Preferential Induction of CYP1A1 and CYP1B1
in CCSP-Positive Cells
Han Chang,* Louis W. Chang,† Ya-Hsin Cheng,† Wen-Tin Tsai,‡ Ming-Xian Tsai,† and Pinpin Lin†
,‡
,1*Institute of Medicine; Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan, R.O.C.; †Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Kaoshiung, Taiwan, R.O.C.; and ‡Institute of Medical and Molecular
Toxicology, Chung Shan Medical University, Taichung, Taiwan, R.O.C. Received August 14, 2005; accepted October 8, 2005
Both benzo[a]pyrene (BaP) and
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors
(AhR). Although animal studies indicate that both compounds
induce pathological changes in the peripheral lung, the specific
cell type involved remains unclear. Clara cells, expressing Clara
cell specific protein (CCSP) and abundant in cytochrome P450,
are nonciliated bronchiolar epithelial cells in the peripheral lung.
Here we explore the hypothesis that CCSP-positive Clara cells are
highly responsive to AhR ligands and are the primary cell type
involved in BaP- and TCDD-induced toxicities. The
responsive-ness to AhR ligands was evaluated by measuring the respective
mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and
1B1 (CYP1B1) using real-time RT-PCR and
immunocytochemis-try assays. Two in vitro models were used: primary cultures of
human small airway epithelial (SAE) cells and rat lung slice
cultures. In the presence of calcium, human SAE cells
differen-tiated into CCSP-positive cells. BaP- and TCDD-induced mRNA
and protein levels of CYP1A1 and CYP1B1 levels were
signifi-cantly elevated in CCSP-positive cell cultures. Similarly, AhR
mRNA and protein levels were increased in CCSP-positive cell
cultures, as determined by real-time RT-PCR and Western blot
analysis. When rat lung slice cultures were treated with BaP or
TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly
induced in Clara cells. These results indicate that, in the
pe-ripheral lung of both rats and humans, CCSP-positive cells (Clara
cells) may be more sensitive to AhR ligands than other cell types.
Key Words: BaP; TCDD; Clara cells; CYP1A1; CYP1B1; lung.
Exposure to environmental factors such as dioxins,
poly-cyclic aromatic hydrocarbons (PAH), tobacco smoke, and
various air pollutants have been suggested to enhance the risk
of chronic obstructive pulmonary diseases (COPD) and lung
cancer (Bertazzi et al., 2001; Boffetta et al., 1997; Kogevinas,
2000). Benzo[a]pyrene (BaP) is the major PAH found in
to-bacco smoke, which is strongly associated with an increased
risk of COPD and lung cancer (Hecht, 1999).
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin.
In animal studies, TCDD and BaP both induce pathological
changes in the peripheral lungs (Brix et al., 2004; Hecht et al.,
2002; Stoner et al., 1984; Tritscher et al., 2000). For example,
TCDD induces bronchiolar metaplasia in bronchioloalveolar
interfaces (Brix et al., 2004; Tritscher et al., 2000), and
BaP-induced lung adenoma was identified in the peripheral lung in
a sensitive strain of mice (Hecht et al., 2002; Stoner et al.,
1984). Thus, certain cell types in the peripheral lung are likely
to be highly susceptible to BaP and/or TCDD.
Both BaP and TCDD are ligands for the aryl hydrocarbon
receptor (AhR). Ligand interaction induces AhR translocation
to the nuclei and heterodimerization with the AhR translocator
(Arnt), which subsequently transactivates genes of several
drug-metabolizing enzymes, such as cytochrome P4501A1
(CYP1A1) and cytochrome P4501B1 (CYP1B1) (Kress and
Greenlee, 1997; Whitlock, 1999). There is ample evidence
showing that BaP- and TCDD-induced toxic effects are AhR
dependent in vivo (Fernandez-Salguero et al., 1996; Shimizu
et al., 2000). BaP-induced carcinogenesis is abolished in
AhR-deficient mice (Shimizu et al., 2000). Therefore, AhR
expres-sion and CYP1A1/CYP1B1 induction are important indicators
for susceptibility to BaP and TCDD. In human lung tissues, our
studies (Lin et al., 2003) and others (Saarikoski et al., 1998)
both show that AhR and CYP1A1 are mainly expressed in
bronchiolar epithelial cells of the peripheral lung. Similarly,
AhR is expressed in bronchiolar Clara cells and ciliated cells of
rats (Tritscher et al., 2000). TCDD treatment increased
CYP1A1 expression in the cells showing alveolar-bronchiolar
metaplasia (Tritscher et al., 2000). These data provide evidence
that bronchiolar epithelial cells of both humans and rats are
responsive to TCDD.
