JNK1 and AP-1 regulate PMA-inducible squamous differentiation
marker expression in Clara-like H441 cells
Hue
Vuong1,
Tricia
Patterson1,
Pavan
Adiseshaiah1,
Paul
Shapiro2,
Dhananjaya V.
Kalvakolanu3, and
Sekhar P. M.
Reddy1
1 Department of Environmental Health Sciences, The Johns
Hopkins University School of Public Health, Baltimore 21205;
2 University of Maryland School of Pharmacy, Baltimore 21201;
and 3 Greenbaum Cancer Center, University of Maryland
School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
Exposure of distal bronchiolar region to
various toxicants and pollutants suppresses Clara cell differentiation
marker expression and greatly enhances the induction of squamous cell
differentiation (SCD). Here, we demonstrate for the first time phorbol
13-myristate 12-acetate (PMA)-inducible expression of SCD markers,
SPRRs, in Clara-like H441 cells. The transcriptional
stimulation of human SPRR1B expression is mainly mediated by
a
150- to
84-bp region that harbors two critical activator protein
(AP)-1 sites. In unstimulated cells, the
150- to
84-bp region is
weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA
prominently induced the binding of JunB and Fra1. Consistent with this,
overexpression of wild-type Jun proteins upregulated the
SPRR1B promoter activity. Conversely, a c-jun mutant
suppressed both basal and PMA-inducible reporter gene expression.
Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly
enhanced basal level activity, indicating an opposing role for these
proteins in SPRR1B expression in a manner similar to that
observed in proximal tracheobronchial epithelial cells (BEAS-2B clone
S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun
NH2-terminal kinase (JNK) 1 mutant significantly reduced
the PMA-inducible SPRR1B promoter activity in H441 cells.
Thus either temporal expression and/or spatial activation of AP-1
proteins by JNK1 might contribute to the induction of SCD in Clara cells.
small proline rich proteins; airway epithelium; transcriptional
regulation; mitogen-activated protein kinases; Jun/Fos; c-Jun
NH2-terminal kinase 1; activator protein 1
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INTRODUCTION |
PHORBOL 13-MYRISTATE
12-ACETATE (PMA), a stable functional analog of diacylglycerol, is
produced in vivo by hydrolysis of phospholipids, which induces diverse
biological responses. PMA mainly activates several protein kinase C
(PKC) isoenzymes, which in turn initiate a signaling cascade to
regulate the expression of genes that are involved in cell growth and
differentiation in response to environmental stimuli including cellular
injury repair, inflammation, and acute phase response. Clara
cells are the progenitor cell type for the distal bronchiolar
epithelium during injury and repair (16). Administration
of PMA, which induces acute and progressive lung injury
(12), downregulates the expression of human surfactant
protein gene expression in both human fetal lung implants and in
Clara-like bronchiolar epithelial cell line H441 (9, 13, 15, 19,
20). PMA also potently induces squamous cell differentiation
(SCD) in tracheobronchial epithelial (TBE) cells in vivo and in vitro.
Previous studies have shown a close relationship between an early
induction of human small proline-rich protein type I (SPRR1)
and SCD in TBE cells (27, review). Tobacco smoke, which potently
induces SCD in airway epithelium, enhances the expression of
SPRR1 in nasal (28) and bronchial epithelium
(22). Subsequently, it was shown that PMA (1,
21) and tobacco smoke (22) induce expression of
SPRR1 (now referred to as SPRR1B) in TBE cells,
which occurs primarily at the transcriptional level and is mediated by
the TRE sites and activator proteins (AP)-1.
The distal bronchiolar region represents a major target for pulmonary
toxicity induced by various environmental toxicants and pollutants
(16). Several studies have demonstrated that the exposure
of animals to toxicants, such as naphthalene, selectively induces
cellular injury in distal bronchiolar region (25, 29). For
example, naphthalene at lower levels (50-100 mg/kg body wt) induces Clara cellular injury in the distal airway region, whereas proximal airways require a substantially higher dose (>300 mg/kg) (17). Later studies attributed this difference to a higher
level of cytochrome P-450 isoenzyme 2F2, which converts
naphthalene to an active metabolite, 1R, 2S-naphthalene, in distal
airway region compared with proximal airways (3).
Conversely, the content of glutathione, which plays a protective role
in cellular detoxification, is higher in upper airways compared with
distal bronchiolar region (18). Together these studies
indicate certain differences in the mechanisms governing cellular
injury and repair process in upper and distal airway regions.
