MAPK/AP-1 signal pathway in tobacco smoke-induced cell proliferation and squamous metaplasia in the lungs of rats

Cai-Yun Zhong {dagger}, Ya-Mei Zhou {dagger}, Gordon C. Douglas 1, Hanspeter Witschi and Kent E. Pinkerton *

Center for Health and the Environment and 1 Department of Cell Biology and Human Anatomy, University of California, Davis, CA 95616, USA

* To whom correspondence should be addressed. Tel: +1 530 752 8334; Fax: +1 530 752 5300; Email: kepinkerton{at}ucdavis.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Overwhelming evidence has demonstrated tobacco smoke (TS) is causally associated with various types of cancers, especially lung cancer. Sustained epithelial cell hyperplasia and squamous metaplasia are considered as preneoplastic lesions during the formation of lung cancer. The cellular and molecular mechanisms leading to lung cancer due to TS are not clear. Mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1) can be activated by various stimuli and play a critical role in the control of cell proliferation and differentiation. To date, information on the response of the MAPK/AP-1 pathway during hyperplasia and squamous metaplasia induced by TS is lacking. We therefore investigated the effects of TS on the development of epithelial hyperplasia and squamous metaplasia, regulation of MAPK/AP-1 activation, and expression of AP-1-regulated cell cycle proteins and differentiation markers in the lungs of rats. Exposure of rats to TS (30 mg/m3 or 80 mg/m3, 6 h/day, 3 days/week for 14 weeks) dramatically induced cell proliferation and squamous metaplasia in a dose-dependent manner, effects that paralleled the activation of AP-1-DNA binding activity. Phosphorylated ERK1/2, JNK, p38 and ERK5 were significantly increased by exposure to TS, indicating the activation of these MAPK pathways. Expression of Jun and Fos proteins were differentially regulated by TS. TS upregulated the expression of AP-1-dependent cell cycle proteins including cyclin D1 and proliferating cell nuclear antigen (PCNA). Among the AP-1-dependent cell differentiation markers, keratin 5 and 14 were upregulated, while loricrin, filaggrin and involucrin were downregulated following TS exposure. These findings suggest the important role of MAPK/AP-1 pathway in TS-induced pathogenesis, thus providing new insights into the molecular mechanisms of TS-associated lung diseases including lung cancers.

Abbreviations: AP-1, activator protein-1; BrdU, bromodeoxyuridine; DAB, diaminobenzidine; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinases; FA, filtered air; JNK, Jun N-terminal kinases; MAPK, mitogen-activated protein kinases; PCNA, proliferating cell nuclear antigen; SAPK, stress-activated protein kinase; TS, tobacco smoke; TSP, total suspended particulates


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Compelling evidence has demonstrated tobacco smoke (TS) is causally associated with various types of human cancers. Both epidemiological and experimental studies have revealed the positive link between TS and risk of lung cancer development as well as oral, esophageal, laryngeal, pancreatic, bladder, kidney, cervical and skin cancers (14). It is estimated TS causes ~80–90% of all lung cancer. In the USA, up to 90% of deaths from lung cancer in men and 95% in women are attributable to smoking (5). TS contains >4000 constituents including at least 60 known carcinogens. These potential carcinogens include both initiators (e.g. tobacco smoke-specific nitrosamine and polycyclic aromatic hydrocarbons) and promoters (e.g. acetaldehyde and phenol derivatives). Carcinogenic metals (e.g. arsenic, nickel, cadmium and chromium) and radioactive elements (e.g. polonium-210) are also found in TS. TS also contains numerous free radicals and oxidative agents. Inhaled TS is known to induce an inflammatory response, which can produce further reactive oxygen species (ROS) in tissues.

The vast majority of lung tumors are bronchogenic carcinomas which account for 90–95% of all lung tumors. Bronchogenic carcinomas arise most often in and about the hilus of the lung. Approximately three-quarters of these cancers originate in the major bronchi, while a smaller proportion of these cancers arise in the periphery of the lung from alveolar or bronchiolar cells (6). The cellular and molecular mechanisms for bronchial carcinogenesis by TS are not clear. It is speculated that sustained epithelial cell hyperplasia, altered cellular differentiation as well as genetic instability lead to tumor formation. Cell signal pathways that sense external insults and govern critical cellular process such as cell proliferation, differentiation, apoptosis and transformation are believed to play critical roles in the pathogenesis of toxicant-induced cancer.

