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Published ahead of print on November 4, 2004, doi:10.1165/rcmb.2004-0198OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 72-81, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0198OC

Matrix Metalloproteinase/Epidermal Growth Factor Receptor/Mitogen-Activated Protein Kinase Signaling Regulate fra-1 Induction by Cigarette Smoke in Lung Epithelial Cells

Qin Zhang, Pavan Adiseshaiah and Sekhar P. Reddy

Department of Environmental Health Sciences, Bloomberg School of Public Health; and Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland

Correspondence and requests for reprints should be addressed to Sekhar P. Reddy, The Johns Hopkins University, Department of Environmental Health Sciences, Division of Physiology, Rm. E7547, 615 North Wolfe Street, Baltimore, MD 21205. E-mail: sreddy{at}jhsph.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Exposure to cigarette smoke (CS) can lead to the development of lung cancer, but the molecular mechanisms underlying this process remain unclear. Given that activator protein 1 (AP-1) regulates genes involved in both physiologic and pathophysiologic processes, we have investigated the effects of CS on Jun and Fos family member expression and regulation using a nonmalignant human bronchial epithelial cell line, 1HAEo. Exposure to CS caused a marked upregulation of c-Jun, c-Fos, and Fra-1, but not of Fra-2, Jun-B, and Jun-D expression. Because Fra-1 is overexpressed in various tumors and upregulates genes associated with tumor progression, we further elucidated the mechanisms that control CS-stimulated fra-1 induction. CS stimulated fra-1 induction primarily at the transcriptional level. However, epidermal growth factor receptor (EGFR)-specific inhibitor, AG1478, completely suppressed CS-stimulated fra-1 expression. Similarly, the specific inhibitors of extracellular signal–regulated kinase (ERK), c-Jun NH2 terminal kinase (JNK), and p38 kinase signaling markedly suppressed fra-1 induction. Consistent with this finding, AG1478 blocked CS-stimulated ERK, JNK, and p38 phosphorylation. These results suggest that EGFR-activated multiple kinase signaling is essential for fra-1 induction. Furthermore, treatment of cells with GM6001, which inhibits matrix metalloproteinase activity, significantly suppressed CS-stimulated EGF shedding, EGFR and ERK kinase phosphorylation, and subsequent fra-1 induction. Collectively, our findings indicate an obligatory role for metalloproteinase-EGFR–mediated mitogen-activated protein kinase signaling in controlling CS-induced fra-1 expression.

Key Words: activator protein-1 • cigarette smoke • epidermal growth factor receptor • lung • matrix metalloproteinase


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Cigarette smoke (CS) is a complex mixture that contains both volatile components such as acrolein, napthalene, and aldehydes, and nonvolatile components such as aflatoxin B1, benzo[a]pyrene, and phorbol ester analogs (1–3). CS exposure causes lung cancer (2, 4, 5), a leading cause of cancer deaths in the United States and other developed countries. In addition, exposure to CS also increases the incidence of asbestos-associated lung carcinogenesis in human populations (6–8). Prior exposure to CS also impairs toxicant naphthalene-induced bronchiolar epithelial repair, with persistence of squamous cells in the terminal bronchioles in rodents (9). Although the pathogenic effects of smoking are well known, the molecular mechanisms underlying CS-promoted respiratory pathogenesis remain elusive.

Considerable experimental evidence generated in both tissue culture and animal models has indicated that as part of a protective response after toxic injury, bronchial epithelial (BE) cells rapidly undergo changes in their structure and function to repair their epithelium (10–12). This phenomenon is a very dynamic and multistep process in which epithelial cells rapidly migrate to the injured area, proliferate, and finally differentiate into a normal phenotype to restore regular bronchial functions (13, 14). However, an aberrant proliferation and differentiation of BE cells following toxic injury can lead to the development of various respiratory diseases, including lung cancer (15). For example, exposure to CS results in epithelial cell hyperplasia and squamous metaplasia, which have been considered to be among the preneoplastic stages in the transformation of bronchial epithelium (15, 16), but the mechanisms underlying this process remain unclear.

Activator protein–1 (AP-1), primarily composed of the Jun (c-Jun, Jun-B, and Jun-D) and Fos (c-Fos, Fos-B, Fra-1, and Fra-2) protein families, acts as one of the environmental biosensors that participate in cellular switching of the genetic program involved in various cellular processes in response to various oxidant and toxic stimuli (17, 18). This switching has mainly been attributed to differences in the spatiotemporal expression, protein structure, post-translational modifications, and turnover of Jun and Fos family member (19). Genetic mouse models and in vitro cell culture approaches have shown that aberrant activation of AP-1 proteins causally linked to pathogenesis (18), suggesting that an abnormal expression and/or activation of AP-1 proteins by toxins can lead to disease development. Recently, we have shown that AP-1 proteins distinctly regulate the expression of the squamous differentiation marker SPRR1B in BE cells (20, 21). The Fra-1–based AP-1 complex markedly upregulated SPRR1B transcription, whereas that the complex containing Fra-2 suppressed it (20). Consistent with this finding, fra-1 is overexpressed in squamous cell carcinomas of the esophagus and stomach (22, 23) as well as in breast cancer cells (24). Other studies have demonstrated a differential activation of AP-1 family member expression by other toxicants (reviewed in Ref. 25), such as asbestos, a known lung carcinogen (7). Collectively, these findings suggest that differential expression and/or activation of AP-1 family members by CS may contribute to respiratory pathogenesis.

We hypothesized that CS distinctly activates AP-1 family member expression in BE cells, resulting in an altered phenotype in the cells of the lung. Although AP-1 family members are known to be activated by disparate stimuli in a wide variety of cell types that includes the lung, the mechanisms that control their expression by CS are unclear. Therefore, the present study was designed to investigate the effects of CS on Jun and Fos family member expression and regulation using a nonmalignant human BE cell culture as a model system.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture and CS Exposure
The 1HAEo cell line, an SV40-transformed nonmalignant human BE cell line (26), was maintained in MEM containing 10% fetal calf serum. Research-grade 2R4F cigarettes without filters were obtained from the University of Kentucky Tobacco Research and Development Center (Lexington, KY). Cells were grown in either 12.5 cm2 (for transfections) or 25 cm2 (for protein and RNA isolation) Falcon flasks (Becton Dickinson) to 90% confluence, serum-starved for 14 h, and then exposed to mainstream CS as described previously (27, 28): CS (30 cc/4 puffs) was drawn into a syringe-driven device fitted with a tube. CS was delivered via the tube into cell culture flasks that were inverted to expose cells directly to smoke for 2 min. The flasks were restored to their original orientation, so that the cells were covered with culture medium devoid of serum, and the flasks were placed in the 37°C incubator. Throughout the study, we used CS at a dose of 1 cc for 12.5-cm2 and 2 cc for 25-cm2 flasks. No cell cytoxicity was observed under these exposure conditions.