Naphthalene suppresses the expression of Clara cell differentiation
(CCD) markers and greatly enhances the expression of squamous cell
functions (29). Moreover, exposure of mice to naphthalene
and tobacco smoke greatly enhances the expression SPRR1B in
distal bronchiolar region (Reen Wu, University of California at Davis,
Davis, CA; personal communication). However, the exact molecular
mechanisms governing the regulation of cell proliferation and
differentiation in response to injury, in particular the induction of
SCD, in the distal bronchiolar region, are not clearly
understood. Based on certain differences involved in injury and
repair mechanisms, we hypothesized that the induction of SCD in the
distal bronchiolar region is regulated by a mechanism different from
those that operate in the proximal airway epithelium. In the present
study, we used Clara-like bronchiolar epithelial cell culture (H441) as
a model system to study the regulation of CCE precursor protein gene
expression and to understand the molecular mechanisms involved in the
induction of SCD in the distal bronchiolar region. Here, we
present for the first time evidence for a PMA-inducible expression of
SPRRs in distal bronchiolar cells H441. Promoter
analysis indicates that, similar to proximal TBE cells (S-6),
PMA-stimulated SPRR1B expression in H441 cells requires the
same TREs (PMA-responsive elements or AP-1 sites) and combination of
AP-1 proteins. However, unlike in S6 cells, c-Jun
NH2-terminal kinase (JNK) 1, a mitogen-activated protein kinase (MAPK), is critical for PMA-inducible SPRR1B
expression in H441 cells. Thus the activation of different signaling
pathways might contribute to the induction of SCD in the distal
bronchiolar region.
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MATERIALS AND METHODS |
Cell culture, Northern blot, RT-PCR, and Western blot analyses.
NCI-H441 (Clara-like bronchiolar pulmonary adenocarcinoma) cell line
was obtained from ATCC. All cells were maintained in RPMI culture
medium supplemented with 5% serum, 1% streptomycin and penicillin,
gentamicin (250 ng/ml), and fungizone (125 ng/ml). Cells were treated
with either vehicle (DMSO) or PMA (25 ng/ml) for the indicated time
periods. Total RNA (20 µg/lane) was separated on a 1.2% agarose gel
and transblotted. Northern blot analysis was carried out using
32P-labeled cDNAs of SPRR1B as previously
described (31). RT-PCR was performed as previously
described (14). Briefly, total RNA (750 ng) was reverse
transcribed into cDNA, and PCR amplification was performed with an
aliquot of cDNA using gene-specific primer pairs (see Table
1). The indicated number of
cycles were employed to amplify the product in a linear range.
The amplified cDNA fragments were separated and quantified with the Gel
Doc 2000 System (Bio-Rad). Western blot analysis of cellular extracts
isolated from untreated and PMA-treated H441 cells was performed using
polyclonal antibodies of human SPRR1B (10).
Transient transfections and reporter gene assays.
The wild-type (wt), dominant negative (dn), and constitutively active
(ca) expression vectors were kindly provided by the following: the
dn-mitogen-activating protein kinase kinase (MAPKK) 1 and
ca-MAPKK1 cloned in pCMV vector, Dennis Templeton, Case Western Reserve
University, Cleveland, OH; dn-JNK1 cloned in pCDNA3 vector, Roger
Davis, Howard Hughes Medical Institute, Rochester, NY; dn-extracellular
signal-regulated kinase (ERK) 1 and dn-ERK2 cloned in pCEP4 vector,
Melanie Cobb, University of Texas Southwestern Medical Center, Dallas,
TX; wt-c-Jun and dn-c-Jun (has mutations in transactivation domain)
cloned in pCMV vector, Michael Birrer, National Cancer Institute,
Bethesda, MD; wt-JunB and wt-JunD cloned in SR-alpha vector, Michael
Karin, University of California at San Diego, San Diego, CA; wt-Fra1
cloned in pCMV vector, Eugene Tulchinsky, Institute of Cancer Biology,
Copenhagen, Denmark; and wt-Fra2 cloned pCMV vector, Donna Cohen,
Australian National University, Canberra, Australia; dn-Raf (Raf-C4)
cloned in pRSV vector (Stephen Ludwig, Institut für Medizinische
Strahlenkunde und Zellforschung, Würzburg, Germany) were
obtained. Dn-Ras (Ras-N17) and ca-Ras (Ha-Ras-V12) were
generated as previously described (31). Several deletion
and site-directed mutational fragments of the
150 to +12-bp
SPRR1B promoter fused to the luciferase (Luc) gene as
described previously (21). DNA transfections were performed using a Fugene transfection reagent (Roche Biochemical). Cells were grown on 48-well plates at 70-80% confluence and
then transfected with 100 ng of promoter construct, 50 ng of
CMV-
-galactosidase (
-gal) DNA, and 50-200 ng of empty or
expression plasmid vectors. After 18-20 h posttransfection, cells
were treated with either DMSO or PMA (25 ng/ml) for 14-16 h. Cells
were lysed, and Luc activity was measured using a commercially
available kit (Promega). Luc activity of individual samples was
normalized against
-gal activity and/or total protein as described
previously (31). Luc activity for every construct was
analyzed in duplicate samples, and all experiments were
repeated three to four times.
Electrophoretic mobility shift assay.
