Since the overexpression of iNOS has been reported in esophageal SCC and PAHs may be a causative agent for esophageal cancer (1719), the ability of different PAHs to induce iNOS in cultured RE-149 cells stably transfected with the iNOS luciferase reporter gene was determined. Results showed that exposure of cells to non-cytotoxic concentrations of either B[a]P or CDE induced a marked increase in the expression of iNOS, while B[a]PDE and 5-MCDE had no effect on the induction of iNOS (Figure 1). The induction of iNOS expression by B[a]P and CDE was confirmed by both time-course and doseresponse studies (Figure 2a and b). However, the ability of CDE to induce iNOS is much lower compared with that of B[a]P. These results suggest that among the four PAHs tested, B[a]P is a potent inducer of the expression of iNOS in RE-149 cells, even though CDE exposure is also able to increase the expression of iNOS. In addition, induction of iNOS by B[a]P and CDE, but not by B[a]PDE and 5-MCDE, has also been found in mouse epidermal Cl41 cells, while Cox-2 induction has shown opposing effects (C. Huang and J. Li, data not shown).

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Fig. 1. Effects of PAHs on iNOS induction. RE-149 iNOS mass1 cells (8 x 103) were seeded into each well of 96-well plates, and cultured in PFMR-4 medium containing 2% FBS at 37°C. After the cell density reached 8090%, cells were treated with B[a]P, B[a]PDE, CDE or 5-MCDE at the concentrations indicated. After 48 h, the cells were extracted with lysis buffer, and luciferase activity was measured using the Promega luciferase assay kit as described in the previous studies (76,77). Results are presented as iNOS induction in treated versus untreated control (relative iNOS induction). Each bar indicates the mean and SD of four repeat wells. The asterisk indicates a significant increase relative to the DMSO control (P < 0.05).
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Fig. 2. Time-course and dose-dependent iNOS induction by PAHs. RE-149 iNOS mass1 cells (8 x 103) were seeded and cultured as described in Figure 1. (a) For time-course studies, the cells were treated with 1 µM of either B[a]PDE, B[a]P, CDE or 5-MCDE for the time period indicated. The luciferase activity was measured, and results are presented as relative iNOS induction. (b) For doseresponse studies, the cells were treated with different concentrations of B[a]P, B[a]PDE, CDE or 5-MCDE for 48 h. Each bar indicates the mean and SD of four repeat wells. The asterisk indicates a significant increase from DMSO control (P < 0.05).
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Role of ERKs in iNOS induction by B[a]P
Previous studies have shown that the mitogen-activated protein kinase (MAPKs) pathway is involved in the regulation of iNOS gene expression (31). To evaluate the role of MAPKs activation in the induction of iNOS by B[a]P, specific inhibitors for MAPKs pathways, including MEK1/2-ERKs pathway (PD98059), p38 kinase pathway (SB202190) or JNKs pathway (JNK inhibitor II) were used. As shown in Figure 3, pretreatment of RE-149 iNOS mass1 cells with 100 µM (non-cytotoxic concentration) of PD98059 dramatically reduced the expression of B[a]P-induced iNOS, while SB202190 (1 µM) and JNK inhibitor II (50 µM) had no inhibitory effects on the induction of iNOS (Figure 3). Results from the time-course and doseresponse studies showed that PD98059 inhibited the expression of B[a]P-induced iNOS in a dose- and time-dependent manner (Figure 4a and c), whereas both SB212090 and JNK inhibitor II slightly increased the expression of iNOS (Figure 4). Furthermore, the essential role of ERKs in B[a]P-induced expression of iNOS was demonstrated using a dominant negative ERK2 mutant (DN-ERK2). Overexpression of DN-ERK2 blocked the activation of B[a]P-induced ERKs (Figure 5a), while it did not show any inhibitory effect on B[a]P-induced p38K activation (Figure 5a). Specific impairment of ERKs activation by an overexpression of DN-ERK2 resulted in the total loss of induction of iNOS by B[a]P in RE-149 cells (Figure 5bd). In addition, wortmannin, a PI 3-kinase specific inhibitor, and rapamycin, a mTOR/p70S6K pathway inhibitor, did not inhibit the induction of iNOS by B[a]P (Figures 3, 6a and b). These data indicate that the MEK1/2-ERKs pathway is one of the major cell signaling transduction cascades leading to the expression of B[a]P-induced iNOS in RE-149 cells. It should be noted here that the concentrations of all the chemical inhibitors used in this study did not have any observed cytotoxic effect on RE-149 cells. These concentrations are widely used in our laboratory and other laboratories for published studies, in which they have shown their specificity for their targets (29,3235).

