HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 290/5/L1028    most recent 00479.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (19) Citing Articles via Google Scholar Google Scholar Articles by Kim, Y.-M. Articles by Samet, J. M. Search for Related Content PubMed PubMed Citation Articles by Kim, Y.-M. Articles by Samet, J. M.

Zn²⁺-induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells

¹Department of Environmental Sciences and Engineering, ²Center for Environmental Medicine, Asthma, and Lung Biology, ³Department of Pharmacology, University of North Carolina, Chapel Hill; and ⁴Human Studies Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

Submitted 10 November 2005 ; accepted in final form 20 December 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zinc. In this study, we examined the cellular mechanisms responsible for Zn²⁺-induced IL-8 expression. Zn²⁺ stimulation resulted in pronounced increases in both IL-8 mRNA and protein expression in the human airway epithelial cell line (BEAS-2B). IL-8 promoter activity was significantly increased by Zn²⁺ exposure in BEAS-2B cells, indicating that Zn²⁺-induced IL-8 expression is transcriptionally mediated. Mutation of the activating protein (AP)-1 response element in an IL-8 promoter-enhanced green fluorescent protein construct reduced Zn²⁺-induced IL-8 promoter activity. Moreover, Zn²⁺ exposure of BEAS-2B cells induced the phosphorylation of the AP-1 proteins c-Fos and c-Jun. We observed that Zn²⁺ exposure induced the phosphorylation of ERK, JNK, and p38 MAPKs, whereas inhibition of ERK or JNK activity blocked IL-8 mRNA and protein expression in BEAS-2B cells treated with Zn²⁺. In addition, we investigated the role of protein tyrosine phosphatases in the activation of signaling by Zn²⁺. Zn²⁺ treatment inhibited ERK- and JNK-directed phosphatase activities in BEAS-2B cells. These results suggested that Zn²⁺-induced inhibition of phosphatase activity is an initiating event in MAPK and AP-1 activation that leads to enhanced IL-8 expression by human airway epithelial cells.

interleukin-8; zinc; activating protein-1; c-Jun NH₂-terminal kinase; extracellular signal-regulated kinase; mitogen-activated protein kinase phosphatase; metal; particulate matter

Interleukin (IL)-8 is a pivotal chemotactic cytokine in the human lung that is responsible for the recruitment of neutrophils and macrophages to the site of inflammation. Elevated levels of IL-8 are associated with several pathophysiological states in the lung, including bronchoconstriction, edema, and neutrophilia (39, 55). In humans, the airway epithelium is a major source of IL-8. Levels of IL-8 expression by human airway epithelial cells (HAEC) increase in response to ambient PM and PM components such as zinc (12, 19, 21, 26, 49, 60). The level of IL-8 expression is under the control of multiple signaling cascades in HAEC, including NF-B and the MAPK pathways ERK, JNK, and p38. MAPK regulation of IL-8 expression is mediated through the activation of the transcription factors Fos, Jun, and activating transcription factor, which are categorized as activating protein (AP)-1 family proteins (24, 36, 47, 48).

The MAPKs have previously been described as regulators of the AP-1 transcription factor and linked to IL-8 gene expression (48). Fos proteins including c-Fos, FosB, and Fra1 are known to be phosphorylated by ERK on serine and/or threonine residues in the COOH-terminal domain (40, 47). JNK is well characterized as a specific kinase that binds and phosphorylates Jun proteins including c-Jun, JunB, and JunD (47). Several lines of evidence indicate that phosphorylation of c-Jun and c-Fos by MAPK contributes to both the abundance and activity of AP-1 proteins. Phosphorylation of c-Jun on Ser⁶³ and Ser⁷³ by JNK increases the stability of c-Jun by reducing its ubiquitination and degradation. In addition, it increases the transactivating potential, enhancing AP-1-dependent transcriptional activity (24). In the case of c-Fos, ERK-dependent phosphorylation of c-Fos in the COOH-terminal domain results in a marked increase in its transactivating potential (40).

Zinc exposure has been reported to activate several MAPKs and their associated downstream transcription factors in human airway epithelial cells (49, 58). Levels of MAPK phosphorylation are regulated by the kinase activity of the dual-specificity kinases known as the MAPK kinases (MAPKKs) and the dephosphorylating activity of the MAPK phosphatases (MKPs) (8). In contrast to MAPKKs, MKPs negatively regulate MAPK activity by dephosphorylating tyrosine, serine/threonine, or both tyrosine and threonine (dual specificity) residues of the TXY motif in MAPK. The activity of tyrosine phosphatases can be inhibited by exposure to a number of extracellular stimuli including vanadium, arsenite, and oxidative stress in a various cell types (3, 34, 50, 51). Exposure to zinc has also been shown to inhibit phosphatase activity in various cell types, including HAEC (38, 50).