Gene Expression Analysis
After CS exposure, cells were washed extensively with cold phosphate-buffered saline containing 1 mM sodium orthovanadate (Na3VO4) and harvested into a mitogen-activated protein (MAP) kinase lysis buffer. Protein samples (~ 40 µg) were resolved by 10% SDS-PAGE gel and transferred to PVDF membranes. Membranes were blocked overnight at 4°C in Tris buffer solution containing 0.1% Tween and 5% nonfat milk, and were incubated at room temperature for 1 h with antibodies specific for individual members of the Jun and Fos families (all obtained from Santa Cruz Biotech, Santa Cruz, CA). Membranes were then incubated with horseradish peroxidase–conjugated secondary antibody for 1 h, and immunoreactive bands were detected using enhanced chemiluminescene (ECL) reagents (Amersham Biosciences, Piscataway, NJ). Blots were stripped and probed with anti-ß-actin antibodies. The total amount of AP-1 protein in each sample was quantified with a Bio-Rad Gel Doc 2,000 system (Bio-Rad Laboratories, Hercules, CA) and normalized to that of ß-actin.

For Northern blot analysis, after the exposure to CS, total RNA was isolated from cells using Trizol reagent (Invitrogen, Carlsbad, CA). RNA (~ 15 µg) was separated on a 1.2% agarose gel, blotted onto a Nytran membrane, and sequentially hybridized with 32P-labeled fra-1 and a ß-actin cDNA probe as described previously (29). Fra-1 mRNA expression was quantified with a Gel Doc system, using ß-actin as a reference.

Electrophoretic Mobility Shift Assays
Cells were exposed to CS, nuclear extracts were prepared, and electrophoretic mobility shift assays were performed using a 32P-labeled double-stranded consensus TPA response element (TRE) (5'-CGCTTGATGACTCAGCCGGAA-3') probe as described previously (29). In competition assays, nuclear extracts were incubated for 10–15 min with a 50-fold molar excess of the unlabeled double-stranded oligo before the addition of labeled probe. In supershift assays, nuclear extracts were incubated with 1–2 µg of specific antibodies against c-Jun (sc-45X), Jun-B (sc-46X), Jun-D (sc-74X), c-Fos (sc-7202X), Fos-B (sc-48X), Fra-1 (sc-605X), or Fra-2 (sc-604X) (all obtained from Santa Cruz Biotechnology) for 2 h before addition of the labeled probe.

Plasmids and Transient Reporter Gene Assays
DNA transfections were performed using FuGENE reagent according to the manufacturer's protocol (Roche Biochemical Corporation, Indianapolis, IN). Various lengths of the –379 to +32 bp 5'-flanking region of human fra-1were cloned into a pGL3 basic vector upstream of the luciferase gene (see Ref. 29 for more details). The AP-1 reporter bearing seven copies of the consensus TPA response element (TRE) and the nuclear factor (NF)-{kappa}B-reporter bearing five copies of the {kappa}B response sequence cloned upstream of a TATA box fused to Luc were obtained from Stratagene (La Jolla, CA). Cells were transfected with 100 ng of promoter reporter constructs in the presence of a pRL-TK reference plasmid containing the Renilla luciferase gene. After 18–24 h of incubation, cells were exposed to CS for 5 h, and luciferase activities were measured using a dual luciferase kit (Promega Corp., Madison, WI). Firefly luciferase activity of individual samples was normalized to that of Renilla luciferase as described previously (29). All assay samples were performed in triplicate, and each experiment was repeated at least three times. Data are expressed as the means ± SE. The statistical significance of the differences between groups was determined using Student's t test, and P < 0.05 was considered statistically significant.

Kinase Immunoblot Analysis
Cells were exposed to CS for various time points, washed three times with chilled phosphate-buffered saline containing 1 mM Na3VO4, and then lysed in an MAP kinase lysis buffer. Cell lysates were separated on an SDS-PAGE gel and transferred to a PVDF membrane. Blots were incubated overnight at 4°C with primary antibodies and washed three times with TBST before probing with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Blots were then visualized with enhanced chemiluminescence reagent (Amersham Biosciences). Densitometric analysis was used to quantify protein levels. Kinase phosphorylation was normalized to that of total protein levels and then expressed as a percentage of the control condition.

MMP Activation Assays
Cells were serum-starved overnight and exposed to CS for the indicated times, and medium was then collected. MMP activity of the samples was determined by gelatin zymogram gels (Invitrogen Corp.) according to the manufacturer's recommendations. Gels were incubated in zymogram developing buffer for 12 h, stained with Simply Blue Safe Stain, and photographed.

Enzyme-Linked Immunosorbent Assays
The levels of human epidermal growth factor (EGF) receptor (EGFR) ligands, transforming growth factor (TGF)-{alpha} (Cat # DTGA00) and EGF (Cat # DEG00) were determined by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol (R&D Systems Inc., Minneapolis, MN). The levels of TGF-{alpha} and EGF (pg/ml) were calculated using a standard curve and were expressed as means ± SEM (n = 3).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
CS Distinctly Upregulates AP-1 Proteins in BE Cells
To determine whether CS stimulates AP-1–mediated transcription, cells were transiently transfected with a TRE-Luc reporter and then exposed to CS (2 cc) for 5 h. It should be noted that CS at this dose caused no cellular toxicity (data not shown). A reporter containing NF-{kappa}B recognition sequences was used as a positive control. As anticipated, exposure to CS strongly stimulated NF-{kappa}B–dependent gene transcription (Figure 1A). Similarly, CS significantly enhanced (~ 3-fold) the reporter activity driven by the TRE (Figure 1A). To determine the effect of CS on AP-1 protein family member expression, cells were exposed to CS (2 cc) for 0–360 min and harvested in lysis buffer. Immunoblot analysis of whole-cell lysates was performed using specific antibodies recognizing the individual members of the AP-1 family. Both c-Fos and Fra-1 protein levels were significantly higher, 2.80- and 2.78-fold, respectively, following CS exposure at 30 min as compared with control values (Figure 1B). The stimulated expression remained higher than basal levels through 180 min after exposure. CS also significantly enhanced c-Jun expression (~ 50%) at 60 and 180 min following the exposure (Figure 1C). In contrast, CS exposure had no significant effect on Fra-2, Jun-B, or Jun-D protein levels. Our results are consistent with previous reports that showed a differential activation of AP-1 family member expression in lung epithelial cells both in vivo and in vitro (30–32).