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Fig. 3. Effects of PD98059, SB202190, JNK inhibitor II, Wortmannin, Rapamycin and NEMO-BDBP on iNOS induction by B[a]P. RE-149 iNOS mass1 cells were seeded and cultured in 96-well plates. Cells were pretreated with various inhibitors as indicated for 30 min, and then exposed to B[a]P (2 µM) for the induction of iNOS. After 48 h, the luciferase activity was measured, and the results are presented as relative iNOS induction. Each bar indicates the mean and SD of four repeat wells. The asterisk indicates a significant decrease from B[a]P treated control (P < 0.05).
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Fig. 4. iNOS induction by B[a]P is inhibited by PD98059, and not by SB202190 and JNK inhibitor II. RE-149 iNOS mass1 cells (8 x 103) were seeded and cultured in 96-well plates. Cells were pretreated with (a and c) PD98059, SB202190, or (b and c) JNK inhibitor II for 30 min and then exposed to B[a]P (2 µM) for various time periods as indicated. The luciferase activity was measured as described in Figure 2. The results are presented as relative iNOS induction and each bar indicates the mean and SD of four repeat wells. The asterisk indicates a significant decrease from B[a]P treated cells (P < 0.05). The symbol ( ) indicates a significant increase from B[a]P control (P < 0.05).
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Fig. 6. No effect of wortmannin and rapamycin on iNOS induction by B[a]P. Re-149 iNOS mass1 cells were cultured in 96-well plates. The cells were pretreated with wortmannin or rapamycin for 30 min and then exposed to B[a]P (2 µM). Cells were extracted with lysis buffer at 48 h after exposure (a) or at the time points as indicated (b). The luciferase activity was measured and the results are presented as relative iNOS induction.
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NF-
B pathway is also required for iNOS induction by B[a]P in Re-149 cells
The expression of iNOS has been shown to be regulated through the NF-
B pathway in some cells (36,37). To investigate the possible involvement of NF-
B in the induction of B[a]P-induced iNOS in RE-149 mass1 cells, we pretreated the cells with NEMO-BDBP (12.550 µM), an NF-
B specific peptide inhibitor, and with its negative control peptide, NEMO-BDBP-NC. As shown in Figures 3 and 7a, pretreatment of the cells with NEMO-BDBP resulted in a dramatic inhibition of the expression of B[a]P-induced iNOS in a dose-dependent manner (P < 0.05), while treatment with NEMO-BDBP-NC had no inhibitory effect (Figure 7a). To further explore the role of NF-
B pathway in the expression of B[a]P-induced iNOS, we also established RE-149 stable transfectants with DN-I
B
and IKK-KM. Dominant negative mutant I
B
is a S32A/S36A mutation that renders I
B
non-phosphorylatable, and consequently not degradable. It therefore stays constitutively in a complex with NF
B and completely ablates the NF
B signaling in cells (24,25). IKK-KM is a kinase inactive mutant of murine IKKß (IKK-KM), in which a lysine at the ATP binding site in the kinase domain was substituted by an alanine (26,27). Co-transfection of IKK-KM also inhibited the NF
B-dependent reporter gene activity (26). Therefore, the I
B
phosphorylation induced by B[a]P was impaired in those RE-149 transfectants (Figure 7b). Blockade of IKK/I
B
/NF
B by either overexpression of IKK-KM or DN-I
B
led to a dramatic reduction in the induction of iNOS by B[a]P (Figure 5bd). These results strongly demonstrated that the IKK/I
B
/NF
B pathway is also critical for the expression of B[a]P-induced iNOS in RE-149 cells.
