* Department of Life Science, Division of Molecular and Life Science, and School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, Kyungbuk, Republic of Korea, and Division of Biology, California Institute of Technology, Pasadena, California 91125
Received September 2, 2004; accepted October 28, 2004
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ABSTRACT |
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Key Words: polychlorinated biphenyl; cyclooxygenase-2; compensation.
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INTRODUCTION |
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Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the oxygenation of arachidonic acid to prostaglandin endoperoxides, which are converted enzymatically into prostaglandins (PGs) and thromboxane A2 (Marnett et al., 1999; Warner and Mitchell, 2004
). Two distinct isoforms of COX have been identified. COX-1 is constitutively expressed in most tissues, but in contrast, COX-2, the product of a related inducible gene, is absent in most normal tissues. However, it is expressed rapidly in response to proliferative and inflammatory stimuli such as growth factors, cytokines, and tumor promoters (Kroll et al., 1999
; Matsuura et al., 1999
; Xie and Herschman, 1996
). It has been well established that COX-2 is linked to cell survival (Adderley and Fitzgerald, 1999
; Subbaramaiah et al., 2000
; Van Putten et al., 2001
; Xie and Herschman, 1995
; Yang et al., 2000
). For example, COX-2 protects (1) macrophages from superoxide-induced apoptosis (Address von et al., 1999
); (2) neuronal cells from nerve growth factor (NGF) withdrawal-induced apoptosis (Chang et al., 2000
; McGinty et al., 2000
); and (3) renal cells from hypertonicity-induced cell death (Yang et al., 2000
). Intriguingly, recent reports have suggested that p53 can induce the sustained activation of the Ras/Raf/ERK cascade. And, such activation induces COX-2 expression, which counteracts p53- or genotoxic stress-induced apoptosis (Han et al., 2002
; Lee et al., 2000
).
In this study, we show that 2,2',4,6,6'-PeCB-induced COX-2 expression is mediated by the CRE site, c-Jun, p53 and ERK1/2 MAPK activation. Moreover, the selective COX-2 inhibitor, NS-398, potentiated the 2,2',4,6,6'-PeCB-induced mitochondrial apoptotic pathway. These results suggest that COX-2 expression by 2,2',4,6,6'-PeCB may be a compensatory mechanism designed to abate its toxicity by reducing apoptotic susceptibility.
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MATERIALS AND METHODS |
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Cell culture. Rat-1 fibroblasts and mouse embryonic fibroblasts (MEFs) were maintained in DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified, 5% CO2-controlled incubator. Before stimulation, the cells were starved with medium without serum for one day.
Plasmids. p53-Luc- and AP-1-Luc plasmid was obtained from Stratagene (La Jolla, CA). COX-2 promoter-luciferase wild type and deletion constructs were kindly provided by Dr. Herschman (UCLA-Los Angeles Center for the Health Sciences, Los Angeles, CA). A c-Jun dominant negative expression vector was a gift of Dr. Tom Curran (St. Jude Children's Research Hospital, Memphis, TN).
Transient transfection. Rat-1 cells were transfected plasmids using FuGENE transfection reagent according to a procedure recommended by the manufacturer. The transfected cells were cultured for one day, starved for one day, and then treated with PCBs for 6 h.
Luciferase reporter gene assay. The cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.8, 1% Triton X-100, 150 mM NaCl, 2 mM DTT). The cell lysate (5 µl) was mixed with luciferase activity assay reagent (25 µl) and luminescence produced for 5 s was measured using luminoskan (labsystems). Data are presented as the fold of relative light unit relative to vehicle-treated control cultures.
Western blot analysis. For Western blot analysis, whole cell lysate were prepared in lysis buffer (20 mM Tri-HCl, pH 7.4, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM PMSF). Proteins (30 µg/lane) were separated on a denaturing 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Primary antibody was incubated with the membranes overnight at 4°C. Secondary antibody linked to horseradish peroxidase was used at 1:10,000, and signals were visualized by the ECL technique.
