Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2008 Jun 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2007 Mar 30;221(3):285–294. doi: 10.1016/j.taap.2007.03.019

2,2′,4,4′-Tetrachlorobiphenyl Upregulates Cyclooxygenase-2 in HL-60 cells via p38 Mitogen-Activated Protein Kinase and NF-κB

Steven A Bezdecny 1, Peer Karmaus 1, Robert A Roth 1, Patricia E Ganey 1,1
PMCID: PMC1950673  NIHMSID: NIHMS25620  PMID: 17482227

Abstract

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2′,4,4′,-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A2 with subsequent release of arachidonic acid (AA), and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 minutes. The increase in COX-2 mRNA was associated with release of 3H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A2, reduced 3H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB-mediated increases in superoxide anion were prevented by the NADPH oxidase inhibitor apocynin or the free radical scavenger 4-hydroxy TEMPO, but neither of these inhibitors affected the 2244-TCB-induced changes in COX-2 mRNA levels or 3H-AA release. Taken together these data suggest that p38 MAP kinase-dependent activation of NF-κB is critical for the 2244-TCB-mediated upregulation of COX-2 mRNA.

Keywords: : polychlorinated biphenyl, HL-60, cyclooxygenase-2, p38, NF-κB

Introduction

Polychlorinated biphenyls (PCBs) are persistent, man-made pollutants that are widespread in the environment. Food is the main source of human exposure, and although PCB levels are declining due to legislation and preventive measures, people are still exposed (Turyk et al. 2006). The 209 PCB congeners differ with regard to the number and the location of the chlorine atoms on the two benzene rings. In the present study, we focused on 2,2′,4,4′-tetrachlorobiphenyl (2244-TCB), a noncoplanar congener. Noncoplanar PCBs are generally considered to be less toxic than coplanar or dioxin-like PCBs, but they occur in the environment in larger concentrations than the dioxin-like PCBs (WHO, 2003).

Noncoplanar PCBs cause a variety of adverse effects, including increased release and decreased synthesis of insulin in rat insulinoma cells (Fischer et al. 1996), changes in the concentrations and distribution of dopamine and 5-hydroxytryptamine in female rat brains (Chu et al. 1996), increases in rat liver weight (Lecavalier et al. 1997), reduced phagocytosis by neutrophils and monocytes in marine mammals (Levin et al. 2004), and modulation of superoxide anion production in rat neutrophils (Brown and Ganey, 1995). PCBs also affect the regulation of a number of genes, the most studied being cytochromes P450 in liver (Chubb et al. 2004). In particular, 2244-TCB upregulates expression of the gene cyclooxygenase-2 (COX-2) in a neutrophil-like cell line (Bezdecny et al. 2005).

COX-2 is generally found in negligible amounts in normal cells, such as quiescent neutrophils, but is induced by a wide variety of inflammatory stimuli (Smith et al. 2000; Vane et al. 1998). COX-2 catalyzes the committed step in the synthesis of prostaglandins and thromboxanes. Once produced, these mediators can cause a range of immunomodulatory effects by acting on G protein-linked prostanoid receptors or, in some cases, on nuclear receptors (Louis et al. 2005). A number of shared or convergent pathways have been described that are involved in transcriptional regulation of COX-2 in response to inflammatory mediators, among which are NF-κB (Ghosh et al. 1998) and elements of the mitogen-activated protein (MAP) kinase cascades (Dean et al. 1999; Lasa et al. 2000; Moon and Pestka, 2002; Steer et al. 2006; Su and Karin, 1996). In addition, reactive oxygen species (ROS; Amma et al. 2005) and activated phospholipase A2 (PLA2) and its product, free arachidonic acid (AA; Pawliczak et al. 2004) have been implicated in the upregulation of COX-2 in various cell systems. Given the observation that PCBs increase COX-2 expression (Bezdecny et al. 2005), it was of interest to determine the signal transduction pathways involved in this alteration. Accordingly, the above-mentioned pathways were examined. Furthermore, since crosstalk among these pathways has been reported (Nakano et al. 2006; Wullaert et al. 2006), their interdependence was evaluated in granulocytic HL-60 cells exposed to 2244-TCB.

Materials and Methods

Chemicals

2,2′,4,4′-Tetrachlorobiphenyl (2244-TCB; >99% pure) was purchased from ChemService (West Chester, PA). [3H-5,6,8,9,11,12,14,15]-Arachidonic acid (3H-AA; 180–240 Ci/mmol) was purchased from DuPont NEN (Boston, MA). PD 98059 and U 0126 were purchased from Calbiochem (San Diego, CA). SB-202190, SB-203580, oleyloxyethyl phosphorylcholine (OP), bromoenol lactone (BEL), Bay 11-7082 and helanalin were purchased from Biomol (Plymouth Meeting, PA). Actinomycin D was purchased from Invitrogen (Carlsbad, CA). 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and apocynin were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were of the highest grade commercially available.

HL-60 cells

HL-60 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells between passage 20 and 50 were grown in Iscove’s Modified Dulbecco’s Medium supplemented with 10% cosmic calf serum (HyClone; Logan, UT), 0.1% gentamicin and 0.9% antibiotic-antimycotic. Cultures were maintained in a humidified incubator at 37°C in a controlled atmosphere of 5% CO2/95% air. Cells were induced to differentiate along the granulocyte pathway by culturing in the presence of 1.25% dimethyl sulfoxide (DMSO) for 5 days. Cells were then maintained an additional two days in the absence of DMSO. After this procedure, approximately 80% of the cells had nuclear appearance characteristic of neutrophils. In addition, they reduced nitroblue tetrazolium, a functional marker of differentiation to a neutrophilic phenotype (Hua et al. 2000).

Exposure to polychlorinated biphenyls

Stock solutions of 2244-TCB were prepared by dissolution in dimethylformamide (DMF). The differentiated HL-60 cells were suspended in Hanks’ balanced salt solution (HBSS) in 12 × 75 mm borosilicate glass test tubes (VWR, Chicago, IL), and 1 μL/mL of the stock solution was added to the cells to achieve the desired concentration. Control cells received 1 μL/mL of DMF. The concentration of 2244-TCB used in the current study was selected based on their previously described effects on HL-60 cells and their minimal cytotoxicities (Bezdecny et al. 2005).

Exposure to inhibitors

Stock solutions of inhibitors were prepared by dissolution in DMSO, and 1 μL/mL of the stock solution was added to the cells to achieve the desired concentration. Control cells received 1 μL/mL of DMSO. The concentrations of inhibitors used in the current study were selected based on their IC50 values and previously described activities (Ackerman et al. 1995; Hsieh et al. 2006; Lee et al. 1994; Lee et al. 2006; Magolda and Galbraith, 1985).

Actinomycin D studies

Differentiated HL-60 cells (5 × 106) were suspended in HBSS and treated with 30 μM 2244-TCB for 10 minutes at 37°C. Samples were then exposed to 10 μg/mL actinomycin D or vehicle. Samples were collected 0, 10, 20, 30, 40, 50 and 60 minutes after exposure to actinomycin D and centrifuged for 10 minutes at 4000 g. RNA was collected, and real-time RT-PCR was performed on the samples as described below.

