p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression
Weidong Wu,1,2
Robert A. Silbajoris,3
Young E. Whang,4
Lee M. Graves,5
Philip A. Bromberg,2 and
James M. Samet3
1Division of Immunology and Infectious Disease, Department of Pediatrics, 2Center for Environmental Medicine, Asthma, and Lung Biology, 4Department of Medicine and Lineberger Comprehensive Cancer Center, and 5Department of Pharmacology, University of North Carolina, Chapel Hill; and 3Human Studies Division, National Health Effects and Environmental Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina
Submitted 29 April 2005
; accepted in final form 22 June 2005
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ABSTRACT
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Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.
zinc; airway epithelial cell; signal transduction
CYCLOOXYGENASE (COX) is a bifunctional enzyme with both cyclooxygenase and peroxidase activities (39). It catalyzes the conversion of membrane phospholipid-released arachidonic acid to prostaglandin H2 (PGH2), a precursor of all PGs, thromboxanes, and prostacyclins, in concert with a series of cell-specific isomerases (25, 35). Three isoforms of COX have been identified, named: COX-1, -2, and -3 (6). COX-1 is expressed constitutively in most tissues and appears to be responsible for the production of PGs that control physiological functions (35). COX-3 is an alternatively spliced form of COX-1 and expressed primarily in brain and heart as a constitutive enzyme. In contrast, COX-2 is undetectable or present at very low levels under basal conditions but is rapidly induced by mitogenic and inflammatory stimuli in a wide variety of cell types (35, 42). COX-dependent pathways are centrally involved in a large array of physiological and pathophysiological processes in the lung, such as the regulation of pulmonary vascular tone (32), vascular and interstitial tissue remodeling (8, 50), the regulation of capillary endothelial and alveolar epithelial permeability (12, 43), the control of bronchial mucous secretion and transport (15, 16, 44), and the regulation of bronchial tone including the pathogenesis of bronchial asthma and chronic obstructive lung disease (4, 45).
The regulation of COX-2 expression varies significantly with stimuli and cell types. For example, UVB-induced expression of COX-2 in HaCaT keratinocytes is associated with activation of EGF receptor (EGFR), extracellular signal-regulated kinase (ERK), p38 kinase, and phosphatidylinositol 3-kinase (PI3K) (3). Agents that interfere with microtubules, including taxanes, induce COX-2 by activating protein kinase C and mitogen-activated protein kinases (MAPKs) in human esophageal adenocarcinoma cells (57). Tumor necrosis factor (TNF)-
induces COX-2 and PGE2 production through the sphingosine kinase 1/sphingosine-1-phosphate pathway in an L929 fibroblast cell line (27). EGF stimulates COX-2 expression and PGE2 production in articular chondrocytes via ERK and p38 kinase signaling in association with differentiation status (18). Interleukin (IL)-1
upregulates COX-2 expression in HT-29 cells through ERK, p38, c-Jun NH2-terminal kinase (JNK), and PI3K pathways (22).
Zinc is an essential micronutrient involved in structural and regulatory cellular functions of a large number of proteins (47). On the other hand, zinc is also a common airborne metallic contaminant that derives from combustion, tire wear, and industrial processes that may contribute to the adverse health effects of ambient pollution (1, 2, 9, 10, 17). Our preliminary studies have shown that exposure of human bronchial epithelial cells to zinc ions (Zn2+) increases expression of proinflammatory mediators including IL-6, IL-8, TNF-
, and intercellular adhesion molecule-1. In addition, we have found Zn2+ exposure to induce COX-2 mRNA and protein expression through transcriptional and posttranscriptional mechanisms in the same cell type (Wu W, Kim Y, Silbajoris RA, Jaspers I, Graves LM, Bromberg PA, and Samet JM, unpublished observations). Although Zn2+ exposure has been shown to activate EGFR, Ras, MAPKs, and the PI3K/Akt pathways in human bronchial epithelial cells (31, 5155), the regulation of COX-2 expression by these activities in Zn2+-treated cells has not been characterized. This study characterizes the association of these intracellular signaling pathways with Zn2+-induced COX-2 expression in human bronchial epithelial cells and their cross talk during this event. Our data suggest that the PI3K/Akt signaling pathway plays a central role in Zn2+-induced COX-2 expression. EGFR, Src, and p38 kinases are involved in Zn2+-induced Akt activation as well as COX-2 expression. In contrast, ERK and JNK are dispensable in this process.