Nuclear extracts from DMSO- or PMA-treated H441 cells were prepared,
and electrophoretic mobility shift assay (EMSA) was performed as
described previously (14) with 0.1-0.5 ng of
32P-end-labeled consensus AP-1 site or
150- to
84-bp
SPRR1B promoter fragment (for sequence see Table
2). For supershift analysis, nuclear
extracts were mixed with 1-2 µg of c-Jun (sc-45X), JunB (sc-46X), JunD (sc-74X), c-Fos (sc-7202X), Fra1 (sc-605x), and Fra2
(sc-604X) antibodies (Santa Cruz Biotechnology) and incubated on ice
for 1-2 h before the labeled DNA probe was added.
In vitro kinase assays and immunoblot analysis.
H441 cells were cultured to 70-80% confluence and treated with
PMA (25 ng/ml) for indicated time periods, and assays were performed as
previously described (31). Briefly, 200 µg of cellular protein were immunoprecipitated using 0.4 µg antibodies derived against JNK1 (C-17) or p38 (C-20) kinases (Santa Cruz Biotechnologies) that were conjugated to protein A-Sepharose (Pharmacia). The
immunoprecipitates were washed extensively, and the kinase activities
of JNK1 and p38 were determined as previously described
(31). Active ERK1/2, p38, and JNK1 immunoblot analysis was
carried out using the phospho-specific antibodies as previously
described (31). Total ERK, p38, and JNK1 content of the
samples was determined by immunoblot analysis using ERK, p38, and JNK1
antibodies, respectively. Protein loading of the membrane was assessed
by Western analysis using
-tubulin antibody (Sigma, T 6557).
Statistical analysis.
Data are expressed as the means ± SE. The StatView program was
used to perform analysis of variance between different samples. Statistical significance was accepted at P < 0.05. All
assay samples were performed in duplicate, and each experiment was
repeated at least two times.
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RESULTS |
PMA upregulates SPRR expression in H441 cells.
Previous studies have demonstrated a close association between the
expression of SPRR1 and induction of airway SCD
(27). To understand the molecular mechanisms of SCD in the
distal bronchiolar region, we treated H441 cells with either DMSO
(vehicle) or PMA (25 ng/ml) for different time periods as indicated.
Total RNA was isolated, and Northern blot analysis was performed with
comparable amount of RNA from each sample. As shown in Fig.
1A, SPRR1B mRNA levels are undetectable in the DMSO-treated control group (lane 1). However, by 3 h, PMA strongly enhanced the
SPRR1B mRNA (lane 2), which reached a maximum by
the 6-h time period and returned to lower levels by 14 h. All
lanes had comparable amounts of 28S RNA, a housekeeping gene (Fig.
1A, bottom). A similar pattern of SPRR1B protein
expression was observed after PMA treatment. As shown in Fig.
1B, H441 cells displayed a very low basal level of SPRR1B
(lane 1), but PMA significantly enhanced the protein levels
(lane 2).

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Fig. 1.
Phorbol 12-myristate 13-acetate (PMA)-stimulated small
proline-rich protein (SPRR) gene expression in H441 cells.
A: cells were grown to 70-80% confluence and treated
with vehicle (DMSO) or PMA (25 ng/ml) for different time periods as
indicated. Total RNA (20 µg/lane) was separated, blotted, and
hybridized with either 32P-labeled SPRR1B cDNA
probe. All lanes had comparable amount of 28S RNA, a housekeeping gene
(bottom). B: an equal amount of cellular protein
isolated from untreated and PMA-treated (25 ng/ml for 16 h) cells
was separated on SDS-PAGE gel, and Western blot analysis was performed
using SPRR1B antibodies. C: total RNA isolated as described
above was subjected to RT-PCR using gene-specific primers (see Table
1).
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To distinguish SPRR1B expression from its ortholog
SPRR1A, which shows significant sequence homology with the
coding region of 1B, we analyzed the SPRR1B
message levels employing a semiquantitative RT-PCR with gene-specific
primers (see Table 1). A good corroboration was observed between the
results of Northern blot and RT-PCR. PMA induced the expression of
SPRR1B message levels in H441 (Fig. 1C) similar
to normal transformed bronchial epithelial cells HBE1 and S6 (1,
21). We next analyzed the expression of other SPRR
family members (SPRR1A, SPRR2A, and SPRR3) in
H441 cells. PMA significantly induced SPRR1A and
SPRR2A but not SPRR3 message levels in H441
cells. Together, these results indicate that PMA differentially induces
the expression of SPRRs in Clara-like bronchiolar epithelial cells.
Induction of SPRR1B expression in H441 cells is regulated mainly at
the transcription level.