Determination of COX-2 mRNA levels

Differentiated HL-60 cells (5 × 106) were suspended in HBSS and pretreated with the appropriate inhibitor or vehicle for 30 minutes. They were then treated with vehicle or 30 μM 2244-TCB for 30 minutes. Previous studies indicated a significant increase in COX-2 mRNA levels after 30 minutes of exposure to 2244-TCB (Bezdecny et al. 2005). At the end of the incubation period, total cellular RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO). The concentration of isolated RNA was determined by spectrophotometry at 260 nm using a Beckman DU 640 spectrophotometer (Beckman Coulter Inc., Fullertom, CA.). cDNA was synthesized by reverse transcription at 42°C for 45 minutes in a 20 μL reaction mixture containing 1 μg total RNA and 100 units Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega Corp., Madison, WI). After heating at 99°C for 5 minutes for denaturing, followed by cooling at 5°C, the cDNA was used for amplification. For PCR reactions, 3 μL of denatured cDNA was amplified in a final volume of 30 μL with 0.75 units of AmpliTaq Gold (Applied Biosystems, Foster City, CA), 1μM of each primer and Sybr green PCR buffer containing 25mM MgCl2 with 10mM of each dNTP (dATP, dCTP, dGTP and dTTP; Applied Biosystems, Foster City, CA). Primers were constructed using primer express. The following primers were used for COX-2: 5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′ (forward primer), 5′-AGATCATCTCTGCCTGAGTATCTT-3′ (reverse primer). The predicted size of the fragment was 301 bp. For β-actin, the primers were 5′-GACGAGGCCCAGAGCAAGAGAG-3′ (forward primer), 5′-ACGTACATGGCTGGGGTGTTG-3′ (reverse primer). The predicted size of the fragment was 284 bp. PCR was performed in a thermal cycler (Applied Biosystems 7500) using a program of 50°C for 2 minutes, 95°C for 5 minutes and 40 cycles at 95°C for 15 seconds, 60°C for 1 minute. Results were analyzed using the program ABI 5700 SDS. All results were compared to β-actin and expressed as fold induction versus vehicle control. Fold induction was calculated by first determining the ratio of COX-2 to β-actin in cells treated only with vehicle. All results from treated cells were then divided by this value to give the fold increase in COX-2 mRNA over that of untreated cells.

Labeling of HL-60 cells with 3H-arachidonic acid

Differentiated HL-60 cells (10 × 106/mL) were suspended in HBSS containing 0.1% bovine serum albumin (BSA) and incubated for 120 minutes at 37°C with 0.5 μCi/mL 3H-AA. At the end of the labeling period cells were washed twice and resuspended in HBSS containing 0.1% BSA. An aliquot of cells was subjected to scintillation counting to determine cellular uptake of radiolabel; uptake was routinely ~70% of added 3H-AA.

Determination of arachidonic acid release from HL-60 cells

Cumulative release of 3H-AA was measured in HL-60 cells (2 × 106) pretreated with the appropriate inhibitor or vehicle for the indicated time, then exposed to 30 μM 2244-TCB or vehicle for 90 minutes at 37°C. Previous studies indicated a significant increase in 3H-AA release after 90 minutes of exposure to 2244-TCB (Bezdecny et al. 2005). At the end of the incubations, samples were placed immediately on ice and then centrifuged, and radioactivity in the cell-free supernatant fluids was determined by liquid scintillation counting. Release is expressed as a percentage of the total radioactivity in the labeled cells.

Western blotting for COX-2 and p38

Differentiated HL-60 cells (50 × 106) were suspended in HBSS and pretreated at 37°C with the appropriate inhibitor or vehicle for 30 minutes, then exposed to 30 μM 2244-TCB or vehicle for 90 minutes. Immediately after treatment, cells were centrifuged for 10 minutes at 4000 g. Supernatant was discarded, and the cell pellet was lysed with 100 μL of 2% SDS. Proteins (30 μL) were separated on 10% Bis-Tris polyacrylamide gels (NuPAGE, Invitrogen Corporation, Carlsbad, CA) and electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). After blocking, the membranes were incubated with a primary anti-human COX-2 monoclonal antibody (1:1000 dilution; Cayman Chemical, Ann Arbor, MI), or a primary anti-human phospho-p38 monoclonal antibody (1:1000 dilution for each; both from Cell Signaling Technology, Beverly, MA) and then a secondary antibody (peroxidase-conjugated goat anti-mouse IgG; 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). The blots were developed using an ECL detection kit (Amersham Biosciences, Little Chalfont, UK). The blots were stripped and successively reprobed with an anti-human β-actin monoclonal antibody (Sigma Chemical Co. St. Louis, MO) then with a corresponding secondary antibody. The intensities of bands of interest were measured densitometrically and corrected using Bio-Rad Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA). β-actin was used as an internal standard.

Cyclooxygenase activity assay

COX activity was measured using a COX Activity Assay (Cayman Chemical, Ann Arbor, MI) according to manufacturer’s instructions. Differentiated HL-60 cells (15 × 106) were suspended in HBSS and pretreated at 37°C with the appropriate inhibitor or vehicle for 30 minutes, then treated for 90 minutes with 30 μM 2244-TCB. Immediately after treatment, cells were centrifuged for 10 minutes at 4000 g. Supernatant was discarded, and the cell pellet was resuspended in 200 μL of assay buffer then lysed via sonication. Activities of COX-1 and COX-2 were differentiated using the isoform-specific inhibitors DuP-697 and SC-560 according to manufacturer’s instructions.

Measurement of phospho-p38 and phospho-ERK

Levels of phosphorylated and total p38 and ERK (extracellular signal-regulated kinase) were determined using FACE cell-based ELISAs from Active Motif (Carlsbad, CA). Differentiated HL-60 cells (20 × 106) were suspended in HBSS and pretreated at 37°C with the appropriate inhibitor or vehicle for 30 minutes. For determination of levels of phospho-ERK, cells were then treated for 45 minutes with vehicle or 30 μM 2244-TCB. For determination of levels of phospho-p38, cells were treated for 90 minutes with vehicle or 30 μM 2244-TCB. Levels of total and phospho-ERK and p38 were then determined according to manufacturer’s instructions and presented as a ratio of phospho-protein to total protein.

Preparation of nuclear extracts

Differentiated HL-60 cells (5 × 106) were suspended in HBSS and pretreated with the appropriate inhibitor or vehicle for 30 minutes. They were then treated with vehicle or 30 μM 2244-TCB. After 30 minutes, nuclear protein was isolated using a nuclear extract kit (Active Motif, Carlsbad, CA) according to manufacturer’s instructions.

Determination of nuclear NF-κB

Differentiated HL-60 cells (10 × 106) were suspended in HBSS and pretreated with the appropriate inhibitor or its vehicle. 30 minutes later they were treated with vehicle or 30 μM 2244-TCB for 30 minutes, after which nuclear protein was isolated. Amounts of nuclear NF-κB were measured using a TransAM NF-κB ELISA kit (Active Motif, Carlsbad, CA) according to manufacturers instructions.

Superoxide anion production

Superoxide anion generation by HL-60 cells was measured as the reduction of cytochrome c in the presence and absence of superoxide dismutase (SOD; Babior et al. 1976). Differentiated HL-60 cells were suspended in Ca2+- and Mg2+-containing HBSS and pretreated with the appropriate inhibitor or vehicle for the indicated time. Cytochrome c (10 mg/mL) and 2244-TCB or vehicle was then added to the pretreated cells. Experiments were performed in 96-well plates, and for every sample two wells were incubated: one to which SOD (840 U/mL) was added and one to which an equal volume of vehicle was added. The amount of superoxide anion produced was estimated from the amount of cytochrome c reduced as determined from the difference in absorbance between the two wells, using an extinction coefficient of 18.5 cm−1 mM−1.