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MATERIALS AND METHODS
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Materials and reagents.
American Chemical Society-grade metal salt zinc sulfate, Triton X-100, and polyacrylamide were purchased from Sigma Chemical (St. Louis, MO). SDS-PAGE supplies such as molecular mass standards and buffers were from Bio-Rad (Richmond, CA). Anti-human COX-2 polyclonal antibody was obtained from Cayman Chemical (Ann Arbor, MI). Phospho-specific and pan antibodies against EGFR, Src, p38, JNK, glycogen synthase kinase (GSK), and Akt were obtained from Cell Signaling Technology (Beverly, MA). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) antibody was obtained from Cascade Bioscience (Winchester, MA).
-Actin antibody was purchased from USBiological (Swampscott, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The EGFR tyrosine kinase inhibitor PD-153035, the mitogen/ERK kinase activity inhibitor PD-98059, the Src kinase inhibitor PP2, the p38 kinase inhibitor SB-203580, the JNK inhibitor SP-600125, and the PI3K inhibitors LY-294002 and wortmannin were purchased from EMD Biosciences (San Diego, CA). FuGENE 6 transfection reagent was obtained from Roche Diagnostics (Indianapolis, IN). Chemiluminescence reagents were from Pierce Biotechnology (Rockford, IL).
Cell culture and in vitro exposure.
The BEAS-2B (subclone S6) cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus (SV) 40 construct (29). BEAS-2B cells (passages 7080) were grown to confluence on tissue culture-treated Costar plates in keratinocyte basal medium (KBM) supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human EGF, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin, as described previously (31). Cells were placed in KBM (without supplements) for 2022 h before treatment with zinc sulfate (Sigma) to reduce the background signaling in the cells.
Immunoblotting.
Cells with or without pretreatment of pharmacological inhibitors were treated with Zn2+, washed twice with cold phosphate-buffered saline (PBS), and then lysed in RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors: 20 µg/ml leupeptin, 20 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 200 µM sodium orthovanadate, and 20 mM sodium fluoride). Cell lysates were subjected to SDS-PAGE, as described before (54). Proteins were transferred onto nitrocellulose membrane. Membrane was blocked with 5% nonfat milk, washed briefly, incubated with primary antibody at 4°C overnight, followed by incubating with corresponding HRP-conjugated secondary antibody for 1 h at room temperature. Immunoblot images were detected using chemiluminescence reagents and the Gene Gynome Imaging System (Syngene, Frederick, MD).
RT-PCR.
BEAS-2B cells grown to confluence were exposed to Zn2+. Cells were washed twice with cold PBS and then lysed with TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was isolated according to manufacturer-provided instructions. RNA (200 ng) was reverse transcribed into cDNA as described previously (55). Quantitative PCR was performed with Platinum Quantitative PCR SuperMix-UDG (Invitrogen, Carlsbad, CA) and an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA). COX-2 mRNA levels were normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Relative amounts of COX-2 and GAPDH mRNA were based on standard curves prepared by serial dilution of cDNA from human BEAS-2B cells. The following oligonucleotide primers and probes were employed: COX-2: 5'-GAA TCA TTC ACC AGG CAA ATT G-3' (sense), 5'-TCT GTA CTG CGG GTG GAA CA-3' (antisense), 5'-TCC TAC CAC CAG CAA CCC TGC CA-3' (probe); GAPDH: 5'-GAA GGT GAA GGT CGG AGT C-3' (sense), 5'-GAA GAT GGT GAT GGG ATT TC-3' (antisense), 5'-CAA GCT TCC CGT TCT CAG CC-3' (probe).