Previous studies have shown that PMA downregulates the CCD marker
expression, surfactant protein (SP)-A and SP-B, in H441 cells at the
transcriptional and posttranscriptional levels, respectively (9,
13, 19, 20). In the present study, we analyzed the regulation of SPRR1B in H441 cells to understand the
molecular mechanisms (transcriptional and/or posttranscriptional)
governing the induction of SCD in distal bronchiolar region. We mainly
focused on SPRR1B regulation, as our previous studies and
those of others have demonstrated a close association of
SPRR1B expression with airway SCD both in vivo and in vitro
(1, 21, 26). H441 cells were pretreated with actinomycin D
(10 µg/ml) for 30 min before PMA exposure. RNA was isolated 6 h
later, and SPRR1B message levels were analyzed by RT-PCR. As
shown in Fig. 2A, PMA
significantly induced the SPRR1B message levels (lane
2). However, pretreatment of cells with actinomycin D before PMA
exposure significantly reduced inducible SPRR1B message
levels (lane 4). To analyze whether PMA has any effect on
SPRR1B mRNA stability, we treated cells with PMA for 6 h and then with actinomycin D. Cells were harvested at indicated time
points thereafter. As shown in Fig. 2B, actinomycin D did
not significantly affect the message levels stimulated by PMA. These
results indicate that PMA-inducible expression of SPRR1B in
H441 cells is regulated at the transcriptional level.

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Fig. 2.
PMA-stimulates SPRR1B expression in H441 cells
mainly at the transcriptional level. A: cells were
pretreated with either actinomycin D (Act D, 10 µg/ml) or vehicle for
40 min and then exposed to PMA. After 6 h, cell cultures were
harvested for RNA isolation, and RT-PCR was performed as in Fig. 1.
B: cells were first treated with PMA for 6 h and then
exposed to Act D. Cell cultures were harvested at indicated time
periods, and RT-PCR was performed using primers specific for
SPRR1B or -actin.
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TREs located between the
150- and
84-bp promoter modulate
PMA-inducible SPRR1B expression in H441 cells.
To characterize the regulatory elements involved in PMA-inducible
SPRR1B expression, several promoter mutants of
SPRR1B linked to the Luc gene (Fig.
3A) were transiently
transfected into H441 cells. We have mainly focused on the region from
150 to
84 bp, as it contains the motifs necessary for basal and
PMA-inducible SPRR1B regulation in TBE cells
(21). Luc expression in the presence (dark bars) or
absence of PMA (light bars) was analyzed. As shown in Fig.
3B, the 113-Luc construct, which bore the proximal TRE (
109), displayed basal level activity, whereas PMA significantly upregulated it. The 150-Luc construct, which contains both the proximal
and distal TREs, exhibited both basal enhanced and a high-level
PMA-inducible activity. As shown in Fig. 3C, mutation of
both TREs [double mutant (DM)] ablated both basal and PMA-inducible expression. Interestingly, mutation of either TRE alone did not have a
significant effect on both basal and PMA-stimulated gene expression
(data not shown). Interestingly, mutation of the promoter region
between the two TREs (IN) also significantly reduced the basal level
activity indicating the importance of these flanking sequences in gene
regulation. These results are consistent with our previous studies that
demonstrated a critical role for both these TREs in regulating
PMA-inducible expression of SPRR1B (21).

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Fig. 3.
PMA-responsive elements (TREs) located between 150 and 84 bp
regulate SPRR1B promoter in H441 cells. A: the
position of functional motifs of SPRR1B promoter.
B: cells were grown to 70-80% confluence and then
transfected with 100 ng of chimeric construct containing
150-SPRR1B promoter linked to a Luciferase (Luc) reporter
gene along with 50 ng of -galactosidase ( -gal) vector, and
transient transfections were carried out as described in
MATERIALS AND METHODS. After 24-h incubation, medium was
replaced with fresh culture medium containing either vehicle or PMA (25 ng/ml) for ~14 h, and Luc gene expression was analyzed.
*P < 0.05 compared with vehicle-treated group
(bar 3). C: transient transfection assays were
performed as described in B, using either wild-type (WT) or
mutated fragments (see Table 3) of SPRR1B promoter-reporter
constructs. *P < 0.05 compared with vehicle-treated
group (bar 1). **P < 0.05 compared with
vehicle- treated group (bar 5). DM, promoter with mutations
in TRE sites; IN, promoter with mutations in between 2 TRE
sites.
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Analysis of AP-1 binding to the consensus AP-1 site and the SPRR1B
promoter.
The above data indicated that the TREs of SPRR1B promoter
are necessary for PMA-stimulated expression in H441 cells. We therefore examined the patterns of AP-1 proteins binding to the consensus AP-1
site (Fig. 4). Nuclear extracts were
isolated from untreated and PMA-treated H441 cells incubated with a
32P-labeled consensus AP-1 site (Fig. 4A). A
basal-level AP-1 protein binding was observed in the unstimulated cells
(lane 1), which was significantly enhanced with PMA
treatment (lane 2). We next examined the composition of AP-1
protein complex in untreated (Fig. 4B, lanes
2-7) and PMA-treated (Fig. 4B, lanes
9-14) H441 cells using antibodies specific for the individual
Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, Fra1, and Fra2) proteins.
PMA significantly enhanced the binding of c-Jun, JunB, JunD, Fra1, and
Fra2, but not c-Fos, as revealed by a supershift of their complexes
with specific antibodies. Thus PMA enhanced the binding of Jun and Fos
family members to a consensus AP-1 site similar to the patterns
observed in HBE1, S6, and A549 cells (14).

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Fig. 4.