Statistical analysis

Data are expressed as mean ± SEM. Results for RT-PCR studies were analyzed by two-way repeated measures analysis of variance. All other results were analyzed by one-way analysis of variance. Group means for all data were compared using Tukey’s post hoc test. Appropriate transformations were performed on all data that did not follow a normal distribution (e.g., percent data). For all studies, the criterion for statistical significance was p < 0.05.

Results

COX-2 mRNA expression in HL-60 cells exposed to 2244-TCB in the absence and presence of actinomycin D

Treatment with 2244-TCB caused a time-dependent increase in COX-2 mRNA levels that peaked at ~4-fold after 30 minutes exposure and declined thereafter (Fig. 1). To evaluate whether an increase in transcription contributes to increased levels of COX-2 mRNA after exposure to 2244-TCB, HL-60 cells were pretreated for 10 minutes with 2244-TCB, then ActD was added. The 2244-TCB-induced increase in COX-2 mRNA was significantly smaller in cells treated with ActD compared to vehicle-treated cells. These results suggest that exposure to 2244-TCB causes transcription of the COX-2 gene in granulocytic HL-60 cells.

Figure 1. Effect of inhibition of transcription on 2244-TCB-mediated COX-2 mRNA expression.

Figure 1

Open in a new tab

Differentiated HL-60 cells (5 × 106) were treated with 30 μM 2244-TCB for 10 minutes. Cells were then treated with 10 μg/mL of the transcriptional inhibitor actinomycin D (ActD) or its vehicle, and samples were collected every 10 minutes. COX-2 mRNA levels were determined by Sybr Green RT-PCR as described in Materials and Methods. All data were normalized to β-actin. N= 4 separate experiments. a= significantly different from vehicle-treated groups at the same time.

Effects of signal transduction inhibitors on 2244-TCB-mediated 3H-AA release

In the absence of 2244-TCB exposure, vehicle-treated HL-60 cells released ~5% of 3H-AA after 90 minutes. Exposure to 2244-TCB produced a significant increase in 3H-AA release (Table 1). BEL, an inhibitor of the calcium-independent PLA2 (Ackermann et al. 1995), attenuated the 2244-TCB-mediated increase in 3H-AA release. Incubation with either OP, an inhibitor of the cytosolic and secretory isoforms of PLA2 (Magolda and Galbraith, 1989), or SB-202190, an inhibitor of p38 MAP kinase (Lee et al. 1994), had no effect on 2244-TCB-induced 3H-AA release.

Table 1.

2244-TCB-mediated 3H-AA release in the presence of inhibitors of p38 and PLA2.

% 3H-Arachidonic Acid Release
Pretreatment Vehicle 2244-TCB
Vehicle 4.9 ± 0.4 11.1 ± 0.3a
BEL 4.5 ± 0.3 6.8 ± 0.5b
OP 4.7 ± 0.4 9.6 ±0 0.9a
SB-202190 4.4 ± 0.4 9.5 ± 0.6a
Open in a new tab

Differentiated HL-60 cells were labeled with 3H-AA as described in Materials and Methods. Labeled cells (1 × 106 cells/mL) were pretreated with vehicle, 25 μM BEL, 15 μM OP, or 5 μM SB-202190 for 30 minutes. They were then treated with vehicle (DMF) or 30 μM 2244-TCB for 90 minutes. 3H-AA release is presented as the percent of total cellular 3H-AA released into culture medium. N = 3 separate experiments. a=significantly different from respective group in the absence of 2244-TCB; b = significantly different from 2244-TCB in the absence of inhibitor.

p38 MAP kinase protein phosphorylation in the presence of 2244-TCB and signal transduction inhibitors

Total levels of p38 protein were similar in untreated cells and 2244-TCB-treated cells and were unaffected by SB-202190, SB-203580, OP or BEL (data not shown). In untreated cells, levels of phosphorylated (active) p38 were negligible. Levels of phospho-p38 increased after 60 (data not shown) or 90 minutes of exposure to 2244-TCB (Fig. 2). The p38 MAPK inhibitors SB-202190 and SB-203580 attenuated this increase. The 2244-TCB-induced increase in phospho-p38 protein level was not affected by exposure to OP or BEL.

Figure 2. 2244-TCB-mediated activation of p38 in the presence of signal transduction inhibitors.

Figure 2

Open in a new tab

Levels of active, phospho-p38 protein were determined by western analysis in HL-60 cells (50 × 106) pretreated for 30 minutes with vehicle, 5 μM SB-202190 (SB1), 15 μM SB-203580 (SB2), 15 μM OP or 25 μM BEL, then treated with 30 μM 2244-TCB for 90 minutes as described in Materials and Methods. β-actin was used as a loading control. Phospho-p38/β-actin ratios were determined by densitometry. Levels of phospho-p38 were negligible in the absence of 2244-TCB. N = 3 separate experiments. a= significantly different from 2244-TCB in the absence of inhibitor.

Effects of signal transduction inhibitors on 2244-TCB-mediated changes in COX-2 mRNA, protein and activity

Exposure to 2244-TCB increased COX-2 mRNA as compared to control (Fig. 3A). Incubation with either SB-202190 or SB-203580 significantly reduced this increase. Neither OP nor BEL treatment affected the 2244-TCB-mediated increase in COX-2 mRNA.

Figure 3. 2244-TCB-mediated COX-2 mRNA expression in the presence of signal transduction inhibitors.

Figure 3

Open in a new tab

(A) HL-60 cells (5 × 106) were pretreated for 30 minutes with vehicle, 5 μM SB-202190 (SB1), 15 μM SB-203580 (SB2), 15 μM OP or 25 μM BEL, then treated with vehicle or 30 μM 2244-TCB for 30 minutes. COX-2 mRNA was determined by RT-PCR as described in Materials and Methods. (B) HL-60 cells (50 × 106) were pretreated as described in panel A, and then treated with vehicle or 2244-TCB for 90 minutes. COX-2 protein was determined as described in Materials and Methods. βactin was used as a loading control. Levels of COX-2 protein were negligible in the absence of 2244-TCB. (C) HL-60 cells (15 × 106) were pretreated as described in Panel A, and then treated with vehicle or 2244-TCB for 90 minutes. COX-2 activity was determined as described in Materials and Methods. NT= no pretreatment. N= 5 (A) or 3 (B and C) separate experiments. a= significantly different from respective group in the absence of 2244-TCB; b= significantly different from 2244-TCB in the absence of inhibitor.

COX-2 protein was not detectable in untreated cells (data not shown). By 90 minutes of exposure to 2244-TCB, COX-2 protein was increased to detectable levels (Fig. 3B; Bezdecny et al, 2005). This increase was reduced by incubation of the cells with SB-202190 or SB-203580. The 2244-TCB-mediated increase in COX-2 protein was unchanged by exposure to OP or BEL.

COX-2 activity in untreated HL-60 cells was ~10 units/mL (Fig. 3C). Treatment with 2244-TCB for 90 minutes increased COX-2 activity approximately 4-fold. Pretreatment with SB-202190 significantly decreased 2244-TCB-stimulated COX-2 activity. Neither OP nor BEL had a significant effect on COX-2 activity.