The activator protein-1 (AP-1) transcription factor is a hetero- or homo-dimeric complex that comprises members of the proto-oncogene Jun proteins (c-Jun, JunB and JunD) and Fos proteins (c-Fos, Fos B, Fra-1 and Fra-2) families (7,8). In response to a broad range of extracellular stimuli, AP-1 proteins bind to the TPA-response element (TRE) to transcriptionally activate target genes that regulate many critical cellular processes, including cell proliferation, differentiation, apoptosis and oncogenic transformation, thus playing the central role in the process of tumorigenesis (8,9). Mitogen-activated protein kinases (MAPK) are responsible for the phosphorylation and activation of Jun and Fos proteins. There are three well-characterized subfamilies of MAPK, including the extracellular signal-regulated kinases (ERK1/2), the Jun N-terminal kinases (JNK)/stress-activated protein kinase (SAPK) and p38 (10,11). A fourth MAPK, ERK5/BMK1 (big MAPK1) has also been identified (12,13). Previous reports have shown TS exposure induces AP-1 and MAPK activation (1418). One in vitro study has examined the differential expression of Jun (c-Jun, JunB and JunD) and Fos (c-Fos, Fos B, Fra-1 and Fra-2 ) protein families (17). In vivo studies, however, primarily examined the effects of TS on c-Jun and c-Fos expression, little is known regarding other members of the AP-1 family. Information on the response of the new member of MAPK, ERK5, to TS is also unknown since previous studies examined only the three classic MAPK pathways. Evidence also reveals that TS induces the expression of cyclin D1 and PCNA, the AP-1 target genes involved in promoting cell cycle progression (15,19). No studies, however, have been done to investigate AP-1 target cell differentiation genes in TS-induced squamous cell metaplasia.

This study was designed to investigate the regulation and role of the MAPK/AP-1 signal pathway in TS-induced lung epithelial hyperplasia and squamous cell metaplasia. We demonstrate that exposure to TS induces pulmonary epithelial cell proliferation and squamous metaplasia in a dose-dependent manner, and that activation of the MAPK/AP-1 pathway and dysregulation of AP-1 target cell cycle proteins as well as differentiation markers are implicated in the induction of these deleterious lesions. These findings indicate the important role of MAPK/AP-1 in the development of TS-induced pathogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
Bromodeoxyuridine (BrdU) was purchased from Sigma-Aldrich (St Louis, MO). Diaminobenzidine (DAB) substrate kit was from Zymed Laboratories (South San Francisco, CA). [{gamma}-32P]dATP was from Amersham Pharma Biotech (Piscataway, NJ). AP-1 consensus oligonucleotides and T4 polynucleotide kinase were from Promega (Madison, WI). Mouse monoclonal anti-BrdU primary antibody was from Roch Applied Science (Indianapolis, IN). Antibodies to phospho-ERK1/2, phospho-JNK/SAPK, phospho-p38 and phospho-ERK5 were from Cell Signaling Technology (Beverly, MA). Antibodies to c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, Fra-2, cyclin D1, PCNA and involucrin were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to keratin 5, keratin 14, loricrin and filaggrin were from Covance Research Products (Berkeley, CA).

Animals
Twelve-week-old male spontaneously hypertensive (SH) rats (derived from Wistar–Kyoto rats by phenotypic segregation of the hypertensive trait and inbreeding) weighing ~260–310 g were purchased from Charles River Laboratories (Raleigh, NC). This strain of rats was selected based on previous studies, completed in our laboratory, to demonstrate these rats to be highly sensitive to the effects of TS exposure, with a significant induction of squamous cell metaplasia in the epithelium of the intrapulmonary airways (20). All rats were allowed to acclimate for 1 week prior to the onset of experimental exposure. The rats were housed in polypropylene cages, maintained on a 12-hour light/dark cycle, and provided water and rat chow ad libitum. Animals were handled in accordance with standards established by the US Animal Welfare Acts as set forth in the National Institutes of Health Guidelines and by the University of California, Davis, Animal Care and Use Committee.