RT-PCR. cDNA was reverse-transcribed from total cellular RNA using oligo (dT) primers and murine leukemia virus reverse transcriptase. cDNA was amplified for 35 cycles using the following rat COX-2 gene-specific primers: 5'-ACTTGCCTCACTTTGTT GAGTCATTC-3' (sense) and 5'-TTTGATTAGTACTGTAGGGTTAATG-3' (antisense) and rat COX-1 gene-specific primers: 5'-TAAGTACAGGTGCTGGATGG-3' (sense) and 5'-GGTTTCCCCTAT AAGGATGAGG-3' (antisense). The cycling parameters were the following: 1 min at 94°C for denaturation, 1 min at 60°C for primer annealing, and 1 min at 72°C for polymerization. Meanwhile, the same amount of cDNA was amplified for 25 cycles using specific ß-actin primers: 5'-GAGACCTTCAACACCCC-3' (sense) and 5'-GTGGTGGTGAAGCTGTAGCC-3' (antisense). The products were visualized after electrophoresis on a 2% agarose gel containing ethidium bromide.
Cyclooxygenase activity. Rat-1 cells were plated in 24-well plate. One day later, the medium was replaced with serum free DMEM for one day, the cells were then incubated with or without PCBs for 6 h. Media were collected from each well and analyzed for PGE2 by enzyme-linked immunoassay as described previously (Yan et al., 2000).
Caspase-3 activity assay. Rat-1 cells were harvested and sonicated. Following centrifugation at 15,000 rpm for 10 min, 20 µg protein of supernatant in buffer (100 mM Hepes, 10% sucrose, 5 mM dithiothreitol, 106% NP-40, and 0.1% CHAPS at pH 7.25) was added to each well of a 96-well plate along with 50 µM DEVD-aminomethylcoumarin (AMC). After incubation at 37°C for 1 h, the cleaved free AMC (excitation of 355 nm, emission of 460 nm) was detected using fluorometer.
Trypan blue exclusion and MTT assay. For the trypan blue exclusion assay, 1 x 106 cells/1 ml were plated and treated with NS398 and PCB. The cells were then harvested and resuspended in medium, and an equal volume of trypan blue was added. More than 200 cells were scored on a hemocytometer. The percentage of cell survival was calculated by taking the number of trypan blue-excluding cells following treatment and dividing it by the number of DMSO-treated control cells and multiplying by 100.
To determine cell viability, 1 x 104 cells/well was subcultured in a 96-well plate. After treatment with chemical in serum-free DMEM for the indicated times, the cells were incubated for 2 h in the presence of 0.5 mg/ml MTT reagent in a 37°C incubator. After removal of MTT reagent and disruption of cells with DMSO, the absorbance was measured at 570 nm using a 96-well plate reader. Data are presented as the percentage of viability relative to vehicle-treated control cultures.
Statistical analysis. The results are expressed as means ± SE. Statistical significance was determined using the Student's t-test.
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RESULTS |
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ERK1/2 MAPK and p53 Are Important for 2,2',4,6,6'-PeCB-Mediated COX-2 Induction
Mitogen-activated protein kinase pathways mediate the regulation of COX-2 expression to a variety of extracellular stimuli (Guo et al., 2001; Subbaramaiah et al., 2000
; Yan et al., 2000
; Yang et al., 2000
). Figure 3A demonstrated that 2,2',4,6,6'-PeCB but not 2,3,3',4,4'-PeCB induced ERK1/2 MAPK phosphorylation in a time-dependent manner. To investigate whether such activation of ERK1/2 MAPK is important for the induction of COX-2, PD98059, a specific inhibitor of MAPK kinase, was used to block the activation of ERK1/2 MAPK. Treatment of PD98059 reduced COX-2 protein induction and PGE2 production in response to 2,2',4,6,6'-PeCB (Fig. 3B and data not shown).
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COX-2 Reduces Apoptotic Susceptibility
We previously reported that among the congeners examined, 2,2',4,6,6'-PeCB induces apoptosis in human monocytic cells (Shin et al., 2000). Furthermore, COX-2 has been shown to be associated with cell survival by many groups (Adderley and Fitzgerald, 1999
; Dowd et al., 2001
; Shinmura et al., 2000
). Therefore, we investigated the effect of COX-2 induction on 2,2',4,6,6'-PeCB-induced apoptosis using the selective COX-2 inhibitor, NS-398. Co-treatment with 2,2',4,6,6'-PeCB and NS-398 further increased cell death and caspase 3 activity, as compared with 2,2',4,6,6'-PeCB treatment alone (Figs. 4A and 4B). To examine how COX-2 reduces apoptosis by 2,2',4,6,6'-PeCB, we analyzed key steps in the mitochondrial apoptotic pathway. NS-398 potentiated the 2,2',4,6,6'-PeCB-stimulated attenuation of Bcl-xL mRNA, an anti-apoptotic gene, and increased mitochondrial cytochrome c release into the cytosol (data not shown), suggesting that COX-2 induction restricts cell death by blocking the mitochondrial apoptotic pathway. Furthermore, the cell death caused by 2,2',4,6,6'-PeCB was reduced about 30% by PEG2 treatment (Fig. 4C). These data imply that 2,2',4,6,6'-PeCB-induced apoptosis is limited by an increase in prostaglandin resulting from COX-2 induction.