The bronchiolar epithelia consist of various cell types,
including basal cells, ciliated cells, and Clara cells. Bronchiolar
Clara cells are a specialized type of nonciliated secretory cells,
which are believed to be progenitor cells or local stem cells of
the bronchiolar epithelium (Otto, 2002). Several lines of
1To whom correspondence should be addressed at Institute of Medical and
Molecular Toxicology, Chung Shan Medical University, 110 Sec 1, Chein–Kuo N. Rd., Taichung, 402 Taiwan. Fax: 886–4–24751101. E-mail: ppl@csmu. edu.tw.
Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
evidence suggested that bronchiolar Clara cells are considered
to be the precursors of human lung adenocarcinoma (AD)
(Dermer, 1982; Herrera et al., 1983; Linnoila et al., 1992;
Takezawa et al., 2002). Some studies show that Clara cells are
rich in cytochrome P450s (Hukkanen et al., 2002). Since the
majority of AhR ligand-induced toxicities are dependent on
CYP1 expression, we hypothesized that Clara cells might be
responsive to AhR ligands, such as TCDD and BaP.
To establish the relationship between exposure to AhR
ligands and the pathological changes in the peripheral lung, it
is essential to evaluate the responsiveness of Clara cells to AhR
ligands, such as TCDD and BaP. However, primary cultures of
human Clara cells have not previously been established. It has
been reported that, while CYP1A1 may be barely detectable, it
was nevertheless inducible by AhR ligands (Whitlock, 1999).
Recently human small airway epithelial (SAE) cells have
become available that can differentiate to express Clara cell
secretory protein (CCSP), a specific marker for Clara cells
(Jyonouchi et al., 1999). Therefore, we compared the levels of
induced CYP1A1/CYP1B1 in SAE cell cultures before and
after differentiating into CCSP-positive cells. Furthermore, in
order to identify the sensitive cell types in the bronchiolar
epithelia, we identified the locations of AhR ligand-induced
CYP1A1/CYP1B1 proteins in rat lung slice cultures. Harrigan
et al. (2004) have demonstrated that BaP induced BaP-DNA
adducts formation both in rat lung slice cultures and rat lung
in vivo, suggesting that rat lung slices had comparable
biotransformation ability for BaP. Although several studies
showed that mouse lungs are highly sensitive to xenobiotics
(Simmonds et al., 2004; West et al., 2001), Lee and Dinsdale
(1995) reported that Aroclor 1254 increased CYP1A1 protein
in rat lungs, but not in mouse lungs in vivo. Therefore, lung
slice cultures were prepared from rats, instead of mice, in our
present study. Tissue slice cultures are an in vitro system, which
retain different cell types in an organ. Tissue slices retain the
biochemical capacity and the metabolic function of the whole
organ, allowing for identification of specific cell types
re-sponsive to toxicants. The results generated from this study
should allow us to determine if Clara cells are highly
re-sponsive to carcinogenic AhR ligands (BaP and TCDD).
MATERIAL AND METHODS
Chemicals. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was purchased from ULTRA Scientific (Kingston, RI). Benzo[a]pyrene (BaP) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). TCDD and BaP were dissolved in DMSO, stored in aliquots, and kept at20°C until used.
Human SAE cell culture and cell differentiation. Human SAE cells (Clonetics,Walkersville, MD) were cultivated in SAE basal medium (Clonetics, Walkersville, MD) and supplemented with growth factors, including bovine pituitary extract, hydrocortisone, recombinant epidermal growth factor, epi-nephrine, insulin, triiodothyonine, transferrin, gentamicin/amphotericin-B, reti-noic acid, and bovine serum albumin (fatty acid free) (Clonetics, Walkersville,
MD). SAE cells were plated at a density of 2500 cells/cm2. The medium was changed every other day. SAE cells were subcultivated when they became confluent. Cells used in these experiments were kept for a maximum of five passages.
Jyonouchi et al. (1999) has reported that a high concentration of Ca2þ (1 mM CaCl2) induced differentiation and expression of Clara cell secretory
protein (CCSP) in SAE cell cultures. SAE cells were seeded on 12- or 24-well plates at a density of 3 3 104cells/well. When cells became 70% confluent, the
medium was changed to one containing 1 mM Ca2þfor 72 h. Cell morphology was examined under a light microscope after hematoxylin staining. We found that SAE cells consisted of two cell types. The majority of these cells were small round cells with scanty to adequate cytoplasm, defined as basal cells. Basal cells are thought to be transient amplifying cells, which can terminally differentiate into epithelial cells (Otto, 2002). A few larger cells showed moderate cytoplasm, defined as epithelial cells. In the presence of Ca2þ, the cell appearance changed from a monolayer to a stratified or overlapping appear-ance, similar to epithelial cells lying on the top of basal cells in vivo (airway epithelium).