TS exposure
Rats were exposed to a mixture of sidestream and mainstream cigarette smoke in a smoking apparatus built in our laboratory (21). The cigarettes were humidified 1R4F research cigarettes (Tobacco Health Research Institute, Lexington, KY). An automatic metered puffer was used to smoke the cigarettes under Federal Trade Commission conditions (35 ml puff, 2 s duration, 1 puff per min). The smoke was collected in a chimney, diluted with filtered air (FA), and delivered to whole-body exposure chambers. The exposures were characterized for the three major constituents of cigarette smoke: nicotine, carbon monoxide and total suspended particulates (TSP). Animals were exposed for 6 h/day, 3 days/week for a total of 14 weeks. This exposure regimen was selected based on earlier studies that demonstrated this strain of rats better tolerated exposure to a high level of particulate matter smoke for prolonged periods of time with exposure being 3 days/week rather than 5 days/week. Carbon monoxide was measured every 30 min, TSP every 2 h and nicotine per day (approximately midway through the exposure period). Experiments consisted of animals exposed to only FA (control), a low concentration of TS (30 mg/m3 TSP) and a high concentration of TS (80 mg/m3 TSP).

Tissue preparation
Two hours prior to necropsy, each rat received an intraperitoneal injection of BrdU 20 mg/kg body wt, a nucleotide analog used to identify cells undergoing DNA synthesis. At necropsy, animals were anesthetized with an overdose of sodium pentobarbital. The trachea was cannulated and the chest cavity opened by a midline incision. The right lung was frozen in liquid nitrogen, and stored at –80°C until use. The left lung was inflation-fixed by intratracheal instillation of 4% buffered zinc formalin (Z-Fix) at 30 cm of water pressure for 1 h, and stored in 70% ethanol before processing. Transverse slices were cut immediately cranial and caudal to the hilus of the left lung, dehydrated in a graded ethanol series, and embedded in paraffin for use in immunohistochemical and morphormetric studies.

This tissue sampling strategy allowed for examination of the central axial airway, distal airways as well as lung parenchyma. Tissue sections were prepared using a HM 355 rotary microtome (Carl Zeiss, Thornwood, NY). A small piece of intestine from each rat was used as a labeling control for BrdU immunostaining. Sections 5 µm thick were placed on Superfrost Plus glass slides.

Immunohistochemistry for BrdU
Tissue sections were deparaffinized in xylene and hydrated in a graded ethanol series, respectively. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min at room temperature, followed by incubation with 0.1% protease for 3 min. Following non-specific blocking in 10% horse serum, sections were incubated with mouse monoclonal anti-BrdU primary antibody for 1 h at 37°C. Sections were subsequently incubated in biotinylated horse anti-mouse secondary antibody followed by ABC reagent from a Vectastain ABC kit (Vector, Burlingame, CA), each for 30 min at room temperature. DAB was used as chromogen substrate, then counterstained with nuclear fast red.

Labeling indices were determined for the central axial airway, distal airways down to terminal bronchioles and lung parenchyma. Terminal bronchioles were defined as the last conducting airway opening into an alveolar duct. Examination of the lung parenchyma was done by selecting non-overlapping fields initiated by random start and subsequent stage movement across parenchymal tissues in a zigzag pattern.

Intestine tissue from each rat was stained for BrdU labeling simultaneously, and the intense staining in the intestine of each animal served as the positive control for BrdU labeling. A negative control was also performed using mouse IgG. In each region, 500–1000 cells per lung were counted. The labeling index was expressed as a percentage of BrdU-positive cells.