Analysis of H441 nuclear extracts binding to the consensus
activator protein (AP)-1 site. Preparation of nuclear extracts and
electrophoretic mobility shift assay (EMSA) were performed as described
in MATERIALS AND METHODS. A:
32P-end-labeled double-stranded AP-1 oligonucleotide was
incubated with 2 µg of nuclear extracts isolated from vehicle (-) or
PMA-treated (+) cells. B: nuclear extracts were incubated in
the presence of 1-2 µg antibodies specific for individual AP-1
family members c-Jun, JunB, JunD, Fra1, Fra2, or c-Fos. The vertical
bar indicates the position of supershifted (SS) bands, arrow indicates
AP-1 protein complex, arrowhead indicates nonspecific protein complex,
and open arrow indicates the position of the free probe (F).
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We next characterized the composition of the protein complex binding to
the
150- to
84-bp region of the SPRR1B promoter, henceforth SPRR-TRE, that contains two critical TREs. As shown in Fig.
5A, vehicle-treated H441
nuclear extracts (lanes 1 and 7) formed a
DNA-protein complex that was enhanced significantly after PMA treatment
(lane 8). The specificity of the DNA-protein complex was
demonstrated by a competitive inhibition of complex formation by the
unlabeled SPRR-TRE (self, lane 3) and consensus AP-1 site
(AP-1, lane 4). However, unlabeled DM did not block the
complex formation (lanes 5 and 6). Furthermore,
mutation of TRE sites (for sequence, see Table 2) completely blocked
AP-1 complex formation (data not shown). We next characterized
the composition of the AP-1 protein complex binding to the SPRR1B promoter using specific antibodies as in Fig. 5B.
Before the PMA treatment, cells displayed binding of JunD and Fra1
(lanes 4 and 5). However, treatment of cells with
PMA predominantly induced binding of JunB and Fra1 (lanes
8-14). A slight enhancement of Fra2 binding was also noticed
after PMA treatment (lane 13). Preincubation of nuclear
extracts with normal rabbit serum showed no supershifting of the bands
(data not shown). These results indicate that the AP-1 dimers,
consisting of JunD and Fra1, bind to SPRR-TRE in the unstimulated
state, whereas JunB and Fra1 bind to it upon PMA treatment.

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Fig. 5.
Analysis of
H441 nuclear extracts binding to the SPRR1B promoter. EMSA
was performed essentially as described in Fig. 4 except that a WT
150- to 84-bp SPRR1B promoter fragment (Table 2) was
used instead of the AP-1 probe. A:
32P-end-labeled probe was incubated with 2 µg of nuclear
extracts isolated from vehicle (-) and PMA-treated (+) H441 cells. For
competition experiments, nuclear extracts were preincubated for 10 min
with 25- or 100-fold molar excess of unlabeled probes before labeled
probe was added. Lane 1, F; lanes 2-7,
nuclear extracts from unstimulated (-) H441 cells; lane 3,
competition with WT promoter (self); lane 4, competition
with consensus AP-1 site (AP-1); lanes 5 and 6,
competition with DM; lane 8, nuclear extracts from
PMA-stimulated (+) H441 cells. B: SS analysis of WT
SPRR1B promoter using antibodies specific for individual
AP-1 family proteins. For label and symbol details, see Fig.
4.
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AP-1 proteins differentially regulate SPRR1B promoter activity.
Band shift profiles indicated a distinct pattern of AP-1 binding to
SPRR-TRE compared with the consensus AP-1 site. Therefore, we used a
panel of wt and/or dn mutants of the Jun (c-jun,
junB, and junD) and Fos
(fra1 and fra2) expression plasmids to
determine the roles for these proteins in the regulation of
SPRR1B. We did not study the role of c-Fos, because band
shifts studies did not reveal any significant binding of c-Fos to
either consensus AP-1 site or SPRR-TRE in both untreated and
PMA-treated cells. Cells were transiently transfected with
150-Luc reporter plasmid along with the indicated expression
vectors at comparable molar quantities, and Luc activity was analyzed.
As shown in Table 3, cotransfection of wt
c-jun, junB, and junD expression
vectors robustly enhanced the promoter activity to levels comparable
with those observed with PMA treatment. Conversely, overexpression of
c-Jun mutant with a defective in transactivation domain strongly
inhibited both basal and PMA-stimulated SPRR1B expression.
As shown in Fig. 5B, Fra1 binds to the SPRR1B
promoter predominantly, whereas a slight increase in Fra2 binding was
noticed after PMA exposure. Therefore, we analyzed the role for Fra1
and Fra2 in SPRR1B promoter regulation. Overexpression of
fra2 strongly suppressed PMA-stimulated Luc expression
driven by SPRR1B promoter. Conversely, overexpression of
fra1 upregulated the basal level activity, whereas it had no significant effect on PMA-stimulated promoter activity (Table 3).