Treatment of human cells. SAE cells were either preincubated with 1 mM calcium or not. After 72 h preincubation, the culture medium was changed and SAE cells were treated with 1 lM BaP, 1 nM TCDD or a01% DMSO (control). Our preliminary study in human lung cell cultures showed that 1 lM of BaP and 1 nM of TCDD are the minimal doses required to significantly increase both CYP1A1 and CYP1B1 expression. After 24-h incubation, cells were harvested for the real-time RT-PCR assay. For immunocytostaining assay, the incubation of 72 h was required.
Immunocytochemistry of human SAE cells. To clarify the characteristics of SAE cells cultivated in Ca2þ-supplemented medium, some markers were used, including anti-human CCSP (UP1, 1:1000 dilution), cytokeratin 7 (CK7) (Clone OV-TL 12/30, 1:50 dilution), cytokeratin 14 (CK14) (Clone 34bE12), chromogranin A (Clone DAK-A3), anti-CYP 1B1 (1:3000 dilution, clone WB-1B1, Gentest, Woburn, MA), and anti-CYP1A1 (1:100 dilution, clone H-70, Santa Cruz Biotechnology, Santa Cruz, CA). The majority of the anti-bodies studied were purchased from DakoCytomation (Glostrup, Denmark). SAE cells on 24-well plates were fixed with cold methanol for 20 min, air dried, and then incubated with adequately diluted antibodies in a moist chamber for 2 h at room temperature. After washing with PBS buffer, an immunoenzymatic reaction was performed using the Universal LSAB2 kit (DakoCytomation, Glostrup, Denmark). Finally, the brown color was developed with 3#-3# diaminobenzidine (DakoCytomation, Glostrup, Denmark), and Gill’s hema-toxylin was used for nuclear counterstaining. The immunopositive cells were then counted and the data represented as the percentage of positive cells over total cell numbers.
Quantitative real-time RT-PCR assay. Total cell RNA was prepared using TRIZOL reagent (Life Technologies, Rockville, MD) and the phenol-chloroform extraction method. Synthesis of cDNA was performed by 2 lg total RNA mixed with 250 ng random primer (BioLabs, Beverly, MA) using M-MLV Reverse Transcriptase (Promega, Madison, WI). Quantitative PCR was carried out using the ‘‘SYBRÒGreen’’ PCR kit (Qiagen, Hilden, Germany) and analyzed on a ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied Biosystem, Foster City, CA). Primers were chosen with the assistance of the computer program Primer Express (Perkin-Elmer Applied Biosystem, Foster City, CA). These primer sequences and optimal concentrations of CCSP, AhR, Arnt, CYP1A1, CYP1B1, and b-actin are shown in Table 1. The PCR reactions consisted of an initial step 2 min at 50°C, a denaturation step for 10 min at 95°C, followed by 40 cycles at 95°C for 15 sec and one at 60°C for 1 min. Quantitative values were obtained from the threshold cycle (CT)
number, the increase in signal being associated with an exponential growth for PCR product when detected. Each sample target gene expression level was normalized to its b-actin mRNA content. Fold change¼ 2DCt, DCt¼
Cttarget gene Ctb-actin.
Western immunoblot. The cytosolic homogenates of cells were prepared and analyzed by Western immunoblot. Equivalent amounts of 1% Nonidet
P-40 or 1% Triton X-100 lysate protein were denatured in SDS buffer (containing 125 mM Tris, 4% SDS, 25% glycerol, 4 mM EDTA, pH 6.8) and separated by 10% SDS–PAGE. Proteins were transferred to PVDF membrane. An AhR antibody (1:1500 dilution, Biomol, Plymouth Meeting, PA) or b-actin antibody (Sigma, Saint Louis, MO) was used. Bands were visualized using an enhanced chemiluminescence kit according to instructions from the manufac-turer (Amersham, Buckinghamshire, UK).
Animals. Male Sprague-Dawley rats (150–250 g) 6 weeks old were purchased from the National Laboratory Animal Center (Taiwan, ROC). The animals were housed in standard cages under a 12 h light/dark cycle and received food and water ad libitum. All procedures and experiments with animals in this study were approved by the Animal Care and Use Committee at the National Health Research Institute, Taiwan, ROC.