Preparation of lung cytosolic and nuclear protein extracts
The method for the preparation of lung cytosolic and nuclear protein extracts has been described previously (22). Briefly, immediately following deep anesthesia, the right lung lobes were removed, frozen in liquid nitrogen, and stored in –80°C. The lungs were homogenized in ice-cold Tris–HCl buffer (25 mM Tris, 1 mM EDTA, 10% glycerol and 1 mM DTT, pH 7.4) with a glass homogenizer. The homogenate was centrifuged at 10 000 g for 20 min at 4°C. The supernatant containing cytosolic protein was aliquoted and stored at –80°C. The crude nuclear fraction in the pellet was washed three times with homogenate buffer containing Triton X-100, followed by washing one time without Triton X-100. Nuclear protein was extracted with buffer C (20 mM HEPES, 25% glycerol, 0.42 M NaCl and 1 mM EDTA) by centrifugation at 50 000 g for 30 min. Protein concentration was measured by a modified Bradford assay according to the manufacturer's instructions (Bio-Rad, Hercules, CA) with bovine serum albumin as standard.

Western blot analysis
Western blot analyses were used to measure the protein levels of phospho-ERK1/2, phospho-JNK, phospho-p38, phospho-ERK5, c-Jun, JunB, JunD, c-Fos, Fos B, Fra-1, Fra-2, cyclin D1, PCNA, keratin 5, keratin 14, loricrin, filaggrin and involucrin. Fifty micrograms of cytosolic or nuclear proteins were loaded and separated on 10–12% SDS–PAGE, followed by transblotting to an ImmunBlot PVDF membrane (Bio-Rad, Hercules, CA). The membrane was subsequently probed with primary antibody at a dilution of 1:1000. Horseradish peroxidase-conjugated secondary antibody was added at a dilution of 1:3000. The blots were subsequently developed using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech). Following exposure on autoradiography film, immunoreactive protein bands were quantified by densitometry. ß-actin served as the loading control.

Electrophoretic mobility shift assay (EMSA)
EMSA was performed to determine AP-1–DNA binding activity. The oligonucleotide used as a probe was double-stranded DNA containing AP-1 consensus sequence labeled with [{gamma}-32P]dATP using T4 polynucleotide kinase. The binding reaction of nuclear proteins to the probe was assessed by incubation of mixtures containing 5 µg nuclear protein, 0.5 µg poly (dI•dC) and 40 000 c.p.m. 32P- labeled probe in the binding buffer (7.5 mM HEPES, pH 7.6, 35 mM NaCl, 1.5 mM MgCl2, 0.05 mM EDTA, 1 mM DTT and 7.5% glycerol) for 30 min at 25°C. For the competitive assay, excessive unlabeled oligonucleotides were incubated with proteins prior to the addition of radiolabeled probe. Protein-DNA binding complex was separated by 5% polyacrylamide gel electrophoresis and autoradiographed overnight.

Examination of epithelial cell squamous metaplasia
Sections of the paraffin-embedded left lung were cut at a thickness of 5 µm. All sections were stained with hematoxylin and eosin. Structures examined included the central axial airway, distal airways down to terminal bronchioles and lung parenchyma. Epithelial composition covering the basal lamina of each airway was classified as either simple-to-pseudostratified columnar or stratified squamous. Simple or pseudostratified columnar epithelium consists of ciliated, mucous and basal cells lining the basal lamina of the airway. Stratified squamous epithelium consisted of mixed flattened, elongated cells of varying degrees in multiple layers with distinct stratification.

Statistical analysis
Statistical analyses were performed using Statview statistical software (SAS Institute, Cary, NC). All data were expressed as mean ± SE. Comparisons among TS-exposed and FA-exposed groups were made by ANOVA followed by Fisher's protected least significant difference post hoc multiple comparisons. A value of P < 0.05 was considered to be significantly different.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
TS induces cell proliferation in the lungs of rats
To examine the effect of TS on cell proliferation in the lungs of rats, immunohistochemistry for BrdU within specific anatomical sites was performed for the central axial airway, distal airways and lung parenchyma. These studies demonstrated a striking induction in cell proliferation of the airways in rats following 14 weeks of TS exposure. The majority of proliferating cells in the central airway were basal cells. In distal airways, the proliferating cells included basal cells and epithelial cells. In the lung parenchyma, the proliferating cells appeared to be primarily epithelial cells (Figure 1). BrdU labeling indices for the central airway, distal airways and lung parenchyma are shown in Figure 2. At a concentration of 80 mg/m3, TS markedly increased BrdU labeling indices in the central airway (40-fold) and distal airways (33-fold). BrdU labeling index in the lung parenchyma was not significantly increased following 80 mg/m3 of TS exposure. In contrast to the dramatic induction of cell proliferation by 80 mg/m3 of TS exposure, no significant increases in BrdU labeling index for all anatomical regions examined were noted following exposure to 30 mg/m3 of TS.