Gene transfection.
BEAS-2B cells were grown to 4050% confluence in 24-well tissue culture plates and cotransfected with COX-2 promoter reporter constructs (p1.5COX-2-luc), kinase-deficient p38 constructs (pcDNA3.1p38-AF), and pSV-
-galactosidase constructs using FuGENE 6 transfection reagent. Cotransfection was conducted according to the manufacturer's instructions. Twenty-four hours after transfection, cultures were incubated with KBM overnight. The cells were then treated with 50 µM Zn2+ for 8 h before being lysed with lysis buffer. Detection of luciferase and
-galactosidase was conducted with the Dual-Light chemiluminescent reporter gene assay system from Tropix and an AutoLumat LB953 luminometer (Berthold Analytical Instruments, Nashua, NH). COX-2 promoter activity was estimated as specific luciferase activity (luciferase count/unit
-galactosidase count).
Adenovirus infection.
BEAS-2B cells grown to confluence were infected with control adenovirus adCMV3, adenovirus expressing dominant negative Akt [ad-DN-Akt(AAA)], or adenovirus expressing wild-type PTEN (ad-wt-PTEN) at a multiplicity of infection (MOI) of 100 plaque-forming units/cell for 24 h (53, 56). DN-Akt(AAA) is a kinase-dead mutant of Akt in which the phosphate transfer residue in the catalytic site (Lys179), and the two major regulatory phosphorylation sites (Thr308 and Ser473) are all replaced with alanine. This adenoviral construct was kindly provided by Dr. Kenneth Walsh (Boston University). The infection medium was removed and replaced with KBM overnight before challenge with 50 µM Zn2+ for 8 h. BEAS-2B cells were lysed, and the cell lysates were subjected to Western blotting or RT-PCR.
Statistics.
Data are presented as means ± SE. COX-2 mRNA and cotransfection data were carried out by one-way ANOVA for multigroup comparison.
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RESULTS
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Requirement of the PI3K/Akt signaling pathway for Zn2+-induced COX-2 expression.
The PI3K/Akt signaling axis plays an important role in cellular proliferation and growth signaling. Activation of the PI3K/Akt signaling pathway has also been associated with allergen-induced pathogenesis of pulmonary inflammation (21). Our previous study has shown that exposure to Zn2+ activates the PI3K/Akt signaling pathway in a human bronchial epithelial cell line, BEAS-2B (55). To examine whether this signaling pathway is involved in Zn2+-induced COX-2 expression, we employed specific pharmacological inhibitors or dominant negative constructs against this pathway before Zn2+ exposure. Exposure of BEAS-2B cells to 50 µM Zn2+ for 8 h did not result in significant alterations in cell viability, as assessed by assay of lactate dehydrogenase activity released into the culture medium (55). As shown in Fig. 1, A and B, LY-294002, a specific PI3K inhibitor, markedly suppresses Zn2+-induced COX-2 mRNA and protein expression in BEAS-2B cells, respectively. Similar results were observed with another PI3K inhibitor, wortmannin (data not shown). To determine whether Akt is also involved in this event, we infected BEAS-2B cells with ad-DN-Akt(AAA) for 24 h before Zn2+ treatment. The infected cells showed overexpression of Akt and significant reduction of Zn2+-induced phosphorylation of GSK-3
/
, a critical downstream component of the PI3K/Akt signaling pathway (36), compared with cells infected with control adenoviruses (data not shown). Overexpression of DN-Akt(AAA) blocked Zn2+-induced COX-2 mRNA expression (see Fig. 1C). These data suggest that the PI3K/Akt signaling pathway is required for Zn2+-induced COX-2 expression in human bronchial epithelial cells.