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  		Figure 1. CS distinctly upregulates AP-1 family member expression in BE cells. (A) CS stimulates AP-1–mediated transcription. Cells were transfected with 100 ng of the TRE-Luc or NF-{kappa}B reporter construct along with the pRL-TK plasmid. Cells were exposed to room air (ctr) or CS, and reporter activity was analyzed. The firefly luciferase activity was normalized to that of Renilla. The luciferase activity of samples exposed to room air was considered equal to one unit. Data shown are means ± SE (n = 4) of a representative experiment. Cells at confluence were exposed to CS at 2 cc for the times indicated. (B) Western blot analysis of whole-cell lysates was performed using anti-Fos protein family specific antibodies. The membrane was stripped and probed with anti–ß-actin antibodies to control for protein loading. A TPA-treated (180 min) cell lysate was used as a positive control. (C) Western blot analysis of whole-cell lysates using anti-Jun protein family specific antibodies. Autoradiograph is a representative blot of two independent experiments. The densitometric value of AP-1 protein levels in six independent samples was normalized against ß-actin protein content. The normalized value of room air exposed samples was designated as one arbitrary unit (AU). *P < 0.05 compared with unexposed (0) controls. The presence of multiple bands for c-Fos, Fra-1, and Jun-D is consistent with nature of these proteins, as reported by several laboratories.

 
We then used electrophoretic mobility shift assays to examine the binding of the various members of the Jun and the Fos families to the TRE using nuclear extracts from cells with our without exposure to CS. Nuclear extracts from TPA-treated cells were used for comparison. At 180 min after exposure, CS markedly increased protein binding when compared with the unexposed control (Figure 2A). However, it should be noted that CS modestly (~ 50%) increased protein binding as compared with TPA, a potent activator of AP-1 binding. As anticipated, incubation of nuclear extracts with a 50-fold molar excess of the unlabeled TRE oligo, but not the GATA-1 oligo, completely blocked complex formation (data not shown). Incubation with anti Fra-1 (Figure 2A), Jun-B, Jun-D, and c-Jun antibodies (Figure 2B) caused a supershifting of the complex. The intensity of the supershifted band after incubation with anti–Fra-1 and anti–c-Jun antibody was significantly higher than that for untreated cells. Collectively, these results suggest that an enhanced level of c-Jun and Fra-1 at least in part contributed to the CS-stimulated AP-1 DNA binding activity.




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  		Figure 2. CS distinctly stimulates the AP-1 family of transcription factors binding to the consensus binding site. (A) Cells were either exposed to CS or treated with TPA for 180 min. Nuclear extracts (2 µg) from control (ctr) and CS-exposed (CS) cells were incubated with nonimmune IgG (Ig) or anti–Fra-1 (F1) antibodies before the addition of a 32P-end-labeled double-stranded oligo bearing a TRE (underlined) (5'-CGCTTGATGACTCAGCCGGAA-3, top strand only shown), and DNA–protein complexes were resolved by electrophoresis. Fra-1 supershifted bands were quantified using a gel documentation system and normalized to the values for IgG (right panel). (B) Nuclear extracts from control and CS-exposed (CS) cells were incubated with antibodies (2 µg) specific to individual members of the AP-1 family. The vertical bars indicate the position of supershifted (SS) bands, solid arrowheads indicate the AP-1–protein complex, open arrowheads indicate the nonspecific protein–DNA complex, and open arrows indicate the free probe. Fp, probe incubated without nuclear extracts. *P < 0.05 compared with unexposed (0) controls.

 
CS Induces fra-1 Expression at the Transcriptional Level
Fra-1 is overexpressed in various tumors and upregulates gene expression associated with both squamous metaplasia (20, 21) and tumor progression (33). We therefore focused the remainder of our studies on elucidating the mechanisms that regulate fra-1 induction by CS. Cells were exposed to CS for 0–360 min, and Northern analysis was performed as described in MATERIALS AND METHODS. CS markedly stimulated fra-1 mRNA expression in both 1HAEo (Figure 3A) and alveolar type-II epithelial cells (data not shown) cells. Induction was noticeable as early as 60 min and reached a maximum at 360 min after exposure. In both cases, two alternatively spliced mRNA transcripts of fra-1 (22) were induced similarly. We next examined whether CS upregulates fra-1 induction by increasing the transcription or the stability of its mRNA. Cells were treated with the transcriptional inhibitor actinomycin D (AD) for 30 min before CS exposure for 3 h, and RNA was isolated for Northern analysis. CS significantly increased the fra-1 message levels (Figure 3B, lane 2). However, AD completely suppressed both basal and CS-inducible fra-1 mRNA expression (lane 4). To determine whether CS affects the stability of fra-1 mRNA, cells were exposed to CS for 3 h and then treated with AD (Figure 3C) for 0–90 min. The elevated fra-1 mRNA levels remained above the basal level, as compared with an AD-treated, unexposed control group (lane 1). This result suggests that CS induced fra-1 expression mainly at the transcriptional level.



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  		Figure 3. CS stimulates fra-1 induction in part at the transcriptional level. (A) Cells at confluence were exposed to CS for 30–360 min. Total RNA was isolated, blotted, and hybridized with a labeled fra-1 or ß–actin cDNA probe. The intensity of fra-1 mRNA transcripts was quantified using ß–actin mRNA as reference. –Fold values represent the mean induction of the normalized ratio of three independent samples. (B) To determine whether CS regulates fra-1 induction mainly at the transcriptional level, cells were treated with actinomycin D (AD, 10 µg /ml) or DMSO (Veh) for 30 min before CS exposure for 3 h. Results shown are a representative blot of two independent experiments. (C) Cells were exposed to CS for 3 h and then treated with AD for the indicated period. (D) Cells were transfected with the indicated fra-1 promoter reporter construct (see Ref. 29 for more details) along with pRL-TK, to monitor transfection efficiency. After overnight incubation, cells were exposed to CS (2 cc) for 5 h, and reporter expression was analyzed. Data are expressed as -fold change over control values for the 68-Luc promoter activity. *P < 0.05, compared with controls. The data represent the values of nine independent samples from three separate experiments.

 
To confirm these observations, fra-1 promoter-reporter constructs were transiently transfected into cells, and luciferase activity was measured after CS exposure (Figure 3D). We have recently shown that the –379 to +32 bp 5'-flanking region of the fra-1 gene contains sufficient information to mediate inducible transcription in response to disparate stimuli, such as TPA (29), EGF, H202, and TNF-{alpha} (unpublished data). We therefore chose the –379 to +32 bp promoter, –379 to +32 (379-Luc), and a shorter fragment that contains –68 to + 32 bp of the fra-1 minimal promoter (68-Luc), each fused to a reporter gene, and analyzed the effects of CS on promoter activity using transient transfection assays. We also used a full-length fra-1 promoter bearing the –861 to +32 bp 5'-flanking region (861-Luc) for comparison. The 68-Luc reporter displayed a low level of promoter activity, and there was no change in luciferase activity in response to CS. However, CS exposure caused a significant increase in the promoter activity of the 379-Luc and 861-Luc promoter constructs. Collectively, these results indicate that CS stimulates fra-1 expression mainly at the transcriptional level.