Effects of inhibitors on p38 and ERK phosphorylation

In untreated cells, the ratio of phospho-ERK to total ERK was small. Treatment with 2244-TCB caused a significant increase in phospho-ERK (Fig. 4). Pretreatment with either MEK (MAP/ERK) inhibitor, PD 98059 or U 0126, significantly decreased 2244-TCB-stimulated phosphorylation of ERK (Fig. 4A). Additionally, pretreatment with either p38 inhibitor significantly decreased 2244-TCB-stimulated phosphorylation of ERK (Fig. 4B). None of these inhibitors affected phosphorylation of ERK in the absence of 2244-TCB. The 2244-TCB-mediated increase in the ratio of phospho-p38 to total p38 was not affected by MEK inhibitors in the absence or presence of 2244-TCB (Fig. 4C). Phosphorylation of JNK (c-Jun N-terminal kinase) was not affected by 2244-TCB (data not shown).

Figure 4. Effects of inhibitors on p38 and ERK phosphorylation.

Figure 4

Open in a new tab

Phosphorylated and total ERK (A,B) and p38 (C) were determined by ELISA in HL-60 cells (20 × 106) pretreated for 30 minutes with vehicle, 10 μM PD 98059, 5 μM U 0126, 5 μM SB-202190 (SB1) or 15 μM SB-203580 (SB2) then with vehicle or 30 μM 2244-TCB for 45 minutes (A,B) or 90 minutes (C). N = 3 separate experiments. a= significantly different from respective group in the absence of 2244-TCB; b= significantly different from 2244-TCB in the absence of inhibitor.

Lack of effect of MEK inhibitors on 2244-TCB-mediated COX-2 mRNA expression

COX-2 mRNA expression was modest in vehicle-treated cells and was unaffected by exposure to either PD 98059 or U 0126 (Fig. 5). As noted above, 2244-TCB increased COX-2 mRNA as compared to control. Pretreatment with either PD 98059 or U 0126 did not affect this increase in COX-2 mRNA.

Figure 5. Effect of MEK inhibitors on 2244-TCB-mediated COX-2 mRNA expression.

Figure 5

Open in a new tab

COX-2 mRNA was determined by RT-PCR in HL-60 cells (5 × 106) pretreated for 30 minutes with vehicle, 10 μM PD 98059 or 5 μM U 0126, then treated with 30 μM 2244-TCB for 30 minutes. β-actin was used as a loading control. N= 4 separate experiments. a= significantly different from respective group in the absence of 2244-TCB.

Effects of 2244-TCB on nuclear NF-κB

Exposure to 2244-TCB increased the amount of nuclear NF-κB approximately 1.7 fold after 15 minutes of exposure (Fig. 6A). It remained greater than the vehicle-treated value for 75 minutes (data not shown). Pretreatment with either SB-202190 or SB-203580 prevented the 2244-TCB-mediated increase in nuclear NF-κB (Fig. 6A). Neither SB-202190 nor SB-203580 affected nuclear NF-κB in the absence of 2244-TCB. Pretreatment with either PD 98059 or U 0126 did not affect the 2244-TCB-mediated increase in nuclear NF-κB or the amount of nuclear NF-κB in vehicle-treated cells (Fig. 6B).

Figure 6. The 2244-TCB-mediated increase in nuclear NF-κB is reduced by p38 inhibitors, but not by MEK inhibitors.

Figure 6

Open in a new tab

Nuclear NF-κB was determined in HL-60 cells (10 × 106) pretreated with vehicle, 5 μM SB-202190 (SB1) or 15 μM SB-203580 (SB2) (A) or 10 μM PD 98059 or 5 μM U 0126 for 30 minutes, then treated with 30 μM 2244-TCB for 30 minutes. N = 3 separate experiments. a= significantly different from respective group in the absence of 2244-TCB; b= significantly different from respective group in the absence of inhibitor.

Effects of NF-κB inhibitors on 2244-TCB-mediated COX-2 mRNA expression

2244-TCB caused an approximately four-fold increase in COX-2 mRNA as compared to control after 30 minutes of exposure (Fig. 7), confirming previous results (Fig. 1 and Bezdecny et al. 2005). Incubation with either BAY 11-7082 or helenalin, inhibitors of NF-κB activity that act via two different mechanisms, significantly reduced the 2244-TCB-mediated increase in COX-2 mRNA. Inhibition of NF-κB did not affect the expression of COX-2 mRNA in the absence of 2244-TCB.

Figure 7. NF-κB inhibitors attenuate the 2244-TCB-mediated increase in COX-2 mRNA expression.

Figure 7

Open in a new tab

COX-2 mRNA was determined in HL-60 cells (5 × 106) pretreated with vehicle, 10 μM Bay 11-7082 or 10 μM helenalin for 30 minutes, then treated with 30 μM 2244-TCB for 30 minutes. mRNA was quantified using real-time RT-PCR as described in Materials and Methods. β-actin was used as a loading control. N= 4 separate experiments. a= significantly different from respective group in the absence of 2244-TCB; b= significantly different from respective group in the absence of inhibitor.

Effect of inhibitors of superoxide anion production on 2244-TCB-induced changes in COX-2 mRNA expression

We have reported previously that exposure of differentiated HL-60 cells to 2244-TCB causes a time- and dose-dependent increase in superoxide anion production (Bezdecny et al. 2005). In the present study, 2244-TCB increased superoxide production relative to control (Fig. 8A), and pretreatment with either the NADPH oxidase inhibitor apocynin or the intracellular free radical scavenger TEMPO prevented the 2244-TCB-mediated increase in superoxide anion concentration.

Figure 8. Lack of effect of reactive oxygen species inhibition on 2244-TCB-induced upregulation of COX-2 mRNA expression.

Figure 8

Open in a new tab

(A) HL-60 cells (1 × 106) were pretreated with vehicle, 1mM apocynin (30 minutes) or 200 μM TEMPO (10 minutes) then exposed to 30 μM 2244-TCB for 30 minutes after which superoxide anion production was determined. (B) HL-60 cells were pretreated with vehicle, apocynin or TEMPO as described, then with vehicle or 2244-TCB for 30 minutes. mRNA was collected for determination of COX-2 mRNA levels. β-actin was used as a loading control. N = 3 separate experiments. a= significantly different from respective group in the absence of 2244-TCB; b=significantly different from 2244-TCB-treated cells in the absence of inhibitor (i.e. vehicle-treated).

2244-TCB increased COX-2 mRNA levels approximately fourfold after 30 minutes of exposure (Fig. 8B). Neither apocynin nor TEMPO pretreatment affected the 2244-TCB-mediated increase in COX-2 mRNA. Inhibition of ROS production also did not affect release of 3H-AA (Table 2).

Table 2.

2244-TCB-mediated 3H-AA release in the presence of inhibitors of ROS.

% 3H-Arachidonic Acid Release
Pretreatment Vehicle 2244-TCB
Vehicle 3.8 ± 0.2 6.5 ± 0.4a
Apocynin 3.0 ± 0.2 5.8 ± 0.3a
TEMPO 3.8 ± 0.2 7.4 ± 0.4a
Open in a new tab

Differentiated HL-60 cells were labeled with 3H-AA as described in Materials and Methods. Labeled cells (1 × 106 cells/mL) were pretreated with vehicle, 1 μM apocynin for 30 minutes or 200 μM TEMPO for 10 minutes. They were then treated with vehicle (DMF) or 30 μM 2244-TCB for 90 minutes. 3H-AA release is presented as the percent of total cellular 3H-AA released into culture medium. N = 3 separate experiments. a=significantly different from respective group in the absence of 2244-TCB.