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Fig. 1. Requirement of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway for Zn2+-induced cyclooxygenase (COX)-2 expression. Confluent BEAS-2B cells were starved and pretreated with 40 µM LY-294002 before 50 µM Zn2+ treatment. Cells treated by Zn2+ for 4 h were lysed with TRIzol reagent. RNA was extracted and reverse transcribed before further analysis of COX-2 mRNA expression by quantitative PCR (A). Cells treated by Zn2+ for 8 h were lysed with RIPA buffer. Cell lysates were subjected to SDS-PAGE and immunoblotting with anti-human COX-2 antibody (B). Confluent cells were infected with adenovirus expressing dominant negative Akt [ad-DN-Akt(AAA)] at a multiplicity of infection (MOI) of 100 plaque-forming units (pfu)/cell for 24 h (C). Then cells were treated with 50 µM Zn2+ for 4 h. COX-2 mRNA expression was analyzed as described above. Ct, control. Data shown are representative of 3 separate experiments. *P < 0.05 compared with Zn(DMSO).
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PTEN protein is a putative antagonist of the PI3K/Akt pathway through dephosphorylation of the D3-phosphate group of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 is specifically produced from phosphatidylinositol 4,5-bisphosphate by PI3K (23). Our previous study has demonstrated that Zn2+ exposure induces PTEN protein degradation in BEAS-2B cells (55). It is assumed that overexpression of wild-type PTEN can block Zn2+-induced COX-2 expression. Overexpression of PTEN protein was detected in cells infected with ad-wt-PTEN (data not shown), and this inhibited Zn2+-induced Akt phosphorylation compared with cells infected with ad-CMV3 (Fig. 2A). As shown in Fig. 2B, overexpression of wild-type PTEN blocked Zn2+-induced COX-2 mRNA expression. These data suggest that PTEN inhibits the signal transduction from PIP3 to Akt in BEAS-2B cells exposed to Zn2+. Thus PI3K/PTEN/Akt signaling plays a crucial role in Zn2+-induced COX-2 expression in human bronchial epithelial cells.

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Fig. 2. Overexpression of wild-type phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks Zn2+-induced COX-2 mRNA expression. Confluent BEAS-2B cells were infected with adenovirus expressing wild-type PTEN (ad-wt-PTEN) at an MOI of 100 pfu/cell for 24 h. Cells were then treated with 50 µM Zn2+ for 4 h (B) or 8 h (A). Cell lysates were subjected to SDS-PAGE and immunoblotting using a phospho-specific antibody against Akt (p-Akt, A). RNA was extracted and reverse transcribed for analysis of COX-2 mRNA expression using quantitative PCR. COX-2 mRNA expression is presented as fold over Ct. Data shown are representative of 3 separate experiments. *P < 0.05 compared with Zn(CMV3).
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Involvement of the Src/EGFR signaling pathway in Zn2+-induced COX-2 expression.
Our previous studies have shown that exposure to Zn2+ results in EGFR phosphorylation in various cell types including BEAS-2B cells (30, 5154). Moreover, Src kinase activity was required for Zn2+-induced EGFR phosphorylation in mouse fibroblasts and A431 cells (30, 51). To examine whether a similar mechanism also exists in human bronchial epithelial cells, the phosphorylation of Src at Tyr416 was determined. Phosphorylation of Src (Tyr416) is part of the enzyme activation mechanism (46). Exposure to 50 µM Zn2+ induced a time-dependent increase in Src (Tyr416) and EGFR (Tyr1068) phosphorylation in BEAS-2B cells (see Fig. 3, A and B). Blockage of Src kinase activity with a specific Src inhibitor PP2 ablated Zn2+-induced EGFR phosphorylation (Tyr1068), implying a requirement for Src kinase activity in Zn2+-induced EGFR activation (Fig. 3C). As expected, the EGFR tyrosine kinase inhibitor PD-153035 completely blocked EGFR phosphorylation by stimulation with 50 µM Zn2+.