Multiple MAP Kinase Pathways Regulate CS-Stimulated fra-1 Expression
It is well documented that extracellular signal–regulated kinase (ERK), c-Jun NH2 terminal kinase (JNK), and p38 MAP kinase modules play a central role in regulating AP-1 family member expression in response to mitogenic or toxic stimuli in a wide variety of cell types (34). Also, an attenuation of mitogen-inducible fra-1 mRNA expression by ERK signaling inhibitors has been reported (35–38). We therefore sought to identify which of these MAP kinase(s) regulate CS-induced fra-1 expression. Cells were exposed to CS for 30–90 min, and immunoblot analysis was performed using phospho-specific MAP kinase antibodies. CS markedly stimulated (by 304 ± 16%) ERK1 and ERK2 phosphorylation within 30 min when compared with a filtered air-exposed control (Figure 4A). ERK1 and ERK2 phosphorylation remained well above basal level at 90 min. CS exposure significantly increased (by ~ 45%, P < 0.05) p38 phosphorylation after 30 min. In addition to the ERK and JNK kinases, CS significantly stimulated the phosphorylation of the JNK1 and JNK2 isoforms. These results suggest that CS potently activates the ERK, JNK, and p38 MAP kinase pathways in BE cells. To determine whether or not MAP kinase pathways regulate CS-induced fra-1 expression, we have used three chemical inhibitors, PD98059, SP600125, and SB202190, that specifically suppress the activation of ERK, JNK, and p38 MAP kinases, respectively. Treatment of cells with any these inhibitors before the CS exposure markedly suppressed fra-1 induction (Figure 5). Together, these observations suggest that CS stimulates the phosphorylation of ERK, JNK, and p38 kinases, which in turn controls inducible fra-1 expression in BE cells.



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  		Figure 4. ERK, JNK, and p38 MAP kinase pathways regulate CS-stimulated fra-1 expression. (A) Cells were exposed to CS for 0–90 min, harvested in a MAP kinase lysis buffer, and immunoblotted using phospho-specific anti-ERK (top), anti-p38 (middle), or anti-JNK (bottom) antibodies. Blots were stripped and probed with total ERK, p38, and JNK antibodies. (B) Quantification of CS-stimulated ERK, p38, and JNK phosphorylation. The results shown are a representative blot of two independent experiments. *P < 0.05 compared with unexposed (0) controls. The autoradiogram was exposed for a shorter and a longer time to allow us to visualize and quantify the two p-JNK isoforms, which were normalized for the band intensity of total JNK. Open bars, control room air; filled bars, CS.

 


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  		Figure 5. Effect of MAP kinase inhibitors on CS-induced fra-1 mRNA expression. (A) Cells were either treated with DMSO (Veh) or with PD98059 (PD, 30 µM), SB202190 (SB, 20 µM), or SP600125 (SP, 20 µM) pharmacologic inhibitors before CS exposure, and fra-1 mRNA expression was analyzed. (B) Quantification of CS-induced fra-1 mRNA expression in the presence or absence of MAP kinase inhibitors. Results shown are a representative blot of two independent experiments. #P < 0.05 and *P < 0.05 compared with control and CS-exposed samples, respectively.

 
EGFR Mediates CS-Stimulated fra-1 Induction
EGFR plays a central role in various cellular processes, and its overexpression and/or activation has been implicated in various pathological diseases, including cancer (39). We therefore hypothesized that EGFR activation is essential for smoke-induced fra-1 transcription. To examine this possibility, cells were treated with AG1478, which specifically inhibits EGFR tyrosine kinase activity, for 40 min before CS exposure for 180 min, and fra-1 expression was analyzed. Indeed, AG1478 completely suppressed CS-induced fra-1 mRNA expression (Figure 6A), suggesting an essential role for EGFR in regulating fra-1 induction by CS in BE cells. To explore the possibility that CS activates MAP kinases through the EGFR, we examined the effects of AG1478 on CS-stimulated ERK, JNK, and p38 kinase phosphorylation. AG1478 significantly suppressed CS-stimulated ERK, JNK, and p38 phosphorylation (Figure 6B). Together, these results indicate that CS stimulates ERK, JNK, and p38 phosphorylation through the activation of the EGFR.



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  		Figure 6. Effect of EGFR inhibitor on CS-stimulated fra-1 mRNA expression. (A) Cells were treated with tyrphostin AG1478 (AG, 2µM) for 40 min before CS exposure for 3 h. Fra-1 mRNA expression was analyzed by Northern blot analysis (left). The normalized data are presented as the mean ± SE of three independent observations (bottom). *P < 0.001 and #P < 0.001, compared with control and CS-exposed samples, respectively. (B) Inhibition of EGFR activity blocks CS-stimulated ERK1/2, JNK1/2, and p38 kinase phosphorylation. Cells were treated with 2 µM AG1478 or vehicle (DMSO) for 40 min and then exposed to CS for 30 min and immunoblotted using anti-ERK1/2, JNK1/2, and p38 antibodies. Quantification of CS-stimulated ERK, JNK, and p38 kinase phosphorylation in the presence of DMSO or AG1478 was performed as described in Figure 4. Data are expressed as the means ± SE of three independent experiments (n = 6). *P < 0.05, compared with CS-exposed samples.

 
MMPs Are Involved in CS-Stimulated fra-1 Induction
Several studies have shown that various MMPs play a critical role in the activation of the EGFR in response to diverse environmental stimuli (39), including CS (40). To examine whether or not MMPs are involved in CS-stimulated fra-1 induction, cells were exposed to GM6001, a compound that broadly inhibits MMP activation prior to exposure, and fra-1expression was analyzed (Figure 7). As comparison, the effect of GM6001 on EGF-inducible fra-1 expression was analyzed. We found that GM6001 significantly suppressed CS-stimulated fra-1 induction. In contrast, EGF-inducible fra-1 mRNA expression was minimally affected. Thus, it is likely that MMP(s) play a critical role in mediating CS inducible fra-1 expression through EGFR activation.



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  		Figure 7. Metalloproteinase inhibitor blocked CS-stimulated fra-1 mRNA expression. Cells were treated with GM6001 (GM, 10 µM) for 1 h before exposure to CS or treatment with EGF (50 ng/ml). After 3 h incubation, cells were harvested and Northern blot analysis was performed. Data shown are results from a representative experiment.

 
NADPH Oxidase Inhibitor and N-Acetyl Cystiene Have No Effect on CS-Stimulated fra-1 Induction
To examine the role of reactive oxygen species (ROS) in mediating CS-enhanced fra-1 induction, cells were treated with H2O2, and mRNA expression was analyzed by Northern blot analysis. Mitogens such as EGF and TPA were used for comparison. As compared with EGF and PMA, H2O2 weakly and transiently stimulated the expression in 1HAEo cells (Figure 8). To determine the role of ROS in mediating CS-enhanced fra-1 induction, we treated cells with DMSO and NADPH oxidase inhibitor diphenylene iodonium (DPI), or with an antioxidant, N-acetyl cysteine (NAC), before CS exposure. Treatment of cells with either DPI or NAC did not attenuate the mRNA expression (Figure 8B), suggesting that oxidative stress plays only a minimal role (if any) in mediating CS-stimulated fra-1 induction under our experimental conditions.