The underlying mechanisms responsible for Zn²⁺-induced expression of IL-8 remain to be understood. In this study, we examined the molecular signaling of Zn²⁺-induced IL-8 expression in HAEC. Herein, we report that ERK, JNK, and AP-1 are responsible for Zn²⁺-induced IL-8 expression in HAEC. In addition, Zn²⁺ inhibits the activities of ERK- and JNK-directed phosphatases. These results suggest that Zn²⁺-induced inhibition of phosphatase activity contributes to the molecular signaling events, including MAPK and AP-1 activation, that lead to Zn²⁺-induced IL-8 expression.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Reagents. Common laboratory chemicals including BSA and 2-mercaptoethanol were purchased from Sigma-Aldrich (St. Louis, MO). SDS-PAGE supplies were obtained from Bio-Rad (Richmond, CA). Kinase inhibitors, such as PD-98059 (2'-amino-3'-methoxyflavone), PD-153035 {4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline}, SP-600125 [anthra(1,9-cd)pyrazol-6(2H)-one], and SB-203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] were purchased from Calbiochem (San Diego, CA). Specific anti-phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵), anti-phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²), anti-phospho-ERK (Thr²⁰²/Tyr²⁰⁴), anti-phospho-c-Jun (Ser⁶³ and Ser⁷³) anti-ERK, anti-JNK, anti-p38, and anti-c-Jun were obtained from Cell Signaling Technology (Beverly, MA). Anti-phospho-c-Fos (Thr²³²) antibody was purchased from Biosource International (Camarillo, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse secondary antibodies and anti-c-Fos antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Active GST-tagged ERK2 or 6xHis-tagged JNK2 and recombinant MKP-1 were purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture. BEAS-2B (subclone S6) cells were obtained from the Human Studies Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and maintained in keratinocyte growth medium (Cambrex Bioproducts, Clonetics Division, San Diego, CA). This cell line was derived by transformation of human airway epithelial cells with an ad12-SV40 adenovirus construct (46).

IL-8 expression analyses. BEAS-2B cells were lysed in a buffer containing guanidine isothiocyanate and purified using an RNeasy kit according to manufacturer's instructions (Qiagen, Valencia, CA). Total RNA (100 ng), 0.5 mM NTP (Pharmacia, Piscataway, NJ), 5 µM random hexaoligonucleotide primers (Pharmacia, NJ), 10 U/µl RNase inhibitor (Promega, CA), and 10 U/µl Moloney murine leukemia virus RT (GIBCO-BRL Life Technologies) were incubated in a 40°C water bath for 1 h in 50 µl of 1x PCR buffer to synthesize first-strand cDNAs. The reverse transcription was inactivated by heating at 92°C for 5 min. Quantitative PCR of IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specimen cDNA and standard cDNA was performed in a 50-µl final volume mixture containing TaqMan master mix (Perkin-Elmer, CA), 1.25 µM probe, 3 µM forward primer, and 3 µM reverse primer. The probe anneals to the template between the two primers. This probe contains both a fluorescence reporter dye at the 5'-end (6-carboxyfluorescein: emission _max = 518 nm) and a quencher dye at the 3'-end (6-carboxytetramethyl rhodamine: emission _max = 582 nm). During polymerization, the probe is degraded by the 5'-3' exonuclease activity of the Taq DNA polymerase, and the fluorescence is detected by a laser in the sequence detector (TaqMan ABI Prism 7700 Sequence Detector System; Perkin-Elmer, CA). Thermal cycler parameters include 2 min at 50°C, 10 min at 95°C, and 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. The abundance of IL-8 and GAPDH transcripts in first-strand cDNAs was measured by TaqMan quantitative PCR using the oligonucleotide primers and probes previously described (20, 21), TaqMan Universal MasterMix and an Applied Biosystems 7700 Sequence Detection System.

Release of IL-8 protein into the cell culture supernatant was assayed with a human IL-8 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Western blotting. Preparation of cytoplasmic and nuclear extracts from BEAS-2B cells for nuclear protein detection was performed as described(20, 21). Cytoplasmic extraction buffer (CEB) consisted of 10 mM Tris·HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM DTT, and protease inhibitors (PI; 1 mM Pefabloc, 50 µg/ml antipain, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 40 µg/ml bestatin, 3 µg/ml E-64, and 100 µg/ml chymostatin; all purchased from Boehringer Mannheim Roche Applied Science, Indianapolis, IN). Cells were scraped into CEB, transferred into a microcentrifuge tube, and placed on ice for 15 min. Nonidet P-40 (NP-40; Sigma-Aldrich, MO) was added to a final concentration of 0.1% and vortexed for 10 s. Nuclei were pelleted by centrifugation at 15,000 g for 30 s; we repeated this after washing the nuclei with CEB. The supernatant was removed, and the nuclei were incubated for 10 min on ice in a nuclear extraction buffer (consisting of 20 mM Tris·HCl, pH 8.0, 400 mM NaCl, 1.5 mM MgCl₂, 1.5 mM EDTA, and 1 mM DTT, 25%) with PI to extract nuclear proteins. After brief centrifugation, the supernatants, containing the nuclear proteins, were stored at –80°C until ready for used.

Gel electrophoresis followed by Western blotting was performed essentially as described by Laemmli (32) and Towbin et al. (56a). Cells were lysed with radioimmunoprecipitation assay lysis buffer containing 1% NP-40, 0.5% deoxycholate, and 0.1% SDS in PBS at pH 7.4, phosphatase inhibitor cocktail set I and II, and protease inhibitor cocktail set III purchased from Calbiochem (San Diego, CA). We normalized each sample for a protein content of 50–100 µg before loading it on a SDS-PAGE gel. Lysates were mixed with one volume of reducing SDS-PAGE loading buffer containing 0.125 M Tris (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.05% bromphenol blue. The samples were heated for 1 min at 90°C, run on 10% SDS-PAGE gels with prestained molecular weight markers (Bio-Rad, CA) in Tris-glycine-SDS buffer (Bio-Rad), and electroblotted on nitrocellulose membranes (Bio-Rad). The blots were blocked with 5% nonfat milk and incubated with primary antibodies. HRP-conjugated goat anti-rabbit antibody was used as a secondary antibody. Protein bands on the membrane were detected with the enhanced chemiluminescence detection solution (Amersham Biosciences, Piscataway, NJ) and exposed on high-performance chemiluminescence film (Amersham Biosciences). The bands were quantified by a Kodak electrophoresis documentation and analysis system (Rochester, NY).