Together, these results demonstrate that AP-1 dimers, with distinct
composition, upregulate SCD in distal bronchiolar region. More
recently, we showed that binding of Fra2 to the SPRR-TRE correlates
with a loss of SPRR1B expression in A549 cells
(14). In contrast, overexpression of Fra1 strongly
upregulates SPRR1B promoter-driven reporter gene expression
in A549 cells (14). These results indicate that Fra1 and
Fra2 exert opposing effects on SPRR1B promoter in H441
cells. They also indicate that PMA-inducible expression of SPRR1B
in distal bronchiolar cells is regulated by TRE sites and AP-1
proteins in a manner similar to that observed in proximal TBE cells, S6
and HBE1 (14).
Ras regulates SPRR1B gene expression through Raf-1 and MAPKK kinase
1.
To understand the signaling pathways that control PMA-inducible
expression, we examined the role of Ras-Raf MAPK pathway. As said
earlier, PMA activates PKC isoforms that in turn activate Ras, one of
the downstream targets of PKC (30, 33). To determine whether PMA upregulates SPRR1B expression through Ras, we
cotransfected cells with ca-Ras or mutant dn-Ras expression vectors
along with reporter construct. After 24 h, cells were treated with
either PMA or vehicle, and the Luc expression was monitored.
Expression of dn-Ras suppressed PMA-stimulated activity very
significantly (Fig. 6A),
whereas it had no effect on basal expression. Conversely, overexpression of ca-Ras significantly stimulated promoter activity (~2.5-fold) compared with vector-transfected controls (compare bar 1 with 5). We next investigated whether
Raf-1, the downstream target of Ras (30), is required for
gene induction (Fig. 6A). Indeed, dn-c-Raf significantly
suppressed PMA-stimulated, but not the basal-level, SPRR1B
expression (compare bars 2 and 7). Together,
these results indicate that PMA-activated Ras and Raf are critical for
activating SPRR1B promoter. Ras also activates MAPKK kinase
(MAPKKK) 1. Expression of dn-MAPKKK1 did not have any effect on basal
gene expression, whereas it had a very significant effect (nearly 50%
reduction) on PMA-stimulated promoter activity (Fig. 6B). On
the other hand, expression of ca-MAPKKK1 alone strongly augmented
(~4-fold) the Luc expression (Fig. 6B).

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Fig. 6.
Ras regulates
SPRR1B promoter activity via c-Raf and mitogen-activated
kinase kinase kinase (MAPKKK) 1. A: cells were transiently
transfected with 150-SPRR1B-Luc (100 ng) and -gal (50 ng) vectors
along with 100 ng of either empty parental (bars 1 and
2), dominant negative (dn)-Ras (bars 3 and
4), constitutively active (ca)-Ras (bar 5) or
dn-c-Raf (bars 6 and 7) expression vectors.
B: SPRR1B-Luc reporter and -gal vectors were
cotransfected into H441 cells along with empty (bars 1 and
2), dn-MAPKKK1 (bars 3 and 4), or
ca-MAPKKK1 mutant (bar 5) expression vectors. Cells were
then treated with either DMSO or PMA for ~14 h and harvested, and Luc
expression was analyzed. The results shown represent the means ± SE of duplicated samples. The experiment was repeated at least two
times, and similar qualitative results were obtained.
*P < 0.001 compared with PMA-treated empty
vector-transfected group (bar 2).
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MAPKK1, but not ERK1/2 MAPKs, regulates SPRR1B promoter
in H441 cells.
Recently, we showed that PMA-stimulated SPRR1B expression in
BEAS-2B cells is mainly regulated by a MAPKK1/ERK-like MAPK pathway. However, ERK1/2, JNK, and p38 MAPKs did not appear to participate in
this pathway (31). We therefore focused our studies on the latter three (ERK, JNK, and p38) MAPKs and used both pharmacological and/or gene-specific dn mutants to evaluate whether PMA-inducible expression of SPRR1B in Clara-like H441 cells is mediated by
the same signaling pathways as in proximal TBE cells. PD-98059, which prevents the activation of MAPKK1/2 (7), strongly
suppressed both the basal (~50%) and PMA-stimulated (~100%)
reporter gene expression (Fig.
7A). These results have been
further confirmed with UO-126 (data not shown), which specifically
inhibits the function of activated MAPKK1 (7). Consistent
with this, expression of a dn-MAPKK1 robustly suppressed PMA-stimulated
reporter gene expression; conversely, ca-MAPKK1 significantly enhanced
it (Fig. 7B). Furthermore, PD-98059 slightly reduced basal
and very significantly suppressed PMA-stimulated endogenous
SPRR1B expression (data not shown). Thus MAPKK1 regulates
both basal and PMA-stimulated expression of SPRR1B. We next
examined the role of ERK1 and ERK2, the well-known downstream targets
of MAPKK1, in SPRR1B expression. As shown in Fig.
7C, overexpression of a dn-ERK1 or dn-ERK2 either alone or in combination (data not shown) did not suppress PMA-stimulated reporter gene expression. Intriguingly, treatment of H441
cells with PMA enhances the phosphorylation of ERK1/2 proteins (three- to fivefold). The ERK1/2 phosphorylation rapidly increased within 5 min
and persisted at later periods (Fig. 7D). Together, these results indicate that the MAPKK1 pathway regulates PMA-stimulated SPRR1B expression. Paradoxically, ERK1 and/or ERK2 do not
participate in it.