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  		Figure 8. Role of oxidative stress on CS-stimulated fra-1 induction. (A) Cells were serum-starved for 14 h and then treated with H2O2 (500 µM), EGF (50 ng/ml), or TPA (100 ng/ml) for 90 or 360 min. Total RNA was isolated, and fra-1 mRNA expression was analyzed as in Figure 3. (B) To determine whether reactive oxygen species mediate CS-stimulated fra-1 induction, cells were treated with diphenyleneiodonium (DPI, 20 µM) or N-acetyl-L-cysteine (NAC, 30 mM) for 30 min before CS exposure for 360 min. DMSO was used as a vehicle control. Results shown are a representative blot of two independent experiments.

 
CS Stimulates EGF Shedding in an MMP-Dependent Manner
To examine whether CS stimulates EGFR ligand shedding, cells were exposed to CS for 0–15 min, and TGF-{alpha} and EGF protein levels in the conditioned medium were determined. As shown in Figure 9A, CS exposure caused shedding of the EGF ligand (left panel) into the medium, whereas it had no such effect on TGF-{alpha} levels (right panel). To determine whether MMP activation is required for EGF shedding, we treated cells with the MMP inhibitor GM6001 before CS exposure and measured EGF levels in the conditioned media by ELISA. GM6001 markedly suppressed CS-stimulated EGF shedding (Figure 9B). To confirm this activation of MMP by CS, conditioned media from filtered air- and CS-exposed cells was tested in zymogram gel assays. As compared with controls (lanes 1 and 2), CS caused a marked activation of MMP9 at 5 min after the exposure, whereas MMP2 was only modestly activated. The activation of other MMPs by CS either was very weak or undetectable under our experimental conditions. Together, these results suggest a critical role for CS-stimulated MMP activation and subsequent shedding of EGF and EGFR activation. Apparently MMP9 and MMP2 regulate this process.



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  		Figure 9. MMP activation is required for CS stimulated EGF shedding. (A) Cells were exposed to CS for 0–15 min, culture medium was harvested, and the levels of EGF and TGF-{alpha} proteins were analyzed in duplicate by ELISA using a standard curve. Data are expressed as the means ± SE of three independent samples. (B) Cells were treated with either DMSO or GM6001 for 30 min and then exposed to CS for 15 min. EGF protein levels in the culture medium were analyzed (n = 3). (C) The culture medium of cells exposed to CS for 0–15 min was used to analyze MMP activation by zymographic analysis. The first lane displays the molecular weight standards (Cat# LC5925; Invitrogen Corp.). The results shown are a representative blot of two independent experiments.

 
Inhibition of MMP Activation Blocks CS-Stimulated EGFR and MAP Kinase Phosphorylation
To confirm the activation by EGFR, we exposed cells to CS for 0–15 min, and the receptor phosphorylation was measured by immunoblot analysis using Tyr845 phospho-specific EGFR antibodies (Figure 10A). Indeed, CS significantly stimulated the phosphorylation of EGFR at 15 min after the exposure, as compared with filtered air–exposed controls. To determine the role of MMPs in EGFR activation, cells were treated with either DMSO or GM6001 for 40 min before CS exposure, and immunoblot analysis was performed using the total cellular lysates. As anticipated, GM6001 markedly attenuated CS-stimulated EGFR phosphorylation, as compared with vehicle-treated control group (Figure 10B). To determine whether the inhibition of EGFR activation also affects at the level of MAP kinase activation, the immunoblot was subsequently probed with antibodies that specifically recognize the phosphorylated forms of both ERK1 and ERK2 (Figure 10C) kinases, well-known downstream effectors of the receptors. As expected, the phosphorylation of ERK1 and ERK2 was markedly enhanced in cells exposed to CS (Figure 10C). However, pretreatment of cells with GM6001 prevented this effect.



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  		Figure 10. The effects of MMP inhibition on CS-stimulated EGFR phosphorylation and MAP kinase activation. (A) Cells were exposed to CS for 0–30 min and then harvested in a MAP kinase lysis buffer and immunoblotted using a phospho-specific EGFR antibody (Tyr845, Cat# 2231; Cell Signaling Technology, Beverly, MA) The blot was stripped and probed with EGFR antibody (Cat# 2232; Cell Signaling Technology) to normalize the protein loading. The lysates isolated from EGF-treated (50 ng/ml, 15 min) cells was used as a positive control. (B) Cells were treated with DMSO or GM6001 (10 µM) before CS exposure for 30 min. After the exposure, cellular lysates were harvested, and the phosphorylation of EGFR (upper panel) and ERK1/2 MAP kinase (lower panel) was analyzed. Quantification of a representative blot of two independent experiments is shown. The normalized value of room air–exposed (Ctr) samples was designated as one arbitrary unit (AU).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
To date, several laboratories have used a variety of CS-exposure protocols to study the effects of cigarette smoke on lung cell types. The most commonly used methods are performed as follows: (1) A cigarette smoke extract, devoid of vapor-phase volatile components, is prepared by bubbling CS through culture medium. The CS extract at a desired dilution is then added to the cell culture (41–44). (2) The particulate phase of smoke is collected on membrane filters inserted in-line with CS exposure chambers. Particles from filters are extracted with a solvent (DMSO) or serum-free cell culture medium before exposure (40, 45). (3) The mainstream CS is directly drawn into a syringe-driven apparatus fitted with a tube, and the desired volume (cc) of CS is then delivered into tissue culture flasks (27, 28). Although there are several advantages and disadvantages to the use of each method, we chose the third one for the present study, as it represents an ideal exposure system in which the BE cells lining the respiratory tract are being exposed to both volatile and nonvolatile CS components.

Our findings indicate that EGFR-mediated MAP kinase activation is an obligatory step in regulating CS-stimulated fra-1 induction in BE cells. Consistent with our observations, an essential role for the EGFR in asbestos-induced lung epithelial cell proliferation and AP-1 family member expression has been demonstrated both in vitro and in vivo (46). Although the EGFR is critical to various cellular processes, an increased expression and/or the activation of this receptor has been associated with toxin-induced pathogenesis and tumor development in various tissues, including the lung (47, 48). It should be noted that fra-1, which is overexpressed in various tumors, regulates gene expression associated with cell motility and invasiveness of tumorogenic cells (49). Several studies have shown that AP-1 dimers composed of c-Jun and Fra-1 upregulates transcriptional programs associated with tumor progression (33) and squamous metaplasia (20). It is noteworthy that AP-1 in the form of a c-Jun/Fra-1 dimer is more stable than that of the Jun/Jun dimer (17). Recently, Zenz and coworkers have demonstrated that c-Jun, a known oncoprotein, controls keratinocyte proliferation and skin tumor formation (50). Intriguingly, mice lacking c-Jun express reduced levels of EGFR and its ligand HB-EGF in keratinocytes (50), suggesting that c-Jun regulates EGFR expression. c-Jun also is a known regulator of fra-1 transcription induced by mitogenic stimuli (29, 51, 52). Based on these observations and because EGFR, c-Jun, and Fra-1 overexpression is tightly correlated with tumor growth and progression, it is likely that fra-1 induction by CS may promote the stabilization of the c-Jun–based AP-1 complex, thereby activating various transcriptional programs associated with CS-induced pathophysiologic processes in the lung.