Promoter-reporter activity assays. An IL-8 promoter-reporter cassette was generated that was composed of the transcription-silencing sequence (bases 2,193–2,689) from pNF-Bluc (Stratagene) upstream of a 1.46-kb fragment of the IL-8 gene (–1,410 to +54) followed by the coding sequence of enhanced green fluorescent protein (EGFP) and an SV40 polyadenylation sequence. This cassette was inserted in the multicloning site of pShuttle (Clontech, Palo Alto, CA). A recombinant plasmid, psh-IL8pro-EGFP, bearing the desired sequence was used by the University of North Carolina (UNC) Vector Core Facility to generate a recombinant adenovirus.

Wild-type IL-8 promoter activity was assayed using the recombinant adenoviral IL-8 promoter-reporter vector, AdIL-8proEGFP, previously described (26). Mutant IL-8 promoter-reporter adenovirus having an inactivated AP-1 response element was generated as follows. The AP-1 response element of the IL-8 promoter (bases –126 to –120) was changed from TGACTCA to TatCTCA by site-directed mutagenesis of psh-IL8pro-EGFP, the wild-type IL-8-EGFP promoter reporter adenoviral shuttle vector (26). Adenoviruses carrying the mutant promoter-reporter gene were generated by the UNC Vector Core Facility.

Transduction of cells with adenovirus was as described previously (26). In brief, BEAS-2B cells grown to 80% confluence in 24-well plates were transduced with AdIL-8pro-EGFP at a multiplicity of infection of 500 focus-forming units/cell for 8 h. The recovery period of 24 h was followed by the challenge with 50 µM ZnSO₄ or TNF- (10 ng/ml). IL-8 promoter activity was measured as follows: cells were put in suspension by brief treatment with trypsin-EDTA at 37°C and assessed for mean EGFP fluorescence intensity using a FACSsort flow cytometer (Becton Dickinson, San Jose, CA).

Phosphatase activity assays. We used a nonradioactive method for determining phosphatase activity in whole cell lysates. Recombinant, phosphorylated substrates of phosphatases including phospho-ERK or phospho-JNK were dephosphorylated by the activity of phosphatases in cell lysates and detected by Western blotting using anti-phospho-ERK or -JNK antibodies. To measure ERK- or JNK-directed phosphatase activity in the cells, BEAS-2B cells were exposed to media, or 50 µM Zn²⁺ or V⁴⁺, for 30 min, and were lysed in low-salt buffer containing 100 mM HEPES, 0.2% NP-40 (Sigma-Aldrich), and PI cocktail (Calbiochem) for the ERK-directed phosphatase activity assay or in high-salt lysis buffer containing 20 mM Tris·HCl (pH 8.0), 400 mM NaCl, 1.5 mM MgCl₂ PI cocktail, and 0.2% NP-40 for the JNK-directed phosphatase activity assay. We diluted 20 or 150 µg of whole cell lysate for ERK- or JNK-directed phosphatase activity assay, respectively, into a total volume of 30 or 50 µl in phosphatase assay buffer (350 mM sucrose, 20 mM HEPES, and 150 mM KCl). We added 10 ng of recombinant, phosphorylated GST-tagged ERK2 or 6xHis-tagged JNK2 (Upstate Biotechnology, Lake Placid, NY) to each sample, and the reactions were incubated at 37°C for 30 min. To measure the activity of recombinant MKP-1 (Upstate) under 50 µM Zn²⁺ or V⁴⁺, 50 units of recombinant MKP-1 were diluted to as total volume of 30 µl in phosphatase assay buffer and reacted with 50 µM Zn²⁺ or V⁴⁺ at 37°C for 5 min. We added 10 ng of recombinant phosphorylated ERK or JNK to each sample and subjected it to further reaction at 37°C for 30 min. We stopped the reactions by adding 10 µl of Laemmli buffer and boiling for 5 min. Western blotting was performed to detect phosphorylated ERK or JNK.

Statistical analysis. Data are presented as means ± SE of at least three separate experiments. Data comparisons were carried out by one-way ANOVAs followed by Dunnett's posttest and two-tailed Student's t-test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Zn²⁺ exposure upregulates IL-8 expression in human airway epithelial cells. To ascertain the effect of an acute, noncytotoxic exposure to soluble Zn²⁺ ions on HAEC, BEAS-2B cells were grown to subconfluence in 24-well plates and exposed to 50 µM ZnSO₄ for 20 h. This exposure dose and duration were found to have no effect on cell viability (data not shown). Exposure of BEAS-2B cells to Zn²⁺ for 12 h significantly induced IL-8 protein release into the cell culture medium in a dose-dependent manner. Exposure to 15 or 50 µM Zn²⁺ induced IL-8 protein release by 1.6- and 4.6-fold, respectively (Fig. 1A). Similarly, Zn²⁺ induced IL-8 mRNA expression was dose dependent. Exposure of BEAS-2B cells to 15, 50, and 100 µM Zn²⁺ for 3 h induced IL-8 mRNA expression 2-, 7-, and 10-fold, respectively (Fig. 1B). Figure 1C shows the time course of IL-8 mRNA expression in BEAS-2B cells exposed to 50 µM Zn²⁺. Increases in Zn²⁺-induced IL-8 mRNA expression reached statistical significance as early as 2 h and continued to increase up to 4 h of exposure.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Zn2+ induces interleukin (IL)-8 mRNA and protein expression in human airway epithelial cells (BEAS-2B). A: BEAS-2B cells were treated with 15 or 50 µM Zn2+ for 12 h. The concentration of IL-8 protein in the culture supernatant was determined by ELISA and expressed as the fold increase over control. B: BEAS-2B cells were treated with 15–100 µM Zn2+ for 3 h. Levels of IL-8 mRNA were analyzed by real-time RT-PCR. IL-8 mRNA induction was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and expressed as the fold increase over control. C: BEAS-2B cells were treated with 50 µM Zn2+ up to 4 h. IL-8 mRNA induction was normalized to GAPDH levels and expressed as the fold increase over control. D: BEAS-2B cells were infected with an adenoviral IL-8-enhanced green fluorescent protein (EGFP) promoter reporter vector and challenged with Zn2+ (50 µM) or tumor necrosis factor (TNF)- (10 ng/ml) for 12 h. IL-8 promoter activity (mean EGFP fluorescence intensity/cell) was determined by flow cytometry. Data represent means ± SE of 3 independent experiments. *P < 0.05 compared with control (Ct).