Rat lung slice preparation and incubation. Rats were anesthetized with sodium thiopental (120 mg/kg) via intra-peritoneal injection. The lungs was perfused with Ringer’s solution containing 500 U sodium heparin through right pulmonary artery for 5 min to wash the blood away and prevent blood coagulation in small vessels. Then, the lungs were precisely excised and immediately inflated with 1.5% (w/v) low-melting agarose dissolved in culture medium at 37°C. The culture medium was RPMI 1640 medium containing 1 lM insulin, 0.1 mM hydrocortisone-21-hemisuccinate, 5% fetal calf serum, 50 lg/ml streptomycin, 50 IU/ml penicillin, and 2.5 lg fungizone per liter. Subsequently, rat lungs were placed in ice-cold V-7 solution (4°C, pH 7.4) until completely gelled (Fisher et al., 1996). Cylindrical tissue cores (8 mm diame-ter) were prepared from the lung tissues, which were cut to form 450-lm-thick lung slices using a Vitron tissue slicer (Vitron Inc., Tucson, AZ). Every two to three lung slices were floated on a titanium mesh of Teflon roller insert. These inserts were placed into 20-ml glass scintillation culture vials containing 2 ml of culture medium. Culture vials were capped (the cap has a central 2-mm hole) and placed horizontally into the dynamic organ culture incubator at 37°C, 5% CO2and 95% O2. After 2 h the culture medium was changed, and the lung
slices were treated with 10 lM BaP, 1 nM TCDD, or 0.1% DMSO (control) for 24 h. Harrigan et al. (2004) demonstrated that BaP-DNA adducts in rat lung slices were not detectable until the dose of BaP was increased to 10 lM. Our preliminary data showed that 10 lM BaP and 1 nM TCDD induced similar levels of CYP1A1 mRNA in vitro. Therefore, these doses were used for lung slices. The lung slices were then fixed with 10% buffered formalin, dehydrated
with graded ethanol, and embedded with paraffin for immunohistochemical analysis.
Immunohistochemistry for rat lung slices. The rat lung slices were cut to sections of 4–5 lm thickness on Silane-coated microscope slides (DakoCytomation, Glostrup, Denmark). Paraffin was removed from the sections by xylene, and they were rehydrated through graded ethanol to distilled water. For antigen detection, sections were placed in a microwave oven in a citrate buffer (pH 6) for 20–30 min, cooled for 20 min at room temperature and then washed with distilled water. To block endogenous peroxidase activity, sections were incubated with 3% hydrogen peroxide in distilled water for 20 min. Antibodies included anti-CYP1B1, anti-CYP1A1, and anti-rat CCSP (1:4000 dilution, Upstate, Charlottesville, VA) for rat lung slices. These antibodies were incubated overnight at 4°C in a moist chamber, following which an immunoenzymatic reaction was performed using the Universal LSAB2 kit. Finally, the brown color was developed and nuclei stained as for cells (see above).
Statistical analysis. Comparisons between treated and control groups were calculated using the Student’s t-test.
RESULTS
Ca
2þ-Induced Differentiation in Human SAE Cell Cultures
CK 7 and CK 14 are specific markers for human respiratory
epithelial cells and basal cells, respectively. Respiratory basal
cells are thought to differentiate into ciliated and nonciliated
(Clara cell) cells, whose CK14 protein is lost (Nakajima et al.,
1998; Otto, 2002). In SAE cell cultures, epithelial and basal
cells were distinguished by cell morphology as described in
Methods. Regardless of Ca
2þsupplementation, CK7
expres-sion was more common in epithelial cells (24.0% and 39.2%)
than in basal cells (1.0% and 1.6%) (Table 2). However, the
prevalence of CK14 expression was similar in epithelial and
basal cells (Table 2), suggesting both cell types had an ability to
differentiate. Chromogranin A and surfactant protein C are
markers for neuroendocrine cells and type II pneumocytes,
respectively. Neither chromogranin A immunostaining, nor the
mRNA level of surfactant protein C, was detectable in SAE
cells cultivated in a Ca
2þ-supplemented medium (data not
TABLE 1
Primer Sequences and Working Concentrations
Primers Sequences Working concentration (nm) b-actin 5#-tcatgaagtgtgacgtggacatc-3# 100 5#-caggaggagcaatgatcttgatct-3# CCSP Assay ID: Hs00171092a 5#-acatcacctacgccagtcgc-3# 400 AhR 5#-tctatgccgcttggaaggat-3# 5#-gctgctgcctaccctagtctca-3# 200 Arnt 5#-gctgtccgtgtctggaattgt-3# 5#-caccatcccccacagcac-3# 200 CYP1A1 5#-acaaagacacaacgcccctt-3# 5#-cggctggatttggagaacgta-3# 200 CYP1B1 5#-tgatccaattctgcctgcact-3#
Note. Abbreviation: CCSP, Clara cell secretory protein; AhR, aryl hydrocarbon receptor; Arnt, Ah-receptor nuclear translocator; CYP1A1, cytochrome P450 1A1; CYP1B1, cytochrome P450 1B1.
aTagManÒ gene expression assay kit of primers and probes (Applied
Biosystems).