Discussion

Exposure of HL-60 cells to 2244-TCB leads to a modest and transient increase in transcription of the cyclooxygenase-2 gene. Inhibition of transcription with ActD reduced COX-2 mRNA levels in cells exposed to 2244-TCB. These results suggest that transcription of the COX-2 gene occurs in response to 2244-TCB exposure (Fig. 1). In the present study, signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB were examined. One signal transduction pathway that was investigated is the PLA2 pathway that leads to release of AA. AA acts as both the substrate for COX-mediated eicosanoid production as well as a cell signaling molecule. 2244-TCB increased 3H-AA release, and this response was attenuated by pretreatment with BEL, an inhibitor of iPLA2, but not by OP, an inhibitor of cytosolic and secretory PLA2 (Table 1). HL-60 cells resemble primary neutrophils in many respects and have, therefore, been used as a model to study neutrophil responses. Many of the effects that 2244-TCB has on neutrophil function are similar in primary rat neutrophils and HL-60 cells. Indeed, iPLA2-mediated increases in 3H-AA release similar to those observed here have been reported in primary rat neutrophils treated with the PCB mixture, Aroclor 1242, as well as with individual, noncoplanar PCB congeners (Olivero and Ganey, 2001; Tithof et al. 1998). Thus, these results suggest that HL-60 cells and rat neutrophils share a common pathway to PCB-stimulated AA release, i.e. one mediated via iPLA2. Nonetheless, the origin and nature of HL-60 cells should be considered when interpreting these results.

Activation of PLA2 contributed to upregulation of COX-2 in murine macrophages and human lung cells (Balsinde et al. 1999; Pawliczak et al. 2002, 2004). Furthermore, free AA activates a variety of proteins involved in the regulation of COX-2 expression, including p38 MAP kinase (Hii et al. 1998), peroxisome proliferator-activated receptor γ (Pawliczak et al. 2002, 2004) and phosphatidyl inositol-3-kinase (Monick et al. 2002). Interestingly, expression of COX-2 was decreased in brains of PLA2-deficient mice relative to wild type mice (Bosetti and Weerasinghe, 2003). These reports suggest that PLA2 or free AA can contribute to increased expression of COX-2. Despite this possibility, this pathway does not appear to be involved in the 2244-TCB-mediated upregulation of COX-2 in HL-60 cells because treatment with BEL, which inhibited release of 3H-AA, had no effect on COX-2 mRNA, protein or enzyme activity (Fig. 3).

The link between p38 activity and changes in COX-2 expression has been well established in a variety of systems, including rat hepatic macrophages (Ahmad et al. 2002), the HeLa cell line (Lasa et al. 2000), human airway myocytes (Singer et al. 2003) and human monocytes (Dean et al. 1999). p38 regulates COX-2 expression both at the transcriptional level, by activating transcription factors, as well as at the level of RNA stability, by activating cytoplasmic proteins that increase the half life of COX-2 mRNA (Dean et al. 1999; Lasa et al. 2000; Singer et al. 2003). In the present study, 2244-TCB exposure increased levels of phosphorylated, active p38 (Fig. 2). Activation of p38 MAP kinase was associated with increased levels of COX-2 mRNA and COX-2 protein in cells treated with 2244-TCB for similar times. Treatment with p38 MAP kinase inhibitors reduced levels of phospho-p38 as well as mRNA levels, protein and enzyme activity of COX-2 (Figs. 2 and 3). These results suggest that 2244-TCB exposure leads to activation of the p38 MAP kinase system, and that this activation is required for the 2244-TCB-mediated induction of COX-2.

Exposure of neutrophils to PCBs leads to increased production of prostaglandins (PGs) and thromboxane (Tx) (Tithof et al. 1998). Two components are necessary for generation of PGs and Tx: availability of free AA as substrate and COX activity. 2244-TCB provides both of these components by apparently independent pathways. Inhibition of iPLA2 diminished 3H-AA release (Table 1) but not the 2244-TCB-mediated increase in COX-2 mRNA or activation of p38 (Figs. 2 and 3A). Conversely, activation of iPLA2 and subsequent release of 3H-AA was not affected by inhibitors of p38 MAP kinase (Table 1), which did reduce the 2244-TCB-mediated upregulation of COX-2 (Fig. 3).

p38 is not the only MAP kinase reported to be involved in induction of COX-2. Activation of ERK and JNK regulates COX-2 expression in several cell types, including mouse macrophages and RAW-264.7 cells (Moon and Pestka, 2002; Steer et al. 2006), cardiac myocytes (Wu et al. 2006), the HeLa cell line (Holzberg et al. 2003) and glomerular epithelial cells (Takano et al. 2001). Both ERK and JNK regulate COX-2 expression at the transcriptional level by activating transcription factors. In the present study, 2244-TCB exposure had no effect on JNK activation at any time up to 90 minutes after exposure (data not shown). 2244-TCB increased levels of phosphorylated ERK, and this increase was prevented by pretreatment with inhibitors of p38, as well as by the MEK inhibitors PD 98059 and U 0126 (Fig. 4). However, pretreatment with MEK inhibitors did not affect the 2244-TCB-mediated increase in COX-2 mRNA (Fig. 5). These data suggest that ERK is activated by p38 in response to 2244-TCB exposure, but it is not involved in the 2244-TCB-mediated induction of COX-2.

Several transcription factors have been implicated in the regulation of COX-2 in different cell systems. In particular, there is a large body of literature supporting a role for NF-κB in the regulation of COX-2. For example, NF-κB is involved in the upregulation of COX-2 in response to infection by Legionella bacteria in the lung and the parasite Cryptosporidium in the heart (Asaad and Sadek, 2006; N’Guessan et al. 2006) and in triptolide-treated astrocytes (Dai et al 2006). In addition, NF-κB is critical to the regulation of COX-2 in HL-60 cells treated with andrographolide (Hidalgo et al. 2005) and in human neutrophils exposed to water-soluble bacterial proteins (Kim et al. 2001). In the present study, exposure to 2244-TCB caused a time-dependent increase in nuclear NF-κB levels (Figure 6 and data not shown) suggesting activation of this transcription factor. Additionally, nuclear levels of NF-κB were elevated at the same times that 2244-TCB-mediated increases in COX-2 mRNA were observed, and inhibition of NF-κB prevented the 2244-TCB-mediated increase in COX-2 mRNA (Fig. 7), suggesting a link between NF-κB activation and increases in COX-2 mRNA.

Inhibition of p38 reduced the 2244-TCB-mediated increase in nuclear NF-κB (Fig. 6A). The p38 MAP kinase is involved in phosphorylation of IκB, the inhibitory subunit of NF-κB, leading to the subsequent activation and nuclear translocation of NF-κB (Sweeney and Firestein, 2004). Activation of p38 and its subsequent activation of NF-κB regulates COX-2 expression in linoleic acid-treated mouse skin (Hwang, et al. 2006), in RAW 264.7 cells treated with dipyridamole (Chen et al. 2006) and in human tracheal smooth muscle cells treated with interleukin-1beta (Lin et al. 2004). Interestingly, in human pulmonary epithelial cells treated with phorbol 12-myristate 13-acetate, ERK and NF-κB, but not p38 MAP kinase, were required for upregulation of COX-2 (Chang et al. 2005).