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Fig. 3. Zn2+-induced EGF receptor (EGFR) phosphorylation is mediated by Src kinase. Cells grown to confluence were starved in keratinocyte basal medium (KBM) overnight before treatment with zinc sulfate. Cells were exposed to 50 µM Zn2+ for 0, 30, 60, 120, and 240 min. Cells were lysed in RIPA buffer, and the lysates were subjected to SDS-PAGE and immunoblotting using phospho-specific antibodies and corresponding pan antibodies. Phosphorylation of Src at Tyr416 (A) and EGFR at Tyr1068 (B) was measured. Cells were pretreated with vehicle control (0.1% DMSO), PD-153035 (1 µM), or PP2 (10 µM) before treatment of 50 µM Zn2+. Cells were lysed, and phosphorylation of EGFR was determined as described above (C). Data shown are representative of 3 separate experiments.
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To ascertain the role of the Src/EGFR pathway in Zn2+-induced COX-2 expression, we pretreated BEAS-2B cells with PD-153035 or with PP2. Both PP2 and PD-153035 suppressed Zn2+-induced COX-2 protein expression (Fig. 4, A and B). Consistently, EGF, a cognate ligand of the EGFR signaling pathway, was shown to induce COX-2 expression in BEAS-2B cells (Fig. 4C). These data suggest that the Src/EGFR signaling is involved in Zn2+-induced COX-2 expression. However, although EGFR tyrosine kinase activity is required for Zn2+-induced ERK phosphorylation (52, 53), the ERK activation inhibitor PD-98059 had minimal effect on Zn2+-induced COX-2 expression. This suggests that EGFR regulates Zn2+-induced COX-2 expression through other downstream pathways.

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Fig. 4. The Src/EGFR signaling pathway is required for Zn2+-induced COX-2 protein expression. Confluent BEAS-2B cells were starved and then pretreated with vehicle control (0.1% DMSO), PP2 (10 µM) (A), or PD-153035 (1 µM) (B) before 50 µM Zn2+ treatment for 8 h. In addition, BEAS-2B cells were also treated with 100 ng/ml of EGF for 8 h (C). Cells were lysed with RIPA buffer. Cell lysates were subjected to SDS-PAGE and immunoblotting using an anti-human COX-2 antibody and a -actin antibody. Data shown are representative of at least 3 separate experiments.
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Requirement of p38, but not JNK kinase activity, for Zn2+-induced COX-2 expression.
Exposure of BEAS-2B cells to Zn2+ was demonstrated to activate p38 kinase in our previous study (31). To examine whether the p38 kinase is involved in Zn2+-induced COX-2 expression, we determined COX-2 protein expression or promoter activity in the presence of p38 kinase inhibitor SB-203580 or of overexpressed dominant negative p38 (p38-AF), doubly mutated at its activation domain. As shown in Fig. 5A, SB-203580 significantly inhibited Zn2+-induced COX-2 protein expression. Overexpression of p38-AF modestly suppressed Zn2+-induced elevation of COX-2 promoter reporter activity (Fig. 5B), implying that p38 may also affect COX-2 mRNA stability. These data suggest that p38 kinase is also a critical kinase in regulation COX-2 expression by Zn2+ exposure.
Activation of JNK has been observed in BEAS-2B cells exposed to Zn2+ previously (31). However, the specific JNK inhibitor SP-600125 failed to block Zn2+-induced COX-2 protein expression.
p38 and EGFR kinase activity are required for Zn2+-induced Akt phosphorylation.