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Fig. 7.
MAPKK1
regulates SPRR1B expression in H441 cells. A: the
effect on MAPKK1 inhibitor PD-98059 on SPRR1B expression.
Cells were transfected with 150-SPRR1B-Luc reporter and
-gal vectors as in Fig. 3. After 24-h incubation, cells were treated
with either vehicle or PD-98059 (20 µM) before PMA exposure.
B: the regulation of SPRR1B promoter by MAPKK1.
Cells were cotransfected with SPRR1B-Luc reporter and
-gal constructs as in A, either in presence of empty
(bars 1 and 2), dn-MAPKK1 (bars 3 and
4), or ca-MAPKK1 (bar 5) mutants. After
transfection, cells were treated with vehicle or PMA. C: the
effect on dn-extracellular signal-regulated kinase (ERK) 1 and dn-ERK2
mutants on SPRR1B promoter activity. Cells were transfected
with SPRR1B promoter and -gal constructs in the presence
of dn-ERK1 or dn-ERK2 mutants. Values of Luc activity of the empty
vector (pCEP4, 100 ng)-transfected cells were taken as one.
D: phosphorylation of ERK1/2 by PMA. Cells were treated with
PMA (25 ng/ml) for indicated time periods, and cellular extracts were
prepared. An equal amount of protein was separated on polyacrylamide
gel, blotted, and analyzed using phospho-specific ERK1/2 antibody
(top). Total ERK content of the samples was determined by
immunoblot analysis using ERK2 antibodies (middle). Protein
loading of the membrane was assessed by Western blot analysis using
antibodies specific for -tubulin (bottom).
|
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JNK1, but not p38 MAPK, regulates SPRR1B promoter in H441 cells.
The JNK and p38 MAPK family members are activated in response to
cellular injury, ultraviolet irradiation, inflammatory cytokines, and
various environmental stresses (8). Therefore, we next examined a role for p38 and JNK MAPKs in SPRR1B promoter
regulation. To examine the role of p38 pathway, we used a chemical
inhibitor, SB-202190 (SB). H441 cells were pretreated with SB
(5-20 µM), and endogenous SPRR1B mRNA levels were
measured by RT-PCR analysis (Fig.
8A). SB did not significantly
suppress the PMA-stimulated mRNA expression of SPRR1B.
Consistent with this, SB did not significantly inhibit either the basal
or PMA-stimulated SPRR1B promoter-driven reporter gene
expression (Fig. 8B). Intriguingly, the kinase activity of
p38, as measured by the phosphorylation of its substrate, activating transcription factor (ATF)-2, was significantly increased after PMA
treatment (Fig. 6C). Kinase activity increased
onefold within 5 min of treatment and reached peak values (sixfold)
after 15 min of treatment. The kinase activity remained
elevated, below the peak level, even after 3 h. Together, these
results indicate that the p38 MAPK pathway, though functionally active,
does not appear to regulate PMA-stimulated SPRR1B expression
in the Clara cell type.

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Fig. 8.
p38 MAPK pathway does not regulate PMA-stimulated SPRR1B
promoter activation. A: the effect of p38 MAPK inhibitor
SB-202190 (SB) on SPRR1B expression. Cells were grown to
80% confluence and then pretreated with SB for 30 min before PMA
exposure. RNA was isolated and subjected to RT-PCR analysis as
described in Fig. 1. B: cells were transfected with
150-SPRR1B-Luc reporter and -gal vectors. After
transfection, cells were pretreated with either vehicle or SB for 30 min before PMA treatment. C: the effect of PMA on p38
activity. Cells were treated with PMA or DMSO, and cellular extracts
were isolated as described in Fig. 7C and immunoprecipitated
using p38 antibodies. The kinase activity of the immunoprecipitates was
analyzed using activating transcription factor-2 as substrate. As a
positive control for p38 activation, H441 cells were treated with
puromycin (Pu, 2 µg/ml for 30 min) or sorbitol (Sor, 10 mg/ml for
1 h). For ultraviolet (UV) treatment, cells were briefly exposed
to UV (30 s) and incubated further for 1 h in the presence of
culture medium.
|
|
We next examined the role of JNK1 in the regulation of
SPRR1B promoter (Fig. 9).
Expression of dn-JNK1 mutant significantly diminished PMA-stimulated
expression (Fig. 9A). JNK1 and JNK2 activation was also
confirmed by immunoprecipitation with isoform-specific antibodies
followed by Western blot analysis with phospho-JNK-specific antibodies.
As shown in Fig. 9B, PMA treatment significantly enhanced phosphorylation of JNK1 and JNK2 proteins in H441 cells. Consistent with this, enzyme activity of JNK1/2 was also enhanced after PMA treatment. PMA significantly increased JNK1 kinase activity (fourfold) that persisted over a longer time period (Fig. 9C).