It has been clearly established that the binding of EGF and its close relatives to the extracellular domain of the EGFR triggers receptor dimerization and subsequent activation of various signaling cascades (47, 48). The metal matrixproteinases-(MMPs) plays a critical role in the processing of EGF and EGF-like ligand precursors, thereby contributing to the EGFR signal transactivation (39). Consistent with these observations, we noted the involvement of MMP activation in controlling CS-stimulated fra-1 induction (Figure 7). We have also demonstrated that CS stimulates the activation of MMPs, such as MMP2 and MMP9, which are known to be activated by CS and are also implicated in CS-induced emphysema (53–55). Blocking of MMP activation with a broad-spectrum small molecule inhibitor GM6001 prevented both EGF shedding and EGFR phosphorylation (Figure 10B). This result was nicely correlated with an attenuation of CS-stimulated MAP kinase activation (Figure 10C) and subsequent fra-1 induction. Inhibition of MMP activation by GM6001 attenuated CS-stimulated, but not EGF-stimulated, fra-1 induction. Our results clearly demonstrated that MMP activation, which occurs as early as 5 min, precedes EGF shedding and subsequent EGFR phosphorylation, which is quite apparent at 15 min after the exposure. Taken together, these results suggest that stimulation of MMPs activity by CS may play a critical role in the cleavage of EGFR ligand precursors, thereby leading to EGFR activation and subsequent MAP kinase activation and fra-1 induction. Although the activation of MMP2 and MMP9 by CS is quite apparent, a role for other MMPs in mediating CS-promoted cellular effects cannot be ruled out in our studies, because it is possible that the activation of other MMPs by CS may be very weak or undetectable under our experimental conditions. Further investigation is needed to evaluate this possibility.

Oxidative stress has been shown to play a prominent role in the activation of EGFR and its downstream effectors, such as MAP kinases, in response to various stress inducers, including CS (40, 56, 57). Interestingly, we found that inhibition of the oxidative stress with DPI and NAC had no significant effect on CS-stimulated fra-1 mRNA expression (Figure 8B). Importantly, oxidative stress-causing agents such as H2O2 weakly stimulated fra-1 mRNA expression (Figure 8A), as compared with potent mitogens, such as EGF and PMA, both in human bronchial (the present study) and in alveolar lung epithelial cells (29). Thus, it appears that a ligand-dependent, but not an oxidative stress-mediated, mechanism mainly contributes to the induction of fra-1 by CS. The discrepancy regarding the effect of oxidative stress between others' studies and ours could be attributed to differences in experimental conditions and the cell types used. For example, several studies have principally studied the effects of CS (40, 57) that was devoid of volatile compounds, such as aldehydes. In contrast, in our study we have used freshly prepared mainstream CS, which contains both volatile and nonvolatile compounds. The activation of EGFR by aldehydes such as acrolein has been reported (58).

The Ras/Raf/ERK signaling axis is a major MAP kinase pathway known to be activated through the EGFR that promotes EGF-induced cellular responses (59). Consistent with these observations, we found that inhibition of ERK signaling markedly attenuated CS-enhanced fra-1 induction. Blocking the EGFR activation also suppressed CS-stimulated ERK phosphorylation, indicating that CS induces fra-1 transcription through EGFR-mediated ERK signaling. These results are in agreement with previous reports showing an attenuation of mitogen-inducible fra-1 mRNA expression by ERK signaling inhibitors (35–38). Our findings indicate that in addition to ERK, the p38 and JNK kinase pathways also contribute to the CS-induced fra-1 transcription. It should be pointed out that fra-1 transcription by mitogenic stimuli such as TPA is controlled by multiple proteins, such as c-Jun, Jun-D, Fra-2 (29), serum response factor, Elk-1, and activating transcription factor (P.A. and S.P.R., unpublished data), which are the downstream effectors of the ERK, p38, and JNK kinases. Although activation of the p38 and JNK pathways by CS components such as benzopyrene and acrolein has been reported (60–63), further studies are needed to delineate the EGFR-triggered downstream signaling events converging on ERK, JNK, and p38 MAP kinase modules in BE cells in response to CS.

In summary, we propose (Figure 11) that CS stimulates the activity of the MMPs (step 1), causing the shedding of the EGF and EGF-like precursors and leading to EGFR activation (step 2). This activation of the EGFR then results in various signaling cascades that converge at the ERK, JNK, and p38 kinases (step 3), which subsequently activate various transcription factors that bind at the fra-1 promoter and stimulate transcription. Blocking the activation of any of these steps or pathways can cause an attenuation of CS-induced fra-1 transcription. Because Fra-1 overexpression is associated with tumor progression and cell motility, future investigations elucidating the upstream specific signaling pathways and the effector transcription factors controlling CS-inducible fra-1 expression and its functional role may suggest potential preventive strategies against EGFR-mediated CS-induced respiratory pathogenesis, including cancer.



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  		Figure 11. Model depicting EGFR-mediated signaling pathways controlling CS-stimulated fra-1 expression in BE cells. Solid arrows indicate the proposed pathways activated by CS which converge at the MAP kinase pathways that control transcription factor activation binding to the fra-1 promoter. TRE is a TPA response element and SRE represents serum response element. Lines with blunted end indicate the inhibition of pathways. pEGF: precursor EGF, or EGF-like ligands, ERK, extracellular regulated kinase; PD98059 is an ERK1/2 inhibitor; SB201290 is a p38 inhibitor; SP600125 is a JNK inhibitor; AG1478 is an EGFR tyrosine kinase inhibitor, and GM6001 is a broad-spectrum inhibitor of MMPs.

 

   Acknowledgments
 
The authors thank Dieter Gruenert for providing us with the BE cell line 1HAEo. They thank Viswanathan Natarajan laboratory for assisting them in the EGFR activation studies. They acknowledge the Johns Hopkins Urban Environmental Health Center for the use of its core facilities.


   Footnotes
 
This work was supported by NIH grants ES11863, HL58122, ES30819, and HL66109 (to S.P.R.).

Conflict of Interest Statement: Q.Z. has no declared conflicts of interest; P.A. has no declared conflicts of interest; S.P.R. has no declared conflicts of interest.