Zn²⁺ exposure induces MAPK phosphorylation in BEAS-2B cells. To determine whether Zn²⁺ increases the phosphorylation of MAPKs in BEAS-2B cells, we exposed the cells to 50 µM Zn²⁺ for 15 min–2 h and analyzed levels of phosphorylated MAPK by Western blotting. We observed that exposure of BEAS-2B cells to 50 µM Zn²⁺ induced a rapid and marked phosphorylation of the MAPKs ERK, JNK, and p38 at Thr²⁰²/Tyr²⁰⁴, Thr¹⁸³/Tyr¹⁸⁵, and Thr¹⁸⁰/Tyr¹⁸², respectively, which was detectable as early as 15 min and continued to increase through 2 h of continuous exposure (Fig. 2, A–C).

View larger version (53K): [in this window] [in a new window]   Fig. 2. Zn2+ induces the phosphorylation of ERK1/2, JNK, and p38 in BEAS-2B cells. BEAS-2B cells were treated with Zn2+ (50 µM) for 15 min–2 h. Cell lysates were separated by SDS-PAGE and immunoblotted with ERK1/2, phospho-ERK1/2(Thr202/Tyr204) (A), JNK, phospho-JNK(Thr183/Tyr185) (B), p38, and phospho-p38(Thr180/Tyr182) (C) antibodies.

View larger version (20K): [in this window] [in a new window]   Fig. 3. ERK and JNK activities are involved in IL-8 mRNA and protein expression induced by Zn2+ exposure. Cells pretreated with DMSO (vehicle), ERK inhibitor (PD-98059, 10 µM), JNK inhibitor (SP-600125, 10 µM), or p38 inhibitor (SB-203580, 10 µM) for 30 min were incubated with Zn2+ (50 µM) for 4 h (mRNA) or 12 h (protein). IL-8 mRNA levels were normalized to levels of GAPDH. Data are expressed as the fold increase over control; shown are means ± SE of 3 independent experiments. Significance from untreated controls (*) and significance from Zn2+-treated IL-8 level (#), P < 0.05 compared with control.

View larger version (66K): [in this window] [in a new window]   Fig. 4. Zn2+ induced nuclear phosphorylation of activating protein (AP)-1 family proteins, c-Fos (A) and c-Jun (B), in BEAS-2B cells. Nuclear extracts from BEAS-2B cells that had been treated with 50 µM Zn2+ for 30 min–4 h were separated by SDS-PAGE and immunoblotted with c-Fos, phospho-c-Fos(Thr232), c-Jun, or phospho-c-Jun(Ser63 and Ser73) antibodies.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Zn2+-induced IL-8 promoter activity depends on the AP-1 response element of the IL-8 promoter. BEAS-2B cells were infected with either a wild-type (WT) adenoviral IL-8 promoter EGFP construct or the same vector with the AP-1 response element mutated. After a 24-h recovery period, the cells were challenged with Zn2+ (50 µM) or TNF- (10 ng/ml) for 10 h. IL-8 promoter activity (mean EGFP fluorescence intensity/cell) was determined by flow cytometry. Data shown represent means ± SE of 3 independent experiments. Significance from untreated controls (*) and significance from Zn2+-treated WT IL-8 promoter (#), P < 0.05 compared with control.

We first measured the rate of dephosphorylation of recombinant phospho-ERK or phospho-JNK by lysates prepared from BEAS-2B cells that were exposed to 50 µM Zn²⁺ or V⁴⁺ for 30 min. As shown in Fig. 6, Zn²⁺ or V⁴⁺ exposure to BEAS-2B cells for 30 min significantly retarded the dephosphorylation rate of recombinant phospho-ERK (Fig. 6A) or JNK (Fig. 6B). In our experiments, V⁴⁺ was used as a reference protein tyrosine phosphatase (PTP) inhibitor for comparison with the effects of Zn²⁺.

View larger version (40K): [in this window] [in a new window]   Fig. 6. Zn2+ inhibits ERK- and JNK-directed phosphatase activities in BEAS-2B cells. BEAS-2B cells were treated with 50 µM Zn2+ or 50 µM V4+ for 30 min, and then whole cell extracts were analyzed for MAPK phosphatase activity by incubation with phosphorylated recombinant ERK and JNK. Dephosphorylation of phospho-ERK (A) or phospho-JNK (B) by cell lysates was monitored by Western blotting over a 60-min assay period.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

View larger version (16K): [in this window] [in a new window]   Fig. 7. Proposed model responsible for Zn2+-induced IL-8 expression in BEAS-2B cells. Zn2+ induces the inhibition of ERK- and JNK-directed phosphatases activities, resulting in the accumulation of phosphorylated ERK or JNK. These events are followed by the activation of AP-1 family proteins including c-Fos and c-Jun, leading to IL-8 promoter activation and subsequent IL-8 transcription.