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Discussion
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In the present study, we investigated the effects of PAHs on the induction of iNOS and the signaling pathways mediating this induction in RE-149 cells. Treatment of the cells with B[a]P led to a marked increase in the expression of iNOS, while exposure of the cells to CDE resulted in a marginal induction. However, neither B[a]PDE nor 5-MCDE showed any induction of iNOS. Further studies indicated that induction of iNOS by B[a]P in the cells is mediated through both MEK/ERKs- and IKK/I
B
/NF
B-dependent pathways.
PAHs are those compounds that can be absorbed into the body through the skin, lungs and gastrointestinal tract from various sources, such as occupational and environmental exposure, mainstream and sidestream tobacco smoke, as well as dietary intakes (3844). However, it is difficult to determine the relevant concentrations in blood and tissue owing to their very rapid metabolism in the body, and lack of sensitive and reliable detecting methods (4044). In addition, since PAHs are never present in the exposure environment as a single component, but as mixtures of several hundred compounds, this poses another difficulty to determine the physiologically relevant concentrations for PAHs exposure. Although the relative contribution to human exposure of each component of PAHs may vary from source to source, and the relevant concentrations of each component in PAHs are unclear, there is still some evidence to estimate the human exposures. For example, total PAHs were found to be present in barbecued meat at levels up to 164 p.p.b. with B[a]P levels as high as 30 p.p.b. (45). The daily intake of B[a]P from the diet is estimated to range from 120 to 2800 ng/day (46). Mainstream smoke of a cigarette was reported to yield
2040 ng B[a]P, leading to an intake of
400800 ng/day for a pack-a-day cigarette smoker (47). A B[a]P concentration of 1 µM was used for the maximal induction of iNOS in the present studies. This concentration may not be achievable in vivo; however, it did not show any cytotoxicity to RE-149 cells, and was much lower compared with the concentrations used in many previous studies (4850).
PAHs are important environmental contaminants and potentially hazardous to human health (11). PAHs and their derivatives appear to be involved in the development of smoking-associated cancers in human, and they induce a spectrum of tumor types in animal models. For example, B6C3F1 mice developed tumors in multiple organ sites when fed with coal tar containing various PAHs (9). In addition, B[a]P alone induced carcinomas of the esophagus in B6C3F1 mice (10). Thus, B[a]P is a complete carcinogen of the esophagus. The PAHs, B[a]PDE, CDE and 5-MCDE, have been shown to induce mutations in oncogenes and tumor suppressor genes through the formation of DNA adducts, which leads to the initiation of tumor development (2,3). While the tumor initiation effects of PAHs are well demonstrated, the mechanisms by which PAHs induce the activation of signal transduction pathways leading to gene expression are unclear. Results from the present study showed that B[a]P could markedly induce the expression of iNOS in RE-149 cells, while CDE had only a marginal effect. In contrast, B[a]PDE and MCDE had no effect on the expression of iNOS. These results indicate that B[a]P is more active than other PAHs in inducing iNOS in RE-149 cells. Since it is well known that the DNA binding potential and adduct formation of B[a]PDE is much higher than that of B[a]P, we assume that the induction of iNOS by B[a]P is not associated with overall DNA damage. An alternative explanation is that B[a]P exposure leads to the induction of p450 enzymes, which in turn induces the reactive oxygen species (ROS), and consequently leads to the induction of iNOS. This notion was supported by previous findings that the expression of IL-1-induced iNOS was totally impaired by pretreatment of cells with SOD in bovine articular chondrocytes (51), and that ROS was generated in the metabolic sequence of B[a]P converted to B[a]PDE (52).
The family of nitric oxide synthases (NOS) comprises three isoforms, including iNOS, endothelial NOS (eNOS) and neuronal NOS (nNOS) (12). Regulation of the expression of eNOS and nNOS depends on the intracellular calcium, while the induction of iNOS is not associated with intracellular calcium. NOS can catalyze the oxidative deamination of L-arginine to produce nitric oxide and citrulline. Nitric oxide has multifaceted roles in physiological and pathophysiological processes. Nitric oxide is capable of producing DNA damage through its conversion to peroxynitrite, as well as inhibiting DNA synthesis, ribonucleotide reductase and the DNA repair enzyme, poly(ADP-ribose) polymerase (53,54). All these biological effects may lead to initiation of a tumor (12). In addition, it has been shown that nitric oxide is involved in the induction of neoplastic cell transformation (55,56). Nitric oxide has also been shown to affect tumor angiogenesis by stimulating the production of vascular endothelial growth factor (57,58). iNOS is likely to be more important for cancer development than eNOS and nNOS since the latter isoforms are constitutively expressed in a variety of cell types and produce only a low level of nitric oxide, while the induction of iNOS results in a high, sustained level of nitric oxide production (12). Aberrant or excessive expression of iNOS leads to an accumulation of nitric oxide, which can participate in cancer development. Experimental studies have shown that the ablation of iNOS or inhibition of its activity inhibits the development of rodent lung and colon cancer (5860). Collectively, these studies indicate that iNOS and its products play important roles in the development of cancer. Current studies showed that B[a]P exposure led to significant induction of iNOS in RE-149 cells, suggesting that iNOS may be involved in the B[a]P-induced carcinogenic effect in esophagus.