In contrast to p38, MEK inhibitors did not affect the amount of nuclear NF-κB in the present study (Fig. 6B). Activation of ERK and subsequent activation and nuclear translocation of NF-κB regulates COX-2 expression in resveratrol-treated mouse skin (Kundu et al. 2006), in RAW 264.7 cells exposed to Mycobacterium avium proteins (Pathak et al. 2004) and in human pulmonary epithelial cells (Chang et al. 2005). However, results presented here suggest that ERK does not participate in the 2244-TCB-mediated activation of NF-κB. Taken together, the results suggest that 2244-TCB activates NF-κB in a p38-dependent manner, and that this is an important component in the 2244-TCB-mediated upregulation of COX-2.

Another pathway examined involved production of ROS. ROS, such as superoxide anion, contribute to changes in COX-2 gene regulation induced by a number of different stimuli in a variety of cell systems. In human mesangial cells exposed to high glucose, in human monocytes undergoing differentiation, and in human fibroblasts subjected to cyclic stretch stimuli, increased COX-2 expression was dependent on generation of ROS (Amma et al. 2005; Barbieri et al. 2003; Kiritoshi et al. 2003). These changes occurred through activation of p38 MAP kinase and a variety of transcription factors including NF-κB (Amma et al. 2005; Kim et al. 2005; Singer et al. 2003). In the present study, 2244-TCB caused an increase in superoxide anion production, similar to results reported previously for rat neutrophils (Ganey et al. 1993). This increase was prevented by apocynin as well as by TEMPO (Fig. 8A), however, neither of these treatments affected 2244-TCB-mediated upregulation of COX-2 mRNA (Fig. 8B). These results suggest that ROS do not play a critical role in this response in HL-60 cells. However, they could still play a role in eicosanoid formation by COX-2, since this enzyme requires ROS and/or peroxides for full activity (Smith et al. 2000; Vane et al. 1998).

In summary, several different signal transduction pathways are affected in HL-60 cells by exposure to 2244-TCB; however, not all of these pathways play a role in 2244-TCB-mediated upregulation of COX-2 mRNA in HL-60 cells. Neither reactive oxygen species, PLA2 nor free AA appear to be involved in increased expression of COX-2 by 2244-TCB, though increased levels of free AA can still serve as a substrate for eicosanoid production. ERK was activated in response to 2244-TCB exposure, but this activation is not needed for the upregulation of COX-2. In contrast, the modest and transient increases in COX-2 mRNA, protein and activity that were observed were associated with phosphorylation of p38 MAP kinase in 2244-TCB treated HL-60 cells, and these effects were attenuated by pretreatment with p38 MAP kinase inhibitors. p38 activation also led to an increase in nuclear levels of NF-κB, and inhibition of NF-κB prevented 2244-TCB-induced upregulation of COX-2. Taken together, these results support a mechanism (Fig. 9) in which activation of neutrophils by 2244-TCB enhances p38 MAP kinase-dependent biosynthesis of COX-2 enzyme via an NF-κB-dependent mechanism and also activates iPLA2 to provide AA as substrate for prostaglandin synthesis.

Figure 9. Working hypothesis for the 2244-TCB-mediated upregulation of the COX-2 gene.

Figure 9

Open in a new tab

2244-TCB exposure causes phosphorylation and activation of p38 MAP kinase. This activation leads to increases in COX-2 mRNA, protein and activity. These increases are caused by the activation of transcription factors such as NF-κB. In addition, 2244-TCB activates iPLA2 by a pathway independent of p38 MAP kinase, leading to the release of free AA, which can then be converted by COX-2 into prostaglandins and thromboxane.