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Fig. 1. Micrograph of lung tissue sections with BrdU immunohistochemical staining of proliferating cells. Regions include the central airway (A), distal airway (B) and lung parenchyma (C). Lung tissues were from rats exposed to FA, 30 or 80 mg/m3 TS. Proliferating cells were identified as darkly stained with BrdU, as indicated by arrow. Magnification: 250x.

 


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Fig. 2. BrdU labeling index within the regions of the central airway, distal airway and parenchyma of the lungs. The labeling index is expressed as the percentage of BrdU-positive cells. Data are shown as means ± SE (n = 6). *P < 0.05, compared with FA; **P < 0.01, compared with FA.

 
AP-1-DNA binding activity is increased by TS
AP-1 is the major transcription factor that is crucial in the control of a number of signal transduction cascades including cell growth. To examine the effect of TS exposure on AP-1 activation, AP-1-DNA binding activity was measured in lung tissue nuclear extract by EMSA. Consistent with BrdU immunohistochemical staining, exposure to 30 mg/m3 of TS did not significantly change AP-1-DNA binding activity in lung tissues. In contrast, exposure of animals to 80 mg/m3 of TS resulted in a significant increase in AP-1-DNA binding activity (P < 0.01) (Figure 3).



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Fig. 3. Effects of TS on AP-1 activation. (A) Electrophoretic mobility shift assay of AP-1-DNA binding activity in rat lungs. Lane 1, competition assay; lanes 2–5, FA; lanes 6–9, 30 mg/m3 TS; lanes 10–13, 80 mg/m3 TS. (B) Densitometry of AP-1-DNA binding activity. Data are expressed as mean ± SE (n = 4). **P < 0.01, compared with FA.

 
Differential changes of AP-1 subunits by TS
AP-1 is composed of either homo- or hetero-dimers between members of Jun and Fos families. Expression of AP-1 subunits is differentially regulated in response to various stimuli. To examine the effects of TS exposure on the expression of AP-1 subunits, the nuclear protein levels of Jun and Fos family members were measured by western blot analysis. As shown in Figure 4, significant increases in c-Jun (P < 0.05) and Jun D (P < 0.01) were noted in animals exposed to 80 mg/m3 of TS when compared with FA controls, while the level of Jun B was decreased in these animals. The expression of Fos family members, including c-Fos, Fos B and Fra-2 were upregulated following 80 mg/m3 of TS exposure (Figure 5). The expression level of Fra-1 was undetectable in animals exposed to either TS or FA. No significant changes were observed for Jun and Fos proteins in animals exposed to 30 mg/m3 of TS.



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Fig. 4. Effects of TS on the expression of Jun proteins. (A) Western blot analysis of c-Jun, Jun B and Jun D in rat lungs. Lanes 1–3, FA; lanes 4–6, 30 mg/m3 TS; lanes 7–9, 80 mg/m3 TS. (B) Densitometry of western blot for Jun proteins. Values are presented as mean ± SE (n = 3). *P < 0.05, compared with FA; **P < 0.01, compared with FA.

 


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Fig. 5. Effects of TS on the expression of Fos proteins. (A) Western blot analysis of c-Fos, FosB and Fra-2 in rat lungs. Lanes 1–3, FA; lanes 4–6, 30 mg/m3 TS; lanes 7–9, 80 mg/m3 TS. (B) Densitometry of western blot for Fos proteins. Values are presented as mean ± SE (n = 3). *P < 0.05, compared with FA; **P < 0.01, compared with FA.