AP-1, comprising Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra1, and Fra2) family members, plays a central role in regulating gene transcription involved in cell proliferation and differentiation, apoptosis, inflammation, and the immune response. Various toxic stimuli including PM, ultrafine particles, diesel extracts, arsenite, tobacco smoke, and hydrogen peroxide have been reported to activate AP-1 proteins and increase its transactivating potential and binding to DNA (3, 11, 23, 33, 43, 52). In this study, we demonstrate that the AP-1 family proteins c-Jun and c-Fos are phosphorylated in response to Zn²⁺ exposure. These data are consistent with the finding that Zn²⁺-induced IL-8 expression is dependent on the presence of a functional AP-1 response element in the IL-8 promoter.

In addition to AP-1, other transcription factors such as NF-B and NF-IL-6 are important transcription factors activating the IL-8 promoter (45, 48). In a separate study, we have observed that Zn²⁺ induces the activity of a reporter construct governed by a 5X tandem repeat of the NF-B response element in BEAS-2B cells, suggesting that NF-B might be involved in Zn²⁺-induced IL-8 promoter activity (Y.-M. Kim, unpublished observations). Therefore, Zn²⁺-induced IL-8 promoter activity appears to be activated by the coordination of a number of transcription factors including AP-1 and NF-B.

Intracellular levels of phosphorylated MAPKs are regulated by the opposing activities of the MAPKKs and the MAPK phosphatases. MAPKKs including MEK1/2, MKK4/7, and MKK3/6 phosphorylate TXY motifs in the activation loops of ERK, JNK, and p38, respectively. On the other hand, MAPK phosphatases negatively regulate MAPK activity by dephosphorylating tyrosine, serine/threonine, or both tyrosine and threonine (dual specificity) residues of TXY motif in MAPK. The tyrosine-specific MKPs include PRP-SL, STEP, and HePTP, whereas serine/threonine-specific MKPs are PP2A and PP2C. Dual-specificity MKPs include MKP-1, MKP-2, hVH3/B23, MKP-3, MPK-X, MKP-4, MKP-5, hVH-5/M/3/6, PAC-1, and MKP-6. MKPs differ in terms of substrate specificity, tissue distribution, subcellular localization, and target gene regulation (8, 22, 51, 57).

In the present study, we demonstrate that Zn²⁺ inhibits the activities of endogenous phosphatases directed at phospho-ERK or phospho-JNK in BEAS-2B cells. These data suggest the existence of specific phosphatases that normally function to dephosphorylate ERK or JNK and lose their activities upon exposure of BEAS-2B cells to Zn²⁺. One potential phosphatase involved in this effect is MKP-1, a dual-specificity MPK that is known to dephosphorylate MAPKs including ERK and JNK (8, 10, 54). The expression of MKP-1 induced by glucocorticoids at the promoter level has been proposed as a mechanism for inactivation of ERK1/2 and subsequent anti-inflammatory effects of glucocorticoids (25). We have observed that MKP-1 is expressed in BEAS-2B cells and demonstrated that the activity of recombinant MKP-1 is inhibited by Zn²⁺ and V⁴⁺ in vitro, suggesting that Zn²⁺ inhibits the activities of ERK- or JNK-directed phosphatases such as MKP-1 in BEAS-2B cells (Y.-M. Kim, unpublished observations). Additional studies will be required to identify the specific PTPs the inhibition of which leads to the activation of MAPKs and the expression of IL-8 in response to Zn²⁺ treatment.

Zn²⁺ has been reported to inhibit PTPs possibly through the binding to the thiol group of the essential catalytic cysteine residue in the phosphatase active site (37, 38). Most MKPs have an essential cysteine residue in the catalytic domain that might be the target of Zn²⁺-induced inhibition (8, 22). Therefore, it is possible to speculate that thiol binding of Zn²⁺ is the mechanism of Zn²⁺-induced inhibition of ERK or JNK-directed phosphatase activity, leading to unopposed MAPKK activity, an accumulation of phospho-ERK and phospho-JNK, and ensuing activation of AP-1-dependent IL-8 expression in human airway epithelial cells.

In summary, we show here that Zn²⁺-induced IL-8 expression involves ERK, JNK, and AP-1 activities in HAEC. In addition, we show that Zn²⁺ induces the phosphorylation and activation of ERK and JNK, likely through the inhibition of ERK- and JNK-directed phosphatase activities. Our data suggest a mechanism for the initiation of signaling processes that underlie adverse responses induced by exposure to PM constituents.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The research described herein has been reviewed by the National Health and Environmental Effects Research Laboratory and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. EPA, nor does mention of trade names constitute endorsement of recommendation for use.

	ACKNOWLEDGMENTS

 
We acknowledge Dr. R. Jude Samulski and the UNC Vector Core for preparation of the AdIL8pro-EGFP and AdIL-8MAP-1pro-EGFP.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. M. Samet, 104 Mason Farm Rd., US EPA, Human Studies Facility, Chapel Hill, NC, 27514 (e-mail: Samet_James{at}epa.gov)