As demonstrated before, Zn2+ stimulation activates the signaling pathways mediated by Src/EGF, PI3K/PTEN/Akt, and p38. It is assumed that these signaling pathways may cross talk during COX-2 expression in Zn2+-treated cells. To test this hypothesis, we employed specific kinase inhibitors. As shown in Fig. 6, A and B, the EGFR inhibitor PD-153035, the Src kinase inhibitor PP2, and the p38 kinase inhibitor SB-203580 markedly blocked Zn2+-induced Akt phosphorylation. As expected, the PI3K inhibitor wortmannin also ablated this event, but it did not affect Zn2+-induced phosphorylation of Src, EGFR, and p38. The ERK and JNK inhibitors failed to reduce Zn2+-induced Akt phosphorylation. These observations indicate that the PI3K/Akt signaling pathway plays a central role in Zn2+-induced COX-2 expression.

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Fig. 6. Src/EGF and p38 are involved in Zn2+-induced Akt phosphorylation. Confluent BEAS-2B cells were starved and then pretreated with vehicle control (0.1% DMSO), PP2 (10 µM), or PD-153035 (1 µM) before 50 µM Zn2+ stimulation (A) or vehicle control (0.1% DMSO), SB-203580 (20 µM), or LY-294002 (40 µM) before 50 µM Zn2+ treatment (B). Cells were lysed with RIPA buffer, and lysates were subjected to SDS-PAGE and immunoblotting using phospho-specific Akt antibody. Data shown are representative of at least 3 separate experiments.
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DISCUSSION
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Previous studies have suggested that COX-2 expression may be regulated through multiple signaling pathways (11, 22, 24, 37, 48). However, the regulation of COX-2 expression is cell type and stimulus specific. In this present study, Zn2+-induced COX-2 expression in human bronchial epithelial cells presents some novel features: First, multiple kinases including Src/EGFR, p38, and PI3K/Akt, and the phospholipid and protein phosphatase PTEN, but apparently not JNK, are involved in this process; Second, ERK, a downstream kinase of EGFR, is dispensable in Zn2+-induced COX-2 expression, implying that other downstream pathway(s) of EGFR is(are) involved; Third, the PI3K/Akt signaling pathway plays a central role in Zn2+-induced COX-2 expression since the Src/EGFR and p38 kinases act upstream of Akt in upregulating COX-2 expression by Zn2+ exposure. The main observations are summarized in Fig. 7.

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Fig. 7. Proposed mechanisms for intracellular signaling pathway-mediated COX-2 expression in Zn2+-treated BEAS-2B cells. Zn2+ exposure induces activation of Src, EGFR, and p38 kinases, and the degradation of PTEN. These signaling events converge to the activation of the PI3K/Akt signaling pathway, leading to the upregulation of COX-2 expression.
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The role of the PI3K/Akt pathway in COX-2 regulation seems controversial from previous reports. The PI3K inhibitor wortmannin was shown to slightly increase COX-2 mRNA expression in HT-29 colon cancer cells after IL-1
treatment (22). In contrast, wortmannin can inhibit COX-2 expression in endometrial cancer cells (37) and UVB-induced COX-2 expression in HaCaT keratinocytes (3). Our data suggest that the PI3K/Akt signaling pathway is required for Zn2+-induced COX-2 expression: the PI3K/Akt pathway is activated by Zn2+ stimulation in BEAS-2B cells (55); PI3K inhibitors such as LY-294002 and wortmannin ablated Zn2+-induced COX-2 protein expression, and overexpression of dominant negative Akt blocks Zn2+-induced COX-2 mRNA expression. Moreover, overexpression of wild-type tumor suppressor PTEN suppresses Zn2+-induced Akt phosphorylation and COX-2 mRNA expression, which is similar to the effect of another tumor suppressor p53 on COX-2 expression (38). The signaling events relaying PI3K/Akt signaling to Zn2+-induced COX-2 expression are unclear.