TABLE 2
Comparison of the Characteristics of SAE Cells Cultivated
in Ca
2þ-Free or Ca
2þ-Supplemented Medium
Ca2þ-free Ca2þ-supplemente
Cell types CK7a CK14a CK7a CK14a
Epithelial cells 24.0 ± 4.4b 59.4 ± 7.1 39.2 ± 26.5 78.1 ± 26.9
Basal cells 1.0 ± 0.7 50.8 ± 15.0 1.6 ± 2.0 80.6 ± 17.7c
aProtein was detected using a immunocytochemical stain in 24-well plates.
Protein expression was scored as the percentage of immunostained cells and is represented as the mean ± SD from four replicates.
bCompared with basal cells in the same type of medium, p < 0.05. cCompared with the same cell type in Ca2þ-free medium, p < 0.05.
shown). This result indicates that there was no neuroendocrine
and type II pneumocyte differentiation in these cultures.
CCSP is a specific marker for Clara cells. After Ca
2þ-supplementation, the CCSP mRNA levels were markedly
elevated in SAE cell cultures (Table 3). Utilizing
immunocy-tochemical staining, CCSP immunoreactivity was not
detect-able in basal cells but detected in 0.6% of epithelial cells in the
Ca
2þ-free condition (Table 3). After Ca
2-supplementation for
72 h, the prevalence of CCSP immunoreactivity was 0.5% in
basal cells and 19.2% in epithelial cells (Table 3).
Approxi-mately 80% of epithelial cells were CCSP-negative but
CK14-positive. These data suggest that Ca
2þinduces SAE cell
cultures to differentiate into CCSP-positive cell cultures.
Comparison of CYP1A1 and CYP1B1 Induction in SAE
and CCSP-Positive Cell Cultures
Several studies have indicated that some cytochrome P450s
are rich in Clara cells (Hukkanen et al., 2002). Therefore,
constitutive and AhR ligand (TCDD and BaP)-induced
expres-sion of CYP1A1 and CYP1B1 were examined and compared in
SAE (Ca
2þ-free medium) and CCSP-positive (high Ca
2þmedium) cell cultures. Regardless of the presence of Ca
2þ,
CYP1A1 mRNA was barely detectable (Figs. 1A and 1C).
CYP1B1 mRNA was readily detectable in SAE and
CCSP-positive cell cultures (Figs. 1B and 1D). After TCDD
treatment, CYP1A1 mRNA levels (4378 3 10
5) in
CCSP-positive cell cultures were much higher than the levels (702 3
10
5) in SAE cell cultures (Fig. 1A). TCDD-induced CYP1B1
mRNA levels were also increased in CCSP-positive cell
cultures (Fig. 1B). Similarly, after BaP treatment, CYP1A1
mRNA levels were 1249 and 99 (3 10
5) in CCSP-positive
and SAE cell cultures, respectively (Fig. 1C). BaP-induced
CYP1B1 mRNA levels increased approximately two-fold
with Ca
2þpresence (Fig. 1D). Taken together, TCDD- and
TABLE 3
Expression of CCSP in SAE Cells Cultivated with Ca
2þ-Free
or Supplemented Medium
Calcium-free Calcium-supplemented Cell types CCSP mRNAa CCSP proteinb CCSP mRNAa CCSP proteinb SAE cells 1.5 ± 0.9 0.1 ± 0.3 586.6 ± 176.5c 10.3 ± 6.7 Epithelial cells 0.6 ± 1.2 19.2 ± 6.6c Basal cells 0.0 ± 0.0 0.5 ± 0.6 aCCSP mRNA molecules relative to 108b-actin molecules was determined using real-time RT-PCR. Each data is the mean of four replicates
bCCSP protein was detected by immunocytochemical stain in 24-well
plates. CCSP expression is scored as the percentage of immunostained cells as the mean ± SD from four replicates.
cCompared with cells in Ca2þ-free medium, p < 0.05.