Received in original form June 19, 2004

Received in final form October 12, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Hoffmann D, Hoffmann I. The changing cigarette, 1950–1995. J Toxicol Environ Health 1997;50:307–364.[CrossRef][Medline]
  2. Witschi H, Joad JP, Pinkerton KE. The toxicology of environmental tobacco smoke. Annu Rev Pharmacol Toxicol 1997;37:29–52.[CrossRef][Medline]
  3. Hecht SS. Biochemistry, biology, and carcinogenicity of tobacco-specific N- nitrosamines. Chem Res Toxicol 1998;11:559–603.[CrossRef][Medline]
  4. Hecht SS. Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 1999;91:1194–1210.[Abstract/Free Full Text]
  5. Shields PG. Molecular epidemiology of smoking and lung cancer. Oncogene 2002;21:6870–6876.[CrossRef][Medline]
  6. Mossman BT, Kamp DW, Weitzman SA. Mechanisms of carcinogenesis and clinical features of asbestos- associated cancers. Cancer Invest 1996;14:466–480.[Medline]
  7. Mossman BT, Churg A. Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med 1998;157:1666–1680.
  8. Nelson HH, Kelsey KT. The molecular epidemiology of asbestos and tobacco in lung cancer. Oncogene 2002;21:7284–7288.[CrossRef][Medline]
  9. Plopper CG, Van Winkle LS, Fanucchi MV, Malburg SR, Nishio SJ, Chang A, Buckpitt AR. Early events in naphthalene-induced acute Clara cell toxicity: II. Comparison of glutathione depletion and histopathology by airway location. Am J Respir Cell Mol Biol 2001;24:272–281.[Abstract/Free Full Text]
  10. Basbaum C, Jany B. Plasticity in the airway epithelium. Am J Physiol 1990;259:L38–L46.
  11. Jetten AM, Harvat BL. Epidermal differentiation and squamous metaplasia: from stem cell to cell death. J Dermatol 1997;24:711–725.[Medline]
  12. Wright JL, Churg A. Animal models of cigarette smoke-induced COPD. Chest 2002;122:301S–306S.[Abstract/Free Full Text]
  13. Rennard SI. Inflammation and repair processes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:S12–S16.[Abstract/Free Full Text]
  14. Knight D. Epithelium-fibroblast interactions in response to airway inflammation. Immunol Cell Biol 2001;79:160–164.[CrossRef][Medline]
  15. Wistuba II, Mao L, Gazdar AF. Smoking molecular damage in bronchial epithelium. Oncogene 2002;21:7298–7306.[CrossRef][Medline]
  16. Sekido Y, Fong KM, Minna JD. Molecular genetics of lung cancer. Annu Rev Med 2003;54:73–87.[CrossRef][Medline]
  17. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1991;1072:129–157.[Medline]
  18. Eferl R, Wagner EF. AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 2003;3:859–868.[CrossRef][Medline]
  19. Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997;9:240–246.[CrossRef][Medline]
  20. Patterson T, Vuong H, Liaw Y-S, Wu R, Kalvakolanu DV, Reddy SP. Mechanism of repression of squamous differentiation marker, SPRR1B, in malignant bronchial epithelial cells: role of critical TRE-sites and its transacting factors. Oncogene 2001;20:634–644.[CrossRef][Medline]
  21. Reddy SP, Vuong H, Adiseshaiah P. Interplay between proximal and distal promoter elements is required for squamous differentiation marker induction in the bronchial epithelium: role for ESE-1, Sp1, and AP-1 proteins. J Biol Chem 2003;278:21378–21387.[Abstract/Free Full Text]
  22. Matsui M, Tokuhara M, Konuma Y, Nomura N, Ishizaki R. Isolation of human fos-related genes and their expression during monocyte-macrophage differentiation. Oncogene 1990;5:249–255.[Medline]
  23. Hu YC, Lam KY, Law S, Wong J, Srivastava G. Identification of differentially expressed genes in esophageal squamous cell carcinoma (ESCC) by cDNA expression array: overexpression of Fra- 1, neogenin, Id-1, and CDC25B genes in ESCC. Clin Cancer Res 2001;7:2213–2221.[Abstract/Free Full Text]
  24. Roy D, Calaf G, Hei TK. Profiling of differentially expressed genes induced by high linear energy transfer radiation in breast epithelial cells. Mol Carcinog 2001;31:192–203.[CrossRef][Medline]
  25. Reddy SP, Mossman BT. Role and regulation of activator protein-1 in toxicant-induced responses of the lung. Am J Physiol Lung Cell Mol Physiol 2002;283:L1161–L1178.[Abstract/Free Full Text]
  26. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 1994;10:38–47.[Abstract]
  27. Sun W, Wu R, Last JA. Effects of exposure to environmental tobacco smoke on a human tracheobronchial epithelial cell line. Toxicology 1995;100:163–174.[CrossRef][Medline]
  28. Spencer JP, Jenner A, Chimel K, Aruoma OI, Cross CE, Wu R, Halliwell B. DNA damage in human respiratory tract epithelial cells: damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals. FEBS Lett 1995;375:179–182.[CrossRef][Medline]
  29. Adiseshaiah P, Papaiahgari SR, Vuong H, Kalvakolanu DV, Reddy SP. Multiple cis-elements mediate the transcriptional activation of human fra-1 by 12-O-tetradecanoylphorbol-13-acetate in bronchial epithelial cells. J Biol Chem 2003;278:47423–47433.[Abstract/Free Full Text]
  30. Muller JM, Cahill MA, Rupec RA, Baeuerle PA, Nordheim A. Antioxidants as well as oxidants activate c-fos via Ras-dependent activation of extracellular-signal-regulated kinase 2 and Elk-1. Eur J Biochem 1997;244:45–52.[Medline]
  31. Chang WC, Lee YC, Liu CL, Hsu JD, Wang HC, Chen CC, Wang CJ. Increased expression of iNOS and c-fos via regulation of protein tyrosine phosphorylation and MEK1/ERK2 proteins in terminal bronchiole lesions in the lungs of rats exposed to cigarette smoke. Arch Toxicol 2001;75:28–35.[CrossRef][Medline]
  32. Liu C, Wang XD, Bronson RT, Smith DE, Krinsky NI, Russell RM. Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets. Carcinogenesis 2000;21:2245–2253.[Abstract/Free Full Text]
  33. Tulchinsky E. Fos family members: regulation, structure and role in oncogenic transformation. Histol Histopathol 2000;15:921–928.[Medline]
  34. Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 1996;74:589–607.[CrossRef][Medline]