Overexpression of COX-2 appears to be a consequence of both increased transcription and enhanced mRNA stability (13, 33). A variety of transcription factors, including activator protein-1 (AP-1), nuclear factor IL-6, nuclear factor
B (NF-
B), nuclear factor of activated T cells, and polyomavirus enhancer activator 3, can modulate the transcription of COX-2, depending on the cell type and stimulus (35, 41). Our preliminary studies have demonstrated that Zn2+ stimulation leads to increase in promoter activity of both NF-
B and AP-1. PI3K/Akt signaling has been proposed to induce NF-
B-mediated upregulation of COX-2 following I
B phosphorylation and degradation (7, 34, 37). Our recent studies, however, showed that Zn2+ exposure triggered NF-
B activation through an I
B-independent mechanism. Furthermore, the PI3K inhibitor wortmannin produced only a slight inhibition of NF-
B promoter activity in Zn2+-exposed BEAS-2B cells. The mechanisms of Zn2+-induced NF-
B and AP-1 activation and their involvement in Zn2+-induced COX-2 expression require further study. In addition, whether PI3K/Akt signaling affects COX-2 mRNA stability remains to be determined.
In comparison to previous studies that characterize the effect of independent kinases or pathways on COX-2 regulation, the present study is the first to demonstrate that p38 and EGFR kinases regulate Zn2+-induced COX-2 expression through modulating the function of the critical serine/threonine kinase Akt. The EGFR inhibitor blocks Zn2+-induced Akt phosphorylation, but the PI3K inhibitor LY-294002 has little inhibitory effect on Zn2+-induced EGFR phosphorylation, implying that EGFR acts upstream of PI3K/Akt activation. The Shc/Grb2/Sos/Ras/ERK cascade is a major mitogenic signaling pathway initiated by the EGFR. In other studies, ERK is proposed as an important mediator in EGFR-regulated COX-2 expression and function by a variety of stimuli (3, 18, 22, 26, 40, 58). However, our data indicate that ERK is not involved in Zn2+-induced Akt phosphorylation and COX-2 expression. Thus the molecules relaying signals from EGFR to Akt must be located upstream of ERK. In fact, growth factor receptor tyrosine kinases including EGFR can directly recruit and activate the PI3K/Akt pathway independently of their downstream signaling components such as ERK (20). Zn2+ exposure can activate Ras in BEAS-2B cells (53). GTP-bound Ras can also directly recruit and activate the catalytic subunit of PI3K (p110), leading to the activation of the PI3K/Akt signaling (14).
In regard to the requirement of Akt phosphorylation for p38 kinase activity in Zn2+-treated BEAS-2B cells, the mechanisms involved remain speculative. Our data tend to support the explanation that p38 kinase induces Akt phosphorylation through PI3K in that the p38 inhibitor SB-203580 inhibits Zn2+-induced Akt phosphorylation and the PI3K inhibitors have minimal effect on Zn2+-induced p38 phosphorylation.
Airway inflammation is a common mechanism for diverse lung diseases, which is driven by a variety of proinflammatory mediators released from inflammatory, structural/resident lung cell types. These mediators include: nitric oxide, metabolites of arachidonic acid, growth factors, cytokines, and chemotactic cytokines. The expression of proinflammatory mediators are regulated by stimulus-specific signaling pathways (28, 49). This study displays an interactive signaling network responsible for Zn2+-induced COX-2 expression in human bronchial epithelial cells. It should be noted that some of the inhibitors used to modulate specific signaling intermediates could have secondary effects on Zn2+ transporters, some of which may interact with these signaling pathways (5, 19). These findings may suggest pharmacological approaches in the prevention and treatment of pulmonary disorders resulting from Zn2+ inhalation.
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GRANTS
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This work was supported by United States Environmental Protection Agency Cooperative Agreement CR#829522 awarded to the Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina; United States Environmental Protection Agency STAR Grant R829214 to L. M. Graves and W. Wu; and National Cancer Institute Grant CA-85772 to Y. Whang.
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ACKNOWLEDGMENTS
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We greatly appreciate Dr. Ilona Jaspers and Lisa Dailey for technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: W. Wu, Center for Environmental Medicine, Asthma and Lung Biology, Univ. of North Carolina at Chapel Hill, NC 27599 (e-mail: Weidong_Wu{at}med.unc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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