FIG. 1. Comparison of CYP1A1 and CYP1B1 induction in SAE (Ca2þ-free) and CCSP-positive (High Ca2þ) cell cultures with TCDD or BaP treatment. SAE cells were cultivated in Ca2þ-free or supplemented (1 mM) medium for 72 h and were then treated with (A and B) 1 nM TCDD, (C and D) 1 lM BaP or 0.01% DMSO (control solvent) for 24 h. The relative gene expression of (A and C) CYP1A1 and (B and D) CYP1B1 was determined using real-time RT-PCR. ‘‘*’’ represents p < 0.05 as compared with DMSO-treated cells. ‘‘#’’ represents p < 0.05 as compared with SAE cell cultures.
BaP-induced CYP1A1 and CYP1B1 levels were much higher
in CCSP-positive cell cultures than in SAE cell cultures.
In order to localize the CYP1A1 and CYP1B1 induced
proteins in CCSP-positive cell cultures, an
immunocytochem-ical assay was performed. We found that CYP1B1, but not
CYP1A1, was detectable in CCSP-positive cell cultures (Table 4).
Furthermore, CYP1B1 immunoreactivity was more prevalent
in epithelial cells (22.3%) than in basal cells (8.6%) within
CCSP-positive cell cultures (Table 4). After TCDD treatment
for 72 h, CYP1A1 and CYP1B1 immunoreactivities were
increased in both epithelial and basal cells. Similarly, these
immunoreactivities were more common in epithelial cells
(65.1% and 47.4%) than in basal cells (10.4% and 17.1%)
(Table 4). Since CCSP immunoreactivity also located in
epithelial cells (Table 3), these results implied that CYP1A1
and CYP1B1 are more inducible by AhR ligands in
CCSP-positive epithelial cells than in basal cells.
Increased AhR Expression in CCSP-Positive Cell Cultures
It is well known that AhR and Arnt regulate AhR
ligand-induced CYP1A1 and CYP1B1 expression (Kress and Greenlee,
1997; Whitlock, 1999). AhR and Arnt were constitutively
expressed in SAE cells (Figs. 2A and 2C). Consistent with
increased CYP1A1/CYP1B1 induction, the AhR mRNA levels
were significantly increased in CCSP-positive cell cultures
(Fig. 2A). The level of AhR also increased two-fold in
CCSP-positive cell cultures (Fig. 2B). Using immunocytostaining,
AhR expression was observed in both epithelial cells and basal
cells. The AhR immunointensity and the percentages of
AhR-positive cells were similar between SAE and CCSP-AhR-positive
cell cultures (data not shown). This inconsistent finding, the
result of immunocytostaining assay compared with the results
of RT-PCR assay and Western immunoblot, may be due to the
limited sensitivity of immunocytochemistry. Levels of Arnt
mRNA were only slightly increased in CCSP-positive cells
(Fig. 2C). These results imply that increased AhR expression
might partially contribute to the elevated induction of CYP1A1
and CYP1B1 in CCSP-positive cell cultures.
Colocalization of CCSP, CYP1A1, and CYP1B1 Proteins
in Rat Lung Slices
In CCSP-positive cell cultures, CYP1A1 and CYP1B1
expression were highly inducible. We further used an in vitro
model of rat lung slice cultures to investigate the locations of
TCDD- and BaP-induced effects. In rat lung slices,
CCSP-positive cells were nonciliated epithelial cells, namely Clara
cells (Figs. 3A, 3D, and 3G). Rat Clara cells were found in the
bronchi and bronchioles, but not in the alveoli. Clara cells were
more common in the bronchioles than in the bronchi (52% vs.
35%). After treatments with TCDD and BaP, increased
CYP1A1 immunoreactivity was detected in the cytoplasm of
nonciliated and ciliated bronchiolar epithelial cells (Figs. 3E
TABLE 4
Expression of CYP1A1 and CYP1B1 on CCSP-Positive Cell
Cultures Treated with TCDD
CYP1A1a CYP1B1a
Cell types DMSO 1 nM TCDD DMSO 1 nM TCDD
Epithelial cells 0.0 ± 0.0 65.1 ± 7.5b,c 22.3 ± 15.8 47.4 ± 16.8c
Basal cells 0.0 ± 0.0 10.4 ± 2.9b 8.6 ± 10.9 17.1 ± 9.9
a
Cells were incubated with DMSO or TCDD for 72 h. Protein was detected by immunocytochemical stain in 24-well plates. Expressions were scored as the percentage of immunostained cells as the mean ± SD from four replicates.
b
Compared with 0.01% DMSO-treated cells, p < 0.05.
cCompared with basal cells, p < 0.05.