  35. Vial E, Marshall CJ. Elevated ERK-MAP kinase activity protects the FOS family member FRA-1 against proteasomal degradation in colon carcinoma cells. J Cell Sci 2003;116:4957–4963.[Abstract/Free Full Text]
  36. Cook SJ, Aziz N, McMahon M. The repertoire of fos and jun proteins expressed during the G1 phase of the cell cycle is determined by the duration of mitogen-activated protein kinase activation. Mol Cell Biol 1999;19:330–341.[Abstract/Free Full Text]
  37. Ramos-Nino ME, Timblin CR, Mossman BT. Mesothelial cell transformation requires increased AP-1 binding activity and ERK-dependent Fra-1 expression. Cancer Res 2002;62:6065–6069.[Abstract/Free Full Text]
  38. Hurd TW, Culbert AA, Webster KJ, Tavare JM. Dual role for mitogen-activated protein kinase (Erk) in insulin-dependent regulation of Fra-1 (fos-related antigen-1) transcription and phosphorylation. Biochem J 2002;368:573–580.[CrossRef][Medline]
  39. Fischer OM, Hart S, Gschwind A, Ullrich A. EGFR signal transactivation in cancer cells. Biochem Soc Trans 2003;31:1203–1208.[Medline]
  40. Lemjabbar H, Li D, Gallup M, Sidhu S, Drori E, Basbaum C. Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-converting enzyme and amphiregulin. J Biol Chem 2003;278:26202–26207.[Abstract/Free Full Text]
  41. Wang H, Liu X, Umino T, Skold CM, Zhu Y, Kohyama T, Spurzem JR, Romberger DJ, Rennard SI. Cigarette smoke inhibits human bronchial epithelial cell repair processes. Am J Respir Cell Mol Biol 2001;25:772–779.[Abstract/Free Full Text]
  42. Hoshino Y, Mio T, Nagai S, Miki H, Ito I, Izumi T. Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line. Am J Physiol Lung Cell Mol Physiol 2001;281:L509–L516.[Abstract/Free Full Text]
  43. Masubuchi T, Koyama S, Sato E, Takamizawa A, Kubo K, Sekiguchi M, Nagai S, Izumi T. Smoke extract stimulates lung epithelial cells to release neutrophil and monocyte chemotactic activity. Am J Pathol 1998;153:1903–1912.[Abstract/Free Full Text]
  44. Hoyt JC, Robbins RA, Habib M, Springall DR, Buttery LD, Polak JM, Barnes PJ. Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells. Exp Lung Res 2003;29:17–28.[CrossRef][Medline]
  45. Shishodia S, Potdar P, Gairola CG, Aggarwal BB. Curcumin (diferuloylmethane) down-regulates cigarette smoke-induced NF-kappaB activation through inhibition of IkappaBalpha kinase in human lung epithelial cells: correlation with suppression of COX-2, MMP-9 and cyclin D1. Carcinogenesis 2003;24:1269–1279.[Abstract/Free Full Text]
  46. Manning CB, Vallyathan V, Mossman BT. Diseases caused by asbestos: mechanisms of injury and disease development. Int Immunopharmacol 2002;2:191–200.[CrossRef][Medline]
  47. Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995;19:183–232.[Medline]
  48. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159–3167.[CrossRef][Medline]
  49. Takeyama K, Jung B, Shim JJ, Burgel PR, Dao-Pick T, Ueki IF, Protin U, Kroschel P, Nadel JA. Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol 2001;280:L165–L172.[Abstract/Free Full Text]
  50. Zenz R, Scheuch H, Martin P, Frank C, Eferl R, Kenner L, Sibilia M, Wagner EF. c-Jun regulates eyelid closure and skin tumor development through EGFR signaling. Dev Cell 2003;4:879–889.[CrossRef][Medline]
  51. Brusselbach S, Mohle-Steinlein U, Wang ZQ, Schreiber M, Lucibello FC, Muller R, Wagner EF. Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos. Oncogene 1995;10:79–86.[Medline]
  52. Schreiber M, Poirier C, Franchi A, Kurzbauer R, Guenet JL, Carle GF, Wagner EF. Structure and chromosomal assignment of the mouse fra-1 gene, and its exclusion as a candidate gene for oc (osteosclerosis). Oncogene 1997;15:1171–1178.[CrossRef][Medline]
  53. Kang MJ, Oh YM, Lee JC, Kim DG, Park MJ, Lee MG, Hyun IG, Han SK, Shim YS, Jung KS. Lung matrix metalloproteinase-9 correlates with cigarette smoking and obstruction of airflow. J Korean Med Sci 2003;18:821–827.[Medline]
  54. Selman M, Cisneros-Lira J, Gaxiola M, Ramirez R, Kudlacz EM, Mitchell PG, Pardo A. Matrix metalloproteinases inhibition attenuates tobacco smoke-induced emphysema in Guinea pigs. Chest 2003;123:1633–1641.[Abstract/Free Full Text]
  55. Seagrave J, Barr EB, March TH, Nikula KJ. Effects of cigarette smoke exposure and cessation on inflammatory cells and matrix metalloproteinase activity in mice. Exp Lung Res 2004;30:1–15.
  56. Seagrave J. Oxidative mechanisms in tobacco smoke-induced emphysema. J Toxicol Environ Health A 2000;61:69–78.[CrossRef][Medline]
  57. Gensch E, Gallup M, Sucher A, Li D, Gebremichael A, Lemjabbar H, Mengistab A, Dasari V, Hotchkiss J, Harkema J, et al. Tobacco smoke control of mucin production in lung cells requires oxygen radicals AP-1 and JNK. J Biol Chem 2004;279:39085–39093.[Abstract/Free Full Text]
  58. Takeuchi K, Kato M, Suzuki H, Akhand AA, Wu J, Hossain K, Miyata T, Matsumoto Y, Nimura Y, Nakashima I. Acrolein induces activation of the epidermal growth factor receptor of human keratinocytes for cell death. J Cell Biochem 2001;81:679–688.[CrossRef][Medline]
  59. Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003;21:2787–2799.[Abstract/Free Full Text]
  60. Yoshii S, Tanaka M, Otsuki Y, Fujiyama T, Kataoka H, Arai H, Hanai H, Sugimura H. Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene. Mol Cell Biol 2001;21:6796–6807.[Abstract/Free Full Text]
  61. Lei W, Yu R, Mandlekar S, Kong AN. Induction of apoptosis and activation of interleukin 1beta-converting enzyme/Ced-3 protease (caspase-3) and c-Jun NH2-terminal kinase 1 by benzo(a)pyrene. Cancer Res 1998;58:2102–2106.[Abstract/Free Full Text]
  62. Ranganna K, Yousefipour Z, Nasif R, Yatsu FM, Milton SG, Hayes BE. Acrolein activates mitogen-activated protein kinase signal transduction pathways in rat vascular smooth muscle cells. Mol Cell Biochem 2002;240:83–98.[CrossRef][Medline]
  63. Finkelstein EI, Nardini M, van der Vliet A. Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease? Am J Physiol Lung Cell Mol Physiol 2001;281:L732–L739.[Abstract/Free Full Text]



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