 
TS activates AP-1 through ERK1/2/JNK/p38/ERK5 pathways
Activation of AP-1 is triggered through distinct pathways in response to various stimuli. In order to better understand the underlying mechanism of TS in inducing cell proliferation and AP-1 activation, AP-1 activation pathways were examined (Figure 6). Results showed that 80 mg/m3 of TS significantly upregulated phosphorylated ERK1/2, indicative of ERK1/2 pathway activation, by 126 and 134% over control values, respectively. In addition, phosphorylated JNK and p38 were also elevated by 148 and 204%, respectively, compared with FA controls. Activation of ERK5, the newer member of MAPK, was also elevated by 190%. The levels of phosphorylated ERK1/2, JNK, p38 and ERK5 in rats exposed to 30 mg/m3 of TS were not significantly different from the FA control group.



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Fig. 6. Effects of TS on the activation (phosphorylation) of MAPK pathways. (A) Western blot analyses of phospho-ERK1/2, phospho-JNK, phospho-p38 and phospho-ERK5 in rat lungs. Lanes 1–3, FA; lanes 4–6, 30 mg/m3 TS; lanes 7–9, 80 mg/m3 TS. (B) Densitometry of western blot. Values are presented as mean ± SE (n = 3). *P < 0.05, compared with FA; **P < 0.01, compared with FA.

 
TS upregulates the expression of AP-1 dependent cell cycle proteins
To further explore the role of AP-1 in TS-induced cell proliferation, the expression of AP-1-dependent cell cycle proteins including cyclin D1 and PCNA were measured. In line with the findings for cell proliferation and AP-1 activation, TS upregulated the expression levels of both cyclin D1 and PCNA in a dose-dependent manner. The level of cyclin D1 was increased by 89% following exposure to 80 mg/m3 of TS. The level of PCNA was increased by 108% following TS exposure at 80 mg/m3. In contrast, 30 mg/m3 of TS did not significantly increase the expression levels of cyclin D1 or proliferating cell nuclear antigen (PCNA) (Figure 7).



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Fig. 7. Effects of TS on the expression of cell cycle proteins. (A) Western blot analyses of cyclin D1 and PCNA in rat lungs. Lanes 1–3, FA; lanes 4–6, 30 mg/m3 TS; lanes 7–9, 80 mg/m3 TS. (B) Densitometry of western blot. Values are presented as mean ± SE (n = 3). **P < 0.01, compared with FA.

 
TS induces airway epithelial squamous metaplasia
A significant consequence of TS exposure is airway epithelial squamous metaplasia. To evaluate the morphologic changes in the airway epithelium, histological analysis was performed. Observations of lungs from rats exposed to 80 mg/m3 of TS for 14 weeks demonstrated dramatic squamous cell metaplasia in the central airway epithelium, in contrast to the columnar epithelium lining the airways of FA control rats (Figure 8). Squamous cell metaplasia in airways consisted of flattened, stratified layering of elongated epithelial cells, some with prominent filamentous-like cytoplasmic inclusions. Areas of airway squamous cell metaplasia demonstrated the absence of ciliated cells and mucous cells within the central airway. Squamous cell metaplasia was most dramatic in the central airway, and became less pronounced entering the distal airways. No squamous cell metaplasia was observed in rats exposed to 30 mg/m3 of TS or to FA.



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Fig. 8. Morphometric analysis of epithelium from the central airway (A), distal airway (B) and lung parenchyma (C) in rats exposed to FA, 30 or 80 mg/m3 TS. Note the striking squamous metaplasia observed in the central airway of rats exposed to 80 mg/m3 TS, as indicated by arrows. Magnification: 250x.