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DISCUSSION |
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We also found that COX-2 expression by 2,2',4,6,6'-PeCB is mediated through the activation of ERK1/2 MAPK and p53 (Fig. 3). Recently, it was reported that a biochemical link between p53 signaling and Ras/Raf/MAPK cascade results in COX-2 expression (Han et al., 2002). Our results also showed that MAPK activation required the p53 transcription function (Fig. 3D). Furthermore, HB-EGF, which activates the EGF receptor and other members of the erbB family, has been shown to be induced in a p53-dependent manner in response to DNA damage and to account, at least in part, for the activation of MAPK in response to p53 (Fang et al., 2001
). In our study, 2,2',4,6,6'-PeCB, but not 2,3,3',4,4'-PeCB increased HB-EGF mRNA in Rat-1 cells (data not shown), suggesting the possibility that 2,2',4,6,6'-PeCB-stimulated COX-2 induction may be mediated by p53-dependent HB-EGF, which activates the Ras/Raf/MAPK pathway.
Several groups have reported that COX-2 and its major final product, PGE2, protect cells against apoptosis (Hoshino et al., 2002; Munkarah et al., 2002
; Sheng et al., 1998
). Rat intestinal epithelial cells overexpressing COX-2 were resistant to butyrate-induced apoptosis and had elevated Bcl2 protein expression, which were reversed by a COX inhibitor (Tsujii and Dubois, 1995
). Also, co-treatment with NS-398 and doxorubicin increased apoptosis by downregulating the anti-apoptotic protein, Bcl-xL, and the cell cycle inhibitor, p27, as compared with cells treated with doxorubicin alone (Han et al., 2002
). Oxidative damage of cardiomyocytes is limited by an increase in prostaglandin formation (Adderley and Fitzgerald, 1999
). In addition, Sheng et al. reported that PGE2 inhibits the apoptosis caused by a COX-2 inhibitor and induces Bcl-2 expression in human colon cancer cells (Sheng et al., 1998
). These findings suggest that COX-2 is a cellular component of a program that favors cell survival rather than apoptosis in response to stresses, and that its induction represents an example of an adaptive response to protect the cell from stress. In the present study, co-treatment with 2,2',4,6,6'-PeCB and NS-398 increased cell death and caspase-3 activity, versus cells treated with 2,2',4,6,6'-PeCB alone. Moreover, the inhibition of COX-2 function by NS-398 enhanced the 2,2',4,6,6'-PeCB-induced Bcl-xL mRNA downregulation, and resulted in increased mitochondrial cytochrome c release (data not shown). Figure 4C shows that treatment with PGE2 reduced the cell death caused by 2,2',4,6, 6'-PeCB, thus demonstrating that PGE2 produced by COX-2 activates anti-apoptotic genes such as Bcl-xL, which may result in reduced apoptosis through the mitochondrial pathway.
In conclusion, our study show that COX-2 is induced by 2,2',4,6,6'-PeCB via its CRE site, and by c-Jun, ERK1/2 MAPK and p53, and that the inhibition of COX-2 activity enhances 2,2',4,6,6'-PeCB-induced apoptosis through the mitochondrial pathway. These results suggest that COX-2 expression by 2,2',4,6,6'-PeCB may be a compensatory mechanism designed to abate its toxicity by reducing apoptotic susceptibility.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Life Science, Division of Molecular and Life Science, POSTECH BIOTECH CENTER, Pohang University of Science and Technology, San 31 Hyoja-Dong, Nam-Gu, Pohang, Kyungbuk 790-784, Republic of Korea. Fax: 82-54-279-0645. E-mail: pgs{at}postech.ac.kr.
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