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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Adamson IY, Prieditis H, Hedgecock C, and Vincent R. Zinc is the toxic factor in the lung response to an atmospheric particulate sample. Toxicol Appl Pharmacol 166: 111–119, 2000.[CrossRef][Web of Science][Medline]
2. Barchowsky A, Soucy NV, O'Hara KA, Hwa J, Noreault TL, and Andrew AS. A novel pathway for nickel-induced interleukin-8 expression. J Biol Chem 277: 24225–24231, 2002.[Abstract/Free Full Text]
3. Cavigelli M, Li WW, Lin A, Su B, Yoshioka K, and Karin M. The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J 15: 6269–6279, 1996.[Web of Science][Medline]
4. Claiborn CS, Larson T, and Sheppard L. Testing the metals hypothesis in Spokane, Washington. Environ Health Perspect 110, Suppl 4: 547–552, 2002.[Medline]
5. Clarke RW, Coull B, Reinisch U, Catalano P, Killingsworth CR, Koutrakis P, Kavouras I, Murthy GG, Lawrence J, Lovett E, Wolfson JM, Verrier RL, and Godleski JJ. Inhaled concentrated ambient particles are associated with hematologic and bronchoalveolar lavage changes in canines. Environ Health Perspect 108: 1179–1187, 2000.[Web of Science][Medline]
6. Councell TB, Duckenfield KU, Landa ER, and Callender E. Tire-wear particles as a source of zinc to the environment. Environ Sci Technol 38: 4206–4214, 2004.[Medline]
7. Dye JA, Lehmann JR, McGee JK, Winsett DW, Ledbetter AD, Everitt JI, Ghio AJ, and Costa DL. Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents. Environ Health Perspect 109, Suppl 3: 395–403, 2001.[Medline]
8. Farooq A and Zhou MM. Structure and regulation of MAPK phosphatases. Cell Signal 16: 769–779, 2004.[CrossRef][Web of Science][Medline]
9. Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, and Beckett WS. Metal fume fever: characterization of clinical and plasma IL-6 responses in controlled human exposures to zinc oxide fume at and below the threshold limit value. J Occup Environ Med 39: 722–726, 1997.[CrossRef][Web of Science][Medline]
10. Franklin CC and Kraft AS. Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein kinase in U937 cells. J Biol Chem 272: 16917–16923, 1997.[Abstract/Free Full Text]
11. Gensch E, Gallup M, Sucher A, Li D, Gebremichael A, Lemjabbar H, Mengistab A, Dasari V, Hotchkiss J, Harkema J, and Basbaum C. Tobacco smoke control of mucin production in lung cells requires oxygen radicals AP-1 and JNK. J Biol Chem 279: 39085–39093, 2004.[Abstract/Free Full Text]
12. Gilmour PS, Rahman I, Donaldson K, and MacNee W. Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles. Am J Physiol Lung Cell Mol Physiol 284: L533–L540, 2003.[Abstract/Free Full Text]
13. Gordon T, Chen LC, Fine JM, Schlesinger RB, Su WY, Kimmel TA, and Amdur MO. Pulmonary effects of inhaled zinc oxide in human subjects, guinea pigs, rats, and rabbits. Am Ind Hyg Assoc J 53: 503–509, 1992.[Web of Science][Medline]
14. Gordon T and Fine JM. Metal fume fever. Occup Med 8: 504–517, 1993.[Medline]
15. Harrison RM and Yin J. Particulate matter in the atmosphere: which particle properties are important for its effects on health? Sci Total Environ 249: 85–101, 2000.[CrossRef][Medline]
16. Hashimoto S, Gon Y, Takeshita I, Matsumoto K, Jibiki I, Takizawa H, Kudoh S, and Horie T. Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am J Respir Crit Care Med 161: 280–285, 2000.[Abstract/Free Full Text]
17. Hashimoto S, Matsumoto K, Gon Y, Nakayama T, Takeshita I, and Horie T. Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase. Am J Respir Crit Care Med 159: 634–640, 1999.[Abstract/Free Full Text]
18. Hill CS and Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell 80: 199–211, 1995.[CrossRef][Web of Science][Medline]
19. Jaspers I, Flescher E, and Chen LC. Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells. Am J Physiol Lung Cell Mol Physiol 272: L504–L511, 1997.[Abstract/Free Full Text]
20. Jaspers I, Samet JM, Erzurum S, and Reed W. Vanadium-induced kappaB-dependent transcription depends upon peroxide-induced activation of the p38 mitogen-activated protein kinase. Am J Respir Cell Mol Biol 23: 95–102, 2000.[Abstract/Free Full Text]
21. Jaspers I, Samet JM, and Reed W. Arsenite exposure of cultured airway epithelial cells activates kappaB-dependent interleukin-8 gene expression in the absence of nuclear factor-kappaB nuclear translocation. J Biol Chem 274: 31025–31033, 1999.[Abstract/Free Full Text]
22. Jia Z. Protein phosphatases: structures and implications. Biochem Cell Biol 75: 17–26, 1997.[CrossRef][Web of Science][Medline]
23. Jimenez LA, Drost EM, Gilmour PS, Rahman I, Antonicelli F, Ritchie H, MacNee W, and Donaldson K. PM₁₀-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-. Am J Physiol Lung Cell Mol Physiol 282: L237–L248, 2002.[Abstract/Free Full Text]
24. Karin M, Liu Z, and Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 9: 240–246, 1997.[CrossRef][Web of Science][Medline]
25. Kassel O, Sancono A, Kratzschmar J, Kreft B, Stassen M, and Cato AC. Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J 20: 7108–7116, 2001.[CrossRef][Web of Science][Medline]
26. Kim YM, Reed W, Lenz AG, Jaspers I, Silbajoris R, Nick HS, and Samet JM. Ultrafine carbon particles induce interleukin-8 gene transcription and p38 MAPK activation in normal human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 288: L432–L441, 2005.[Abstract/Free Full Text]
27. Kodavanti UP, Hauser R, Christiani DC, Meng ZH, McGee J, Ledbetter A, Richards J, and Costa DL. Pulmonary responses to oil fly ash particles in the rat differ by virtue of their specific soluble metals. Toxicol Sci 43: 204–212, 1998.[Abstract/Free Full Text]
28. Kodavanti UP, Mebane R, Ledbetter A, Krantz T, McGee J, Jackson MC, Walsh L, Hilliard H, Chen BY, Richards J, and Costa DL. Variable pulmonary responses from exposure to concentrated ambient air particles in a rat model of bronchitis. Toxicol Sci 54: 441–451, 2000.[Abstract/Free Full Text]
29. Kodavanti UP, Moyer CF, Ledbetter AD, Schladweiler MC, Costa DL, Hauser R, Christiani DC, and Nyska A. Inhaled environmental combustion particles cause myocardial injury in the Wistar Kyoto rat. Toxicol Sci 71: 237–245, 2003.[Abstract/Free Full Text]
30. Kodavanti UP, Schladweiler MC, Ledbetter AD, Hauser R, Christiani DC, Samet JM, McGee J, Richards JH, and Costa DL. Pulmonary and systemic effects of zinc-containing emission particles in three rat strains: multiple exposure scenarios. Toxicol Sci 70: 73–85, 2002.[Abstract/Free Full Text]