 
TS alters the expression of AP-1-regulated cell differentiation markers
AP-1 critically regulates not only the process of cell proliferation, but also cell differentiation as well. To elucidate the role of AP-1 in TS-induced squamous cell metaplasia, the expression of AP-1-regulated cell differentiation markers including keratin (k5 and k14) and late differentiation markers (loricrin, filaggrin and involucrin), were measured. Figure 9 shows that 80 mg/m3 of TS significantly induced the expression of k5 and k14, which were elevated by 140 and 77% over control values, respectively. However, 80 mg/m3 of TS suppressed the expression of late differentiation markers. The levels of loricrin, filaggrin and involucrin were decreased by 68, 53 and 57%, respectively. No significant alterations were observed in rats exposed to 30 mg/m3 of TS.



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Fig. 9. Effects of TS on the expression of cell differentiation markers. (A) Western blot analyses of keratin 5, keratin 14, loricrin, filaggrin and involucrin in rat lungs. Lanes 1–3, FA; lanes 4–6, 30 mg/m3 TS; lanes 7–9, 80 mg/m3 TS. (B) Densitometry of western blot. Values are presented as mean ± SE (n = 3). *P < 0.05, compared with FA; **P < 0.01, compared with FA.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we have demonstrated that TS induces airway epithelial cell hyperplasia and squamous metaplasia in a dose-dependent fashion. In concert with these data, our results also show that TS activates AP-1 through all four distinct MAPK pathways including ERK1/2, JNK, p38 and ERK5, and correspondingly, deregulates AP-1-dependent cell cycle proteins as well as cell differentiation markers. These in vivo findings strongly suggest an important role of the MAPK/AP-1 signal pathway in TS-induced pathogenesis.

It has been established that AP-1 activity plays a central role in the process of tumorigenesis (8,9). Distinct MAPK pathways are responsible for the phosphorylation and activation of AP-1 proteins. ERK1/2 pathway is activated by many different stimuli, including growth factors, viral infection, ligands for G protein-coupled receptors, transforming agents and carcinogens. In addition, Ras mutation persistently activates the Raf/MAPKK/ERK1/2 pathway. Inflammatory cytokines, growth factors, ligands for G protein-coupled receptors and oxidative stresses are the stimuli for the activation of JNK and p38. Oxidative stress and growth factors are also strong stimuli for the ERK5 pathway. Given that TS contains at least 60 known carcinogens with the capacity to mutate oncogenes like Ras (4,2325), it is not surprising that TS induces the activation of the ERK1/2 pathway, as observed in our study. TS can also cleave the proligands for epidermal growth factor receptor (EGFR) which subsequently bind to EGFR resulting in EGFR phosphorylation and activation of ERK1/2 (2629). The fact that TS contains numerous oxidants and that TS is capable of inducing an inflammatory response (16,20,30) makes it very plausible that TS activates both JNK and p38, the pathways predominantly involved in response to oxidative stress and inflammation. These oxidants and the cleavage of EGFR proligands are also able to activate ERK5, as noted in our study. To our knowledge, this is the first report to show that TS activates AP-1 through all four distinct MAPK pathways.

Expression of various AP-1 proteins is differentially regulated in response to TS. We found significantly increased expression of c-Jun, Jun D, c-Fos, Fos B and Fra-2, while decreased expression of Jun B following TS exposure (Figures 4 and 5). Jun and Fos proteins differ significantly in both their DNA binding and transactivation potential as well as their target gene regulation. Overexpression of some of these proteins, such as c-Jun, c-Fos and Fos B, can efficiently transform cells and lead to tumor formation. c-Jun upregulates the promoter activity of cyclin D1, whereas Jun B has an opposite effect (31,32). Because of the difference in the transcriptional properties of various AP-1 proteins, different compositions of the AP-1 complex are critical to the regulation of downstream gene expression. The observation of differential changes of Jun and Fos proteins following TS exposure suggests that abnormal expression of specific AP-1 proteins is implicated in the pathogenesis of TS-induced deleterious effects. Further investigations are warranted to elucidate the individual contribution of AP-1 members in TS-induced pathogenesis of lung diseases.