Acknowledgments

This work was supported by Superfund grant ESO4911 from the NIH. Steven Bezdecny was supported by training grant T32 ES007255 from the NIEHS. The authors thank Lyle Burgoon and Bob Crawford for assistance with real-time PCR.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Ackermann EJ, Conde-Frieboes K, Dennis EA. Inhibition of macrophage Ca(2+)-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem. 1995;270:445–50. doi: 10.1074/jbc.270.1.445. [DOI] [PubMed] [Google Scholar]
  2. Ahmad N, Chen LC, Gordon MA, Laskin JD, Laskin DL. Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic macrophages during acute endotoxemia. J Leukoc Biol. 2002;71:1005–11. [PubMed] [Google Scholar]
  3. Amma H, Naruse K, Ishiguro N, Sokabe M. Involvement of reactive oxygen species in cyclic stretch-induced NF-kappaB activation in human fibroblast cells. Br J Pharmacol. 2005;145:364–73. doi: 10.1038/sj.bjp.0706182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asaad NY, Sadek GS. Pulmonary cryptosporidiosis: role of COX2 and NF-kB. APMIS. 2006;114:682–9. doi: 10.1111/j.1600-0463.2006.apm_499.x. [DOI] [PubMed] [Google Scholar]
  5. Babior BM, Curnutte JT, McMurrich BJ. The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst. J Clin Invest. 1976;58:989–96. doi: 10.1172/JCI108553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Balsinde J, Shinohara H, Lefkowitz LJ, Johnson CA, Balboa MA, Dennis EA. Group V phospholipase A(2)-dependent induction of cyclooxygenase-2 in macrophages. J Biol Chem. 1999;274:25967–70. doi: 10.1074/jbc.274.37.25967. [DOI] [PubMed] [Google Scholar]
  7. Barbieri SS, Eligini S, Brambilla M, Tremoli E, Colli S. Reactive oxygen species mediate cyclooxygenase-2 induction during monocyte to macrophage differentiation: critical role of NADPH oxidase. Cardiovasc Res. 2003;60:187–97. doi: 10.1016/s0008-6363(03)00365-1. [DOI] [PubMed] [Google Scholar]
  8. Bezdecny SA, Roth RA, Ganey PE. Effects of 2,2′,4,4′-tetrachlorobiphenyl on granulocytic HL-60 cell function and expression of cyclooxygenase-2. Toxicol Sci. 2005;84:328–34. doi: 10.1093/toxsci/kfi093. [DOI] [PubMed] [Google Scholar]
  9. Bosetti F, Weerasinghe GR. The expression of brain cyclooxygenase-2 is down-regulated in the cytosolic phospholipase A2 knockout mouse. J Neurochem. 2003;87:1471–7. doi: 10.1046/j.1471-4159.2003.02118.x. [DOI] [PubMed] [Google Scholar]
  10. Brown AP, Ganey PE. Neutrophil degranulation and superoxide production induced by polychlorinated biphenyls are calcium dependent. Toxicol Appl Pharmacol. 1995;131:198–205. doi: 10.1006/taap.1995.1062. [DOI] [PubMed] [Google Scholar]
  11. Chang MS, Chen BC, Yu MT, Sheu JR, Chen TF, Lin CH. Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways. Cell Signal. 2005;17:299–310. doi: 10.1016/j.cellsig.2004.07.008. [DOI] [PubMed] [Google Scholar]
  12. Chen TH, Kao YC, Chen BC, Chen CH, Chan P, Lee HM. Dipyridamole activation of mitogen-activated protein kinase phosphatase-1 mediates inhibition of lipopolysaccharide-induced cyclooxygenase-2 expression in RAW 264.7 cells. Eur J Pharmacol. 2006;541:138–46. doi: 10.1016/j.ejphar.2006.05.002. [DOI] [PubMed] [Google Scholar]
  13. Chu I, Villeneuve DC, Yagminas A, Lecavalier P, Poon R, Feeley M, Kennedy SW, Seegal RF, Hakansson H, Ahlborg UG, Valli VE, Bergman A. Toxicity of 2,2′,4,4′,5,5′-hexachlorobiphenyl in rats: effects following 90-day oral exposure. J Appl Toxicol. 1996;16:121–8. doi: 10.1002/(SICI)1099-1263(199603)16:2<121::AID-JAT320>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  14. Chubb LS, Andersen ME, Broccardo CJ, Legare ME, Billings RE, Dean CE, Hanneman WH. Regional induction of CYP1A1 in rat liver following treatment with mixtures of PCB 126 and PCB 153. Toxicol Pathol. 2004;32:467–73. doi: 10.1080/01926230490483306. [DOI] [PubMed] [Google Scholar]
  15. Dai YQ, Jin DZ, Zhu XZ, Lei DL. Triptolide inhibits COX-2 expression via NF-kappa B pathway in astrocytes. Neurosci Res. 2006;55:154–60. doi: 10.1016/j.neures.2006.02.013. [DOI] [PubMed] [Google Scholar]
  16. Dean JL, Brook M, Clark AR, Saklatvala J. p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J Biol Chem. 1999;274:264–9. doi: 10.1074/jbc.274.1.264. [DOI] [PubMed] [Google Scholar]
  17. Fischer LJ, Zhou HR, Wagner MA. Polychlorinated biphenyls release insulin from RINm5F cells. Life Sci. 1996;59:2041–9. doi: 10.1016/s0024-3205(96)00557-7. [DOI] [PubMed] [Google Scholar]
  18. Ganey PE, Sirois JE, Denison M, Robinson JP, Roth RA. Neutrophil function after exposure to polychlorinated biphenyls in vitro. Environ Health Perspect. 1993;101:430–4. doi: 10.1289/ehp.93101430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol. 1998;16:225–60. doi: 10.1146/annurev.immunol.16.1.225. [DOI] [PubMed] [Google Scholar]
  20. Hidalgo MA, Romero A, Figueroa J, Cortes P, Concha II, Hancke JL, Burgos RA. Andrographolide interferes with binding of nuclear factor-kappaB to DNA in HL-60-derived neutrophilic cells. Br J Pharmacol. 2005;144:680–6. doi: 10.1038/sj.bjp.0706105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hii CS, Huang ZH, Bilney A, Costabile M, Murray AW, Rathjen DA, Der CJ, Ferrante A. Stimulation of p38 phosphorylation and activity by arachidonic acid in HeLa cells, HL60 promyelocytic leukemic cells, and human neutrophils. Evidence for cell type-specific activation of mitogen-activated protein kinases. J Biol Chem. 1998;273:19277–82. doi: 10.1074/jbc.273.30.19277. [DOI] [PubMed] [Google Scholar]
  22. Holzberg D, Knight CG, Dittrich-Breiholz O, Schneider H, Dorrie A, Hoffmann E, Resch K, Kracht M. Disruption of the c-JUN-JNK complex by a cell-permeable peptide containing the c-JUN delta domain induces apoptosis and affects a distinct set of interleukin-1-induced inflammatory genes. J Biol Chem. 2003;278:40213–23. doi: 10.1074/jbc.M304058200. [DOI] [PubMed] [Google Scholar]
  23. Hsieh HL, Wu CY, Hwang TL, Yen MH, Parker P, Yang CM. BK-induced cytosolic phospholipase A2 expression via sequential PKC-delta, p42/p44 MAPK, and NF-kappaB activation in rat brain astrocytes. J Cell Physiol. 2006;206:246–54. doi: 10.1002/jcp.20457. [DOI] [PubMed] [Google Scholar]
  24. Hua J, Hasebe T, Someya A, Nakamura S, Sugimoto K, Nagaoka I. Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage. J Leukoc Biol. 2000;68:216–24. [PubMed] [Google Scholar]
  25. Hwang DM, Kundu JK, Shin JW, Lee HJ, Surh YJ. cis-9, trans-11-Conjugated linoleic acid down-regulates phorbol ester-induced NF-{kappa}B activation and subsequent COX-2 expression in hairless mouse skin by targeting I{kappa}B kinase and PI3K-Akt. Carcinogenesis. 2006 Aug 31; doi: 10.1093/carcin/bgl151. [Epub ahead of print] [DOI] [PubMed] [Google Scholar]
  26. Kim JS, Kim JM, Jung HC, Song IS. Expression of cyclooxygenase-2 in human neutrophils activated by Helicobacter pylori water-soluble proteins: possible involvement of NF-kappaB and MAP kinase signaling pathway. Dig Dis Sci. 2001;46:2277–84. doi: 10.1023/a:1011939704802. [DOI] [PubMed] [Google Scholar]
  27. Kim H, Rhee SH, Kokkotou E, Na X, Savidge T, Moyer MP, Pothoulakis C, LaMont JT. Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J Biol Chem. 2005;280:21237–45. doi: 10.1074/jbc.M413842200. [DOI] [PubMed] [Google Scholar]