31. Kuschner WG, D'Alessandro A, Wong H, and Blanc PD. Early pulmonary cytokine responses to zinc oxide fume inhalation. Environ Res 75: 7–11, 1997.[Medline]
32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[CrossRef][Medline]
33. Lakshminarayanan V, Drab-Weiss EA, and Roebuck KA. H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappaB to the interleukin-8 promoter in endothelial and epithelial cells. J Biol Chem 273: 32670–32678, 1998.[Abstract/Free Full Text]
34. Levinthal DJ and Defranco DB. Reversible oxidation of ERK-directed protein phosphatases drives oxidative toxicity in neurons. J Biol Chem 280: 5875–5883, 2005.[Abstract/Free Full Text]
35. Li J, Johnson XD, Iazvovskaia S, Tan A, Lin A, and Hershenson MB. Signaling intermediates required for NF-B activation and IL-8 expression in CF bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 284: L307–L315, 2003.[Abstract/Free Full Text]
36. Li J, Kartha S, Iasvovskaia S, Tan A, Bhat RK, Manaligod JM, Page K, Brasier AR, and Hershenson MB. Regulation of human airway epithelial cell IL-8 expression by MAP kinases. Am J Physiol Lung Cell Mol Physiol 283: L690–L699, 2002.[Abstract/Free Full Text]
37. Maret W. Zinc and sulfur: a critical biological partnership. Biochemistry 43: 3301–3309, 2004.[CrossRef][Medline]
38. Maret W, Jacob C, Vallee BL, and Fischer EH. Inhibitory sites in enzymes: zinc removal and reactivation by thionein. Proc Natl Acad Sci USA 96: 1936–1940, 1999.[Abstract/Free Full Text]
39. Mills PR, Davies RJ, and Devalia JL. Airway epithelial cells, cytokines, and pollutants. Am J Respir Crit Care Med 160: S38–43, 1999.[Abstract/Free Full Text]
40. Monje P, Marinissen MJ, and Gutkind JS. Phosphorylation of the carboxyl-terminal transactivation domain of c-Fos by extracellular signal-regulated kinase mediates the transcriptional activation of AP-1 and cellular transformation induced by platelet-derived growth factor. Mol Cell Biol 23: 7030–7043, 2003.[Abstract/Free Full Text]
41. Newhook R, Hirtle H, Byrne K, and Meek ME. Releases from copper smelters and refineries and zinc plants in Canada: human health exposure and risk characterization. Sci Total Environ 301: 23–41, 2003.[CrossRef][Medline]
42. Pope CA III, Schwartz J, and Ransom MR. Daily mortality and PM10 pollution in Utah Valley. Arch Environ Health 47: 211–217, 1992.[Web of Science][Medline]
43. Pourazar J, Mudway IS, Samet JM, Helleday R, Blomberg A, Wilson SJ, Frew AJ, Kelly FJ, and Sandstrom T. Diesel exhaust activates redox-sensitive transcription factors and kinases in human airways. Am J Physiol Lung Cell Mol Physiol 289: L724–L730, 2005.[Abstract/Free Full Text]
44. Prieditis H and Adamson IY. Comparative pulmonary toxicity of various soluble metals found in urban particulate dusts. Exp Lung Res 28: 563–576, 2002.[CrossRef][Web of Science][Medline]
45. Rahman I and MacNee W. Role of transcription factors in inflammatory lung diseases. Thorax 53: 601–612, 1998.[Free Full Text]
46. Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, Brash DE, Park JB, Rhim JS, and Harris CC. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res 48: 1904–1909, 1988.[Abstract/Free Full Text]
47. Reddy SP and Mossman BT. Role and regulation of activator protein-1 in toxicant-induced responses of the lung. Am J Physiol Lung Cell Mol Physiol 283: L1161–L1178, 2002.[Abstract/Free Full Text]
48. Roebuck KA. Regulation of interleukin-8 gene expression. J Interferon Cytokine Res 19: 429–438, 1999.[CrossRef][Web of Science][Medline]
49. Samet JM, Graves LM, Quay J, Dailey LA, Devlin RB, Ghio AJ, Wu W, Bromberg PA, and Reed W. Activation of MAPKs in human bronchial epithelial cells exposed to metals. Am J Physiol Lung Cell Mol Physiol 275: L551–L558, 1998.[Abstract/Free Full Text]
50. Samet JM, Silbajoris R, Wu W, and Graves LM. Tyrosine phosphatases as targets in metal-induced signaling in human airway epithelial cells. Am J Respir Cell Mol Biol 21: 357–364, 1999.[Abstract/Free Full Text]
51. Shanley TP. Phosphatases: counterregulatory role in inflammatory cell signaling. Crit Care Med 30: S80–S88, 2002.[CrossRef][Web of Science][Medline]
52. Singal M and Finkelstein JN. Use of indicator cell lines for determining inflammatory gene changes and screening the inflammatory potential of particulate and non-particulate stimuli. Inhal Toxicol 17: 415–425, 2005.[CrossRef][Web of Science][Medline]
53. Smith KR, Kim S, Recendez JJ, Teague SV, Menache MG, Grubbs DE, Sioutas C, and Pinkerton KE. Airborne particles of the California central valley alter the lungs of healthy adult rats. Environ Health Perspect 111: 902–908; discussion A408–909, 2003.[Web of Science][Medline]
54. Sun H, Charles CH, Lau LF, and Tonks NK. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75: 487–493, 1993.[CrossRef][Web of Science][Medline]
55. Takizawa H. Airway epithelial cells as regulators of airway inflammation. Int J Mol Med 1: 367–378, 1998.[Web of Science][Medline]
56. Tebo J, Der S, Frevel M, Khabar KS, Williams BR, and Hamilton TA. Heterogeneity in control of mRNA stability by AU-rich elements. J Biol Chem 278: 12085–12093, 2003.[Abstract/Free Full Text]
56. Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979.[Abstract/Free Full Text]
57. Wu JJ and Bennett AM. Essential role for mitogen-activated protein (MAP) kinase phosphatase-1 in stress-responsive MAP kinase and cell survival signaling. J Biol Chem 280: 16461–16466, 2005.[Abstract/Free Full Text]
58. Wu W, Graves LM, Jaspers I, Devlin RB, Reed W, and Samet JM. Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals. Am J Physiol Lung Cell Mol Physiol 277: L924–L931, 1999.[Abstract/Free Full Text]
59. Wu W, Jaspers I, Zhang W, Graves LM, and Samet JM. Role of Ras in metal-induced EGF receptor signaling and NF-B activation in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 282: L1040–L1048, 2002.[Abstract/Free Full Text]
60. Wu W, Samet JM, Ghio AJ, and Devlin RB. Activation of the EGF receptor signaling pathway in airway epithelial cells exposed to Utah Valley PM. Am J Physiol Lung Cell Mol Physiol 281: L483–L489, 2001.[Abstract/Free Full Text]
61. Wu W, Samet JM, Silbajoris R, Dailey LA, Sheppard D, Bromberg PA, and Graves LM. Heparin-binding epidermal growth factor cleavage mediates zinc-induced epidermal growth factor receptor phosphorylation. Am J Respir Cell Mol Biol 30: 540–547, 2004.[Abstract/Free Full Text]