The AP-1 target genes involved in cell proliferation include cyclin D1 and PCNA. Cyclin D1 is a critical regulator of G1 to S phase transition. Binding of cyclin D1 to cyclin dependent kinase (CDK) 4 and CDK 6 increases the activity of these kinases which phosphorylate and inactivate the tumor suppressor protein retinoblastoma (Rb), thereby unshackling the E2F proteins and transcripting genes whose products are essential for progression through the S phase. The cyclin D1 gene regulatory sequences contain two AP-1 binding sites (33,34). Several AP-1 proteins including c-Jun and c-Fos are shown to bind these sites and activate cyclin D1 expression (31,33,35,36). By maintaining the function of DNA polymerase, PCNA is required for DNA synthesis. The PCNA gene contains AP-1 sites in the promoter region and its expression is regulated by AP-1 activity (37,38). In this study we have shown significant upregulation of cyclin D1 and PCNA following TS exposure. These observations parallel BrdU labeling indices and AP-1 activation data, thus reinforcing the role of AP-1 in TS-induced cell proliferation. Consistent with our results, several studies found that TS as well as nicotine, one of the major components of TS, induce the expression of cyclin D1 and/or PCNA (15,19). It is worth noting that some reports observed TS induced p21 protein is implicated in inhibition of cell proliferation (39,40). Measurements of p53–DNA binding activity by EMSA as well as p53 and p21 protein expression by western blotting did not reveal significant changes in rats following TS exposure in our experiments (data not shown).

The list of AP-1 target genes involved in epithelial cell differentiation includes members of the keratin gene family such as k1, k5, k8, k14, k18 and k19, keratin-associated protein, filaggrin, precursor proteins of the cornified envelopes such as loricrin and involucrin, as well as transglutaminases (4143). The sequential expression of specific differentiation marker genes reflects the molecular and morphological changes that are characteristic for each stage of differentiation. K5 and k14 are indicators of the proliferative basal cells, while loricrin, filaggrin, involucrin and transglutaminases are markers of late differentiation. As expected, the expression levels of k5 and k14 were upregulated by TS (Figure 9), which occurred in concert with elevated AP-1 activity and a striking proliferation of basal cells. In contrast, the expression of late differentiation markers, including loricrin, filaggrin and involucrin, were suppressed following TS exposure, indicating immature cell differentiation. Several possible mechanisms by which AP-1 proteins negatively regulate the expression of late differentiation markers have been proposed: (i) a direct physical interaction between AP-1 proteins and other positive regulators of this gene mutually inhibits these two transcriptional regulators (4446); (ii) a squelching mechanism which involves competition with a common transcriptional co-activator (47); (iii) an indirect mechanism where AP-1 could induce a negative modulator of differentiation; (iv) negative regulation by AP-1 may reflect a combination of changes in all factors that contribute to the levels of AP-1 transcriptional activity (48). Consequently, AP-1 suppresses the expression of late differentiation markers, along with AP-1 promoted cell proliferation, resulting in squamous cell metaplasia in the lungs of rats exposed to TS. Our previous study in which antioxidant inhibited TS-induced squamous cell metaplasia (although its effect on AP-1 was not measured) supports our current findings in TS-induced MAPK/AP-1 activation and squamous cell metaplasia (22). Nevertheless, no previous studies have investigated the effects of TS on AP-1-regulated cell differentiation markers. Therefore, this study provides the first report to explore the role of MAPK/AP-1 pathway in TS-induced squamous cell metaplasia.

In conclusion, the present study demonstrates significant effects of TS on aberrant cell proliferation, squamous cell metaplasia and MAPK/AP-1 pathway activation in the lungs of rats. We postulate that by differential responses to TS components, distinct MAPK pathways lead to AP-1 activation and deregulation of target genes, resulting in TS-induced epithelial hyperplasia and squamous metaplasia. These findings indicate the important role of MAPK/AP-1 in the development of TS-induced pathogenesis, thus providing new insights into the molecular mechanisms for TS-associated lung diseases including lung cancer.


    Notes
 
{dagger} These authors contributed equally to this work. Back


    Acknowledgments
 
The authors thank D.Uyeminami, J.Peake and M.Suffia for technical assistance. This work was supported by grants from NIEHS (R01 ES011634 and P30 ES05707).

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 4, 2005; revised July 18, 2005; accepted July 19, 2005.





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