Carcinogenesis vol.26 no.12 pp.2187–2195, 2005
doi:10.1093/carcin/bgi189
Advance Access publication July 28, 2005
MAPK/AP-1 signal pathway in tobacco smoke-induced cell proliferation and
squamous metaplasia in the lungs of rats
Cai-Yun Zhongy, Ya-Mei Zhouy, Gordon C.Douglas1,
Hanspeter Witschi and Kent E.Pinkerton
Center for Health and the Environment and 1Department of Cell Biology and
Human Anatomy, University of California, Davis, CA 95616, USA
To whom correspondence should be addressed. Tel: þ1 530 752 8334;
Fax: þ1 530 752 5300;
Email: [email protected]
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.
y
These authors contributed equally to this work.
Carcinogenesis vol.26 no.12 # Oxford University Press 2005; all rights reserved.
Introduction
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 (1–4). 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 44000 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). Mitogenactivated 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
(14–18). One in vitro study has examined the differential
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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
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). [g-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
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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 mm 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 phosphoERK1/2, 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. b-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
MAPK/AP-1 pathway in TS-induced pathogenesis
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: 250.
consensus sequence labeled with [g-32P]dATP using T4 polynucleotide kinase.
The binding reaction of nuclear proteins to the probe was assessed by incubation of mixtures containing 5 mg nuclear protein, 0.5 mg poly (dIƒdC) 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 mm.
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 5 0.05 was considered to be significantly different.
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 ¼ 6). P 5 0.05, compared with FA; P 5 0.01, compared with FA.
Results
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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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 5 0.01) (Figure 3).
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 5 0.05) and Jun D
(P 5 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.
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.
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).
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 ¼ 4). P 5 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
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 ¼ 3). P 5 0.05, compared with FA;
P 5 0.01, compared with FA.
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MAPK/AP-1 pathway in TS-induced pathogenesis
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 ¼ 3). P 5 0.05, compared with FA;
P 5 0.01, compared with FA.
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 ¼ 3). P 5 0.05, compared with FA; P 5 0.01, compared with FA.
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 ¼ 3). P 5 0.01, compared with FA.
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.
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,
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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: 250.
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 ¼ 3). P 5 0.05, compared with FA; P 5 0.01, compared with FA.
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.
Discussion
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
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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 (4,23–25), it is not surprising that TS induces the
activation of the ERK1/2 pathway, as observed in our study.
MAPK/AP-1 pathway in TS-induced pathogenesis
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 (26–29).
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 TSinduced 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 (41–43).
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
(44–46); (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.
Acknowledgements
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.
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Received February 4, 2005; revised July 18, 2005;
accepted July 19, 2005
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