This article has been cited by other articles:

				E. Theatre, V. Bours, and C. Oury A P2X Ion Channel-Triggered NF-{kappa}B Pathway Enhances TNF-{alpha}-Induced IL-8 Expression in Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., December 1, 2009; 41(6): 705 - 713. [Abstract] [Full Text] [PDF]

				M. Lazarczyk, P. Cassonnet, C. Pons, Y. Jacob, and M. Favre The EVER Proteins as a Natural Barrier against Papillomaviruses: a New Insight into the Pathogenesis of Human Papillomavirus Infections Microbiol. Mol. Biol. Rev., June 1, 2009; 73(2): 348 - 370. [Abstract] [Full Text] [PDF]

				S. Below, A. Konkel, C. Zeeck, C. Muller, C. Kohler, S. Engelmann, and J.-P. Hildebrandt Virulence factors of Staphylococcus aureus induce Erk-MAP kinase activation and c-Fos expression in S9 and 16HBE14o- human airway epithelial cells Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L470 - L479. [Abstract] [Full Text] [PDF]

				M. Lazarczyk and M. Favre Role of Zn2+ Ions in Host-Virus Interactions J. Virol., December 1, 2008; 82(23): 11486 - 11494. [Full Text] [PDF]

				Y. Ho, R. Samarasinghe, M. E. Knoch, M. Lewis, E. Aizenman, and D. B. DeFranco Selective Inhibition of Mitogen-Activated Protein Kinase Phosphatases by Zinc Accounts for Extracellular Signal-Regulated Kinase 1/2-Dependent Oxidative Neuronal Cell Death Mol. Pharmacol., October 1, 2008; 74(4): 1141 - 1151. [Abstract] [Full Text] [PDF]

				N. Dubi, L. Gheber, D. Fishman, I. Sekler, and M. Hershfinkel Extracellular zinc and zinc-citrate, acting through a putative zinc-sensing receptor, regulate growth and survival of prostate cancer cells Carcinogenesis, September 1, 2008; 29(9): 1692 - 1700. [Abstract] [Full Text] [PDF]

				S. Wiehler and D. Proud Interleukin-17A modulates human airway epithelial responses to human rhinovirus infection Am J Physiol Lung Cell Mol Physiol, August 1, 2007; 293(2): L505 - L515. [Abstract] [Full Text] [PDF]

				L. Zhu, W. Yan, M. Qi, Z. L. Hu, T. J. Lu, M. Chen, J. Zhou, C. H. Hang, and J. X. Shi Alterations of Pulmonary Zinc Homeostasis and Cytokine Production Following Traumatic Brain Injury in Rats Ann. Clin. Lab. Sci., January 1, 2007; 37(4): 356 - 361. [Abstract] [Full Text] [PDF]

				U. Sydlik, K. Bierhals, M. Soufi, J. Abel, R. P. F. Schins, and K. Unfried Ultrafine carbon particles induce apoptosis and proliferation in rat lung epithelial cells via specific signaling pathways both using EGF-R Am J Physiol Lung Cell Mol Physiol, October 1, 2006; 291(4): L725 - L733. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 290/5/L1028    most recent 00479.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (19) Citing Articles via Google Scholar Google Scholar Articles by Kim, Y.-M. Articles by Samet, J. M. Search for Related Content PubMed PubMed Citation Articles by Kim, Y.-M. Articles by Samet, J. M.