FIG. 2. Effect of Ca2þon the expression of AhR and Arnt in SAE cell cultures. SAE cells were cultivated in Ca2þ-free or supplemented medium for 72 h. Total cell RNA and protein were then extracted. Relative gene expression of (A) AhR and (C) Arnt were determined by real-time RT-PCR. (B) The expression of AhR relative to b-actin was quantified using Western immuno-blotting. ‘‘#’’ represents p < 0.05 as compared with SAE cells cultivated in Ca2þ-free medium.
and 3H), in comparison with DMSO-treated lung slices (Fig. 3B).
It should be noted that nonciliated cells (Clara cells) showed
a much stronger intensity than ciliated cells (Figs. 3E and 3H).
CYP1B1 immunoreactivity was weak but constitutively
ob-served in the smooth muscles of the vascular and bronchial
walls (data not shown). The locations for BaP and
TCDD-induced immunoreactions of CYP1B1 were similar to those of
CYP1A1 in rat lung slices (Figs. 3C, 3F, and 3I). Thus, these
results provide evidence that CYP1A1/CYP1B1 induction is
more marked in Clara cells than in other bronchiolar epithelial
cells of rat lung slices.
DISCUSSION
The objective of this study was to determine whether human
Clara cells or CCSP-positive cells are responsive to AhR
ligands. In the primary cultures of human SAE (basal) cells,
AhR ligands (TCDD and BaP)-induced CYP1A1/1B1
expres-sion was highly elevated when cells differentiated into
CCSP-positive cells, indicating human CCSP-CCSP-positive cells are highly
responsive to AhR ligands. However, in the Ca
2þ-induced SAE
differentiating system, human CCSP-positive cells retained the
ability to differentiate (CK14 positive) and would be
consid-ered as stem cells or progenitor cells of the bronchiolar
epithelia. To understand if terminally differentiated Clara cells
are responsive to AhR ligands, we identified the location of
TCDD/BaP-induced CYP1A1/1B1 proteins in the rat lung slice
cultures. It was found that CYP1A1/1B1 proteins were
consis-tently located in Clara cells. These results indicate that the
responsiveness to AhR ligands was highly increased when SAE
(basal) cells had differentiated into CCSP-positive or Clara
cells. In the other words, this finding implies that Clara cells in
the peripheral lung of humans and rats were more sensitive to
AhR ligands than basal and other bronchiolar epithelial cells.
Type II pneumocytes have been demonstrated to be susceptible
to toxicants (Monteil et al., 1999; Tatrai et al., 2001). However,
type II pneumocytes were not present in our present systems.
Thus we cannot ignore the possibility of CYP1A1 and CYP1B1
induction in type II pneumocytes.
The distribution of Clara cells is similar in rats and humans,
being more prevalent in the peripheral than in the central
airways (Boers et al., 1999). Clara cells are believed to
contrib-ute to cell renewal in the peripheral airway epithelium (Aliotta
et al., 2005; Hong et al., 2001; Otto, 2002). In addition to being
progenitor cells for replacing injured epithelium, Clara cells
are known to synthesize and secrete CCSP as well as protease
(Massaro et al., 1994; Singh and Katyal, 1997). CCSP inhibits
phospholipase A2, which is involved in the regulation of the
inflammatory process (Anderson et al., 1994; Johnston et al.,
1997). Protease secreted by Clara cells is trypsin-like and can
inhibit leukocyte protease activity to maintain the
protease-antiprotease balance in the lung (Massaro et al., 1994). TCDD
and BaP have been shown to induce cell apoptosis or inhibit
cell growth in an AhR dependent manner (Lin et al., 2004;
Solhaug et al., 2005). In the other words, CYP1A1/1B1
FIG. 3. Colocalization of Clara cell secretory protein (CCSP) and CYP1A1/CYP1B1 in rat lung slice cultures. Immunohistochemistry, 3400. Rat lung slices were cultivated in medium containing (A to C) 0.1% DMSO, (D to F) 1 nM TCDD, and (G to I) 10 lM BaP for 24 h. Rat lung slices were then fixed and paraffin-embedded. Serial sections were stained with (A, D, and G) anti-CCSP, (D, E, and H) anti-CYP1A1, and (C, F, and I) anti-CYP1B1. Positive immunoreactivity exhibited a cytoplasmic brown coloration. Bronchiolar Clara cells (arrow) were nonciliated cells those partly coexpressed CYP1A1/CYP1B1. Ciliated cells (arrowhead) were CCSP-negative, but CYP1A1/CYP1B1 weakly positive compared with Clara cells.
induction is required for TCDD and BaP-induced cytotoxicity.