Many factors, such as chemical carcinogens, cytokines (IFN-
, IL-1ß and TNF-
), bacterial endotoxin (lipopolysaccharide, LPS) and oxidative stress stimulate the expression of iNOS (37,6062). The regulation of iNOS expression is transcriptionally regulated by certain transcription factors, including NF-
B (20), AP-1 (37), interferon-regulatory protein 1 (63), signal transducer and activator of transcription 1
(64), C/EBPß, and CREB (6567). Various upstream signaling pathways are involved in the regulation process. The MAPKs are important signaling pathways in regulating the expression of iNOS; however, their activity depends on the cell type involved and the agent used to stimulate the expression of iNOS. ERKs and p38 MAPKs are known to participate in the regulation of LPS-induced iNOS expression (68). Lahti et al. (69) reported that the c-Jun NH2-terminal kinase inhibitor, SP600125, reduced the expression of LPS-induced iNOS in mouse macrophages. Han et al. (62) found that the stimulation of iNOS by phorbol ester and IFN-
was mediated through ERKs in microglial cells. Our results indicate that the induction of iNOS by B[a]P in RE-149 cells is regulated predominately through the ERKs-dependent pathway, whereas p38 kinase, JNKs, PI-3K and mTOR/p70S6K are not required for the induction of iNOS by B[a]P in esophageal RE-149 cells.
NF-
B is a critical transcription factor responsible for the regulation of genes involved in intercellular communication, as well as the initiation or promotion of tumorigenesis (70,71). It has been demonstrated that luciferase reporter constructs containing the 4.7 kb 5'-flanking region of the human iNOS gene failed to show any cytokine-inducible activity, whereas constructs containing the 7.2 kb 5'-flanking region were induced 4- to 5-fold in the presence of cytokines (72). Previous studies have found that there are five putative NF-
B cis-regulatory transcription factor binding sites within upstream 7.2 to 4.7 kb in the 5'-flanking region of human iNOS gene (73). Other reports indicate that LPS, IFN-
, IL-1ß and other factors induce the expression of iNOS expression through NF-
B (62,74,75), suggesting that NF-
B plays a role in the regulation of iNOS. Since B[a]P exposure increased in NF-
B DNA binding activity in human hepatoblastoma (HepG2) cells (73), we investigated the induction of iNOS using luciferase reporter constructs containing the human iNOS 5'-flanking gene (7.2 kb) in RE-149 cells. Treatment of cells with specific NF-
B peptide inhibitor or overexpression of either IKK-KM or DN-I-
B blocks the expression of B[a]P-induced iNOS. Our data demonstrated the essential role of NF-
B activation in the expression of B[a]P-induced iNOS in RE-149 cells.
In conclusion, the present study demonstrates that B[a]P, rather than 5-MCDE and B[a]PDE, induces the expression of iNOS in a dose- and time-dependent manner in RE-149 cells, and this induction is mediated through ERKs- and NF-
B-dependent pathways. These studies provide insight into the molecular mechanisms by which B[a]P exposure leads to esophageal cancer.
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Notes
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The first three authors have contributed equally to this work. 
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Acknowledgments
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This work was supported in part by grants from NIH/NCI R01 CA103180, R01 CA094964 and R01 CA112557 and from NIH/NIEHS R01 ES012451, ES 00260. We thank Jane Galvin for secretarial support.
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Received November 4, 2004;
revised January 13, 2005;
accepted February 8, 2005.