  28. Kiritoshi S, Nishikawa T, Sonoda K, Kukidome D, Senokuchi T, Matsuo T, Matsumura T, Tokunaga H, Brownlee M, Araki E. Reactive oxygen species from mitochondria induce cyclooxygenase-2 gene expression in human mesangial cells: potential role in diabetic nephropathy. Diabetes. 2003;52:2570–7. doi: 10.2337/diabetes.52.10.2570. [DOI] [PubMed] [Google Scholar]
  29. Kundu JK, Shin YK, Surh YJ. Resveratrol modulates phorbol ester-induced pro-inflammatory signal transduction pathways in mouse skin in vivo: NF-kappaB and AP-1 as prime targets. Biochem Pharmacol. 2006;72:1506–15. doi: 10.1016/j.bcp.2006.08.005. [DOI] [PubMed] [Google Scholar]
  30. Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J, Clark AR. Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade. Mol Cell Biol. 2000;20:4265–74. doi: 10.1128/mcb.20.12.4265-4274.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lecavalier P, Chu I, Yagminas A, Villeneuve DC, Poon R, Feeley M, Hakansson H, Ahlborg UG, Valli VE, Bergman A, Seegal RF, Kennedy SW. Subchronic toxicity of 2,2′,3,3′,4,4′-hexachlorobiphenyl in rats. J Toxicol Environ Health. 1997;51:265–77. doi: 10.1080/00984109708984026. [DOI] [PubMed] [Google Scholar]
  32. Lee GC, Yi HA, Lee CH. Stimulation of interferon-beta gene expression by human cytomegalovirus via nuclear factor kappa B and phosphatidylinositol 3-kinase pathway. Virus Res. 2006;117:209–14. doi: 10.1016/j.virusres.2005.08.018. [DOI] [PubMed] [Google Scholar]
  33. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature. 1994;372:739–46. doi: 10.1038/372739a0. [DOI] [PubMed] [Google Scholar]
  34. Levin M, Morsey B, Mori C, Guise S. Specific non-coplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure. J Toxicol Environ Health A. 2004;67:517–35. doi: 10.1080/15287390490486761. [DOI] [PubMed] [Google Scholar]
  35. Lin CC, Sun CC, Luo SF, Tsai AC, Chien CS, Hsiao LD, Lee CW, Hsieh JT, Yang CM. Induction of cyclooxygenase-2 expression in human tracheal smooth muscle cells by interleukin-1beta: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB. J Biomed Sci. 2004;11:377–90. doi: 10.1007/BF02254443. [DOI] [PubMed] [Google Scholar]
  36. Louis NA, Hamilton KE, Colgan SP. Lipid mediator networks and leukocyte transmigration. Prostaglandins Leukot Essent Fatty Acids. 2005;73:197–202. doi: 10.1016/j.plefa.2005.05.006. [DOI] [PubMed] [Google Scholar]
  37. Magolda RL, Galbraith W. Design and synthesis of conformationally restricted phospholipids as phospholipase A2 inhibitors. J Cell Biochem. 1989;40:371–86. doi: 10.1002/jcb.240400313. [DOI] [PubMed] [Google Scholar]
  38. Moon Y, Pestka JJ. Vomitoxin-induced cyclooxygenase-2 gene expression in macrophages mediated by activation of ERK and p38 but not JNK mitogen-activated protein kinases. Toxicol Sci. 2002;69:373–82. doi: 10.1093/toxsci/69.2.373. [DOI] [PubMed] [Google Scholar]
  39. Monick MM, Robeff PK, Butler NS, Flaherty DM, Carter AB, Peterson MW, Hunninghake GW. Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA. J Biol Chem. 2002;277:32992–3000. doi: 10.1074/jbc.M203218200. [DOI] [PubMed] [Google Scholar]
  40. Nakano H, Nakajima A, Sakon-Komazawa S, Piao JH, Xue X, Okumura K. Reactive oxygen species mediate crosstalk between NF-kappaB and JNK. Cell Death Differ. 2006;13:730–7. doi: 10.1038/sj.cdd.4401830. [DOI] [PubMed] [Google Scholar]
  41. N’guessan PD, Etouem MO, Schmeck B, Hocke AC, Scharf S, Vardarova K, Opitz B, Flieger A, Suttorp N, Hippenstiel S. Legionella Pneumophila-induced PKC alpha-, MAPK- and NF-{kappa}B –dependent COX-2 expression in human lung epithelium. Am J Physiol Lung Cell Mol Physiol. 2006;292:L267–277. doi: 10.1152/ajplung.00100.2006. [DOI] [PubMed] [Google Scholar]
  42. Olivero J, Ganey PE. Participation of Ca2+/calmodulin during activation of rat neutrophils by polychlorinated biphenyls. Biochem Pharmacol. 2001;62:1125–32. doi: 10.1016/s0006-2952(01)00768-7. [DOI] [PubMed] [Google Scholar]
  43. Pathak SK, Bhattacharyya A, Pathak S, Basak C, Mandal D, Kundu M, Basu J. Toll-like receptor 2 and mitogen- and stress-activated kinase 1 are effectors of Mycobacterium avium-induced cyclooxygenase-2 expression in macrophages. J Biol Chem. 2004;279:55127–36. doi: 10.1074/jbc.M409885200. [DOI] [PubMed] [Google Scholar]
  44. Pawliczak R, Huang XL, Nanavaty UB, Lawrence M, Madara P, Shelhamer JH. Oxidative stress induces arachidonate release from human lung cells through the epithelial growth factor receptor pathway. Am J Respir Cell Mol Biol. 2002;27:722–31. doi: 10.1165/rcmb.2002-0033OC. [DOI] [PubMed] [Google Scholar]
  45. Pawliczak R, Logun C, Madara P, Lawrence M, Woszczek G, Ptasinska A, Kowalski ML, Wu T, Shelhamer JH. Cytosolic phospholipase A2 Group IValpha but not secreted phospholipase A2 Group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells. J Biol Chem. 2004;279:48550–61. doi: 10.1074/jbc.M408926200. [DOI] [PubMed] [Google Scholar]
  46. Singer CA, Baker KJ, McCaffrey A, AuCoin DP, Dechert MA, Gerthoffer WT. p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes. Am J Physiol Lung Cell Mol Physiol. 2003;285:L1087–98. doi: 10.1152/ajplung.00409.2002. [DOI] [PubMed] [Google Scholar]
  47. Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem. 2000;69:145–82. doi: 10.1146/annurev.biochem.69.1.145. [DOI] [PubMed] [Google Scholar]
  48. Steer SA, Moran JM, Christmann BS, Maggi LB, Jr, Corbett JA. Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages. J Immunol. 2006;177:3413–20. doi: 10.4049/jimmunol.177.5.3413. [DOI] [PubMed] [Google Scholar]
  49. Su B, Karin M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol. 1996;8:402–11. doi: 10.1016/s0952-7915(96)80131-2. [DOI] [PubMed] [Google Scholar]
  50. Sweeney SE, Firestein GS. Signal transduction in rheumatoid arthritis. Curr Opin Rheumatol. 2004;216:231–7. doi: 10.1097/00002281-200405000-00011. [DOI] [PubMed] [Google Scholar]
  51. Takano T, Cybulsky AV, Yang X, Aoudjit L. Complement C5b-9 induces cyclooxygenase-2 gene transcription in glomerular epithelial cells. Am J Physiol Renal Physiol. 2001;281:F841–50. doi: 10.1152/ajprenal.2001.281.5.F841. [DOI] [PubMed] [Google Scholar]
  52. Tithof PK, Peters-Golden M, Ganey PE. Distinct phospholipases A2 regulate the release of arachidonic acid for eicosanoid production and superoxide anion generation in neutrophils. J Immunol. 1998;160:953–60. [PubMed] [Google Scholar]
  53. Turyk M, Anderson HA, Hanrahan LP, Falk C, Steenport DN, Needham LL, Patterson DG, Jr, Freels S, Persky V Great Lakes Consortium. Relationship of serum levels of individual PCB, dioxin, and furan congeners and DDE with Great Lakes sport-caught fish consumption. Environ Res. 2006;100:173–83. doi: 10.1016/j.envres.2005.04.005. [DOI] [PubMed] [Google Scholar]
  54. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1 and 2. Annu Rev Pharmacol Toxicol. 1998;38:97–120. doi: 10.1146/annurev.pharmtox.38.1.97. [DOI] [PubMed] [Google Scholar]
  55. WHO, 2003 World Health Organisation (WHO) Polychlorinated Biphenyls: Human Health Aspects. 2003. Concise International Chemical Assessment Document 55; p. 36. [Google Scholar]
  56. Wu G, Luo J, Rana JS, Laham R, Sellke FW, Li J. Involvement of COX-2 in VEGF-induced angiogenesis via P38 and JNK pathways in vascular endothelial cells. Cardiovasc Res. 2006;69:512–9. doi: 10.1016/j.cardiores.2005.09.019. [DOI] [PubMed] [Google Scholar]
  57. Wullaert A, Heyninck K, Beyaert R. Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes. Biochem Pharmacol. 2006;72:1090–101. doi: 10.1016/j.bcp.2006.07.003. [DOI] [PubMed] [Google Scholar]

RESOURCES