This implication is supported by some in vivo studies. For
example, it was reported that the number of Clara cells is
reduced in the lungs of cigarette smokers (Lumsden et al.,
1984; Shijubo et al., 1997). In rats, TCDD exposure caused
pathological changes in the bronchiolar epithelia, primarily in
Clara cells (Brix et al., 2004; Tritscher et al., 2000). Therefore,
AhR ligands may interact with Clara cells to disturb the cell
renewing function.
Previously we showed that TCDD inhibited cell growth, but
failed to induce cell death, in human bronchial epithelial cells
(Lin et al., 2004). In our present study, CCSP-positive cell
cultures were the mixtures of basal cells, CCSP-positive
epi-thelial cells and CCSP-negative epiepi-thelial cells. Furthermore,
in the presence of calcium, epithelial cells were consistently
differentiated from basal cells. Therefore, we were unable to
selectively evaluate effects of TCDD and BaP on growth of
CCSP-positive cells. In lung slice cultures, we failed to observe
morphological change in TCDD and BaP-treated slices 24 h
later. It is likely that the incubation time (24 h) was too short to
cause cytotoxicity in slice cultures.
Using light microscopy, electron microscopy, and
immuno-histochemical methods, earlier studies suggested that
bronchi-olar Clara cells constitute one of the precursor cells for
peripheral lung AD (Dermer, 1982; Kitamura et al., 1997;
Mori et al., 1998). However, several studies showed that CCSP
expression is reduced during the development of lung
carci-nogenesis. CCSP expression is widely observed in normal
bronchiolar epithelia, but less often in human atypical
bron-chiolar lesions and AD (Jensen et al., 1994; Nomori et al.,
1994). Bernard et al. (1992) showed that CCSP levels are
reduced in serum and bronchoalvolar lavage fluids obtained
from lung cancer patients. The loss of CCSP protein also
occurred in a transgenic mouse model for the transformation of
Clara cells to lung AD (Hicks et al., 2003). These data imply
that Clara cells were either damaged or transformed into tumor
cells during lung carcinogenesis. Since CCSP-positive cells are
highly responsive to carcinogenic AhR ligands, the relationship
between exposure of AhR ligands and the development of lung
AD deserves further investigation.
One of our major findings was that CYP1A1/1B1 induction
and AhR expression were increased during Clara cell
differ-entiation. Similarly, CYP1A1 induction and AhR expression
were also increased when proliferating keratinocytes were
induced to differentiate (Wanner et al., 1995). It appeared that
increased AhR expression at least partially explains the
increased CYP1A1/1B1 induction in more differentiated
keratinocytes and CCSP-positive lung cells (Wanner et al.,
1995). By contrast, AhR expression was decreased during
adipose differentiation (Shimba et al., 2001). These findings
suggest that the relationship between AhR expression and
differentiation is cell-type specific. Furthermore, the causal
relationship between AhR expression and differentiation is not
yet established and deserves further investigation.
Although CYP1A1 protein was detected in Clara cells from
rats treated with AhR ligands (PCB or 3-methylcholanthrene)
in vivo (Lee and Dinsdale, 1995), CYP1A1 induction by AhR
ligands was not observed in primary cultures of rat lung cells
(Solhaug et al., 2004, 2005). It is likely that CYP1A1
inducibil-ity in normal lung cells was lost in the in vitro culture system.
CYP1A1 and CYP1B1 are involved in the conversion of BaP
into an ultimate metabolite, which forms DNA adducts (Kim
et al., 1998; Shimada et al., 1999). Recently, Harrigan et al.
(2004) demonstrated that in vitro incubation with BaP
in-creased BaP-DNA adduct levels in rat lung slices, suggesting
that drug metabolizing enzymes were induced by BaP in lung
slice cultures. In our present study, we successfully
demon-strated TCDD/BaP-induced CYP1A1/1B1 proteins in Clara
cells of rat lung slice cultures, which have been maintained
in vitro for 3 days. Our results not only prove that Clara cells
are highly responsive to AhR ligands, but also indicate that rat
lung slice cultures are an excellent in vitro model for studying
toxicant metabolism in the lung.
ACKNOWLEDGMENTS
This work was supported by research grant, DOH94-TD-G-111–030, from the National Research Program for Genomic Medicine and Department of Health, and EO-094-PP-09 from Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan, ROC. The scientific content of this manuscript does not necessarily signify the view and policies of DOH and DEHOM/NHRI or condemn, endorse or recommend any aspects presented. Conflict of interest: none declared.
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