Together, these results highlight the importance of JNK1 in regulation
of PMA-stimulated SPRR1B expression in H441 cells
(Fig. 10).

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Fig. 9.
c-Jun NH2-terminal kinase (JNK)/stress-activated
protein kinase (SAPK) MAPK regulates SPRR1B promoter
activation. A: cells were transfected as in Fig.
6A in the presence of either an empty or dn-JNK1 mutant.
After transfection, cells were treated with or without PMA for ~14 h,
and Luc gene expression was analyzed. B: H441 cellular
extracts were precipitated with JNK1 antibodies and Western blotted
using phospho-specific JNK1/2 antibodies. C: the kinase
activity of the immunoprecipitates (as in B) was analyzed
using c-Jun as substrate. As a positive control, H441 cells were
treated with anisomycin (Ani, 2 µM) for 30 min, and JNK1 activity was
analyzed as described above.
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Fig. 10.
The proposed scheme depicts a signal transduction
pathway that regulates PMA-stimulated SPRR1B expression in
distal (bronchiolar region) and proximal airway epithelial cells. ?,
the putative unidentified kinase.
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|
 |
DISCUSSION |
In response to injury caused by various carcinogens, toxicants,
and pollutants, airway epithelial cells (proximal and distal) lose
their normal secretory functions and express squamous and keratinizing
markers (16). Induction of SCD in nonsquamous airway epithelium is initially thought to be a protective response, acting as
a barrier against toxicants and pollutants; however, if not properly
restored, this phenomenon might lead to airway epithelial cell
transformation and bronchial carcinogenesis (11). An
understanding of the molecular events controlling the SCD process will
help us in delineating the pathways involved in cellular injury and repair processes. It also provides an insight into the toxicant-induced airway disease and bronchial carcinogenesis. Clara cells are
nonciliated epithelial cells that play an important role in respiratory
function by expressing surfactants and a 10-kDa Clara cell protein,
CC10 (16). The present study demonstrates that PMA
selectively induces the expression of SCD markers such as
SPRRs (1A, 1B, and 2A, but not 3) in Clara-like bronchiolar
epithelial cells. These results are in contrast with the previous
studies that demonstrated the downregulation of CCD markers, such as
CC10 and surfactant expression in H441 cells, upon exposure to PMA
(9, 13, 19, 20). Similarly, naphthalene and furan
4-ipomeanol induce cellular injury and suppress the expression of CCD
markers, such as CC10 and cytochrome P-450, and also greatly
enhance the expression of squamous cells (~70-90%) in the
distal bronchiolar region (23). The exact role for SPRRs in the induction of SCD in Clara cells is unclear. However, it is well established that SPRRs act as cross-bridging proteins that
participate in a cross-linking process of several CCE precursor proteins, such as loricrin (constituting 50-90% of CCE proteins) and involucrin (4, 24). It is worth noting that a recent study demonstrated the actual participation of SPRR1B in the
cornification of proximal TBE cells (5). Therefore, we
speculate that expression of SPRRs is an essential feature
of SCD of distal bronchiolar cells and plays a very important role in
providing a physical and chemical barrier against the environment. In
support of this notion, a selective inducible expression of
SPRRs, but not involucrin, was noticed in malignant
keratinocytes that were induced to differentiate (32),
underscoring an important role for SPRRs in epithelial cell
differentiation. Moreover, Chinese hamster ovary cells, a nondifferentiating cell type, express SPRR1 at specific
stages of the cell cycle indicating that SPRRs may play a novel role in
cell cycle progression and growth arrest (26).
Thus, transcriptional regulation of SPRR expression may
provide an additional insight into molecular mechanisms governing the
cellular differentiation of distal bronchiolar cells in response to
toxic stimuli.
Abnormal expression and/or activation of AP-1 (Jun/Fos) proteins in
response to various growth factors and toxicants had been implicated in
the development of various diseases and epithelial cell transformation
(2). The Jun family (c-Jun, JunB, and JunD) proteins can either homo- or heterodimerize with the Fos family (c-Fos,
Fos-B, Fra1, and Fra2), bZIP transcription factors (e.g., ATF/cAMP
response element binding protein, Maf/Nrf family members), as well as
bHLH ZIP proteins (e.g., myoD and upstream stimulatory factor),
resulting in differential DNA binding and transactivation (2). Subsequent AP-1 binding to the TREs
regulates the transcription of a variety of genes that are involved in
cell growth and differentiation and the injury-repair process,
depending on cellular and promoter context (2).
Transcriptional analysis indicates that two functional TREs and AP-1
proteins mediate PMA-inducible SPRR1B expression in H441
cells. Moreover, the composition of AP-1 protein complex (Jun/Fra1
dimers) plays an important role in regulation of SPRR1B expression in H441 cells, in a manner similar to that observed in
proximal S6 cells. PMA stimulates both Fra1 and Fra2 binding to the
consensus AP-1 or TRE site (Fig. 4B), whereas Fra1 more prominently binds to the SPRR1B promoter compared with Fra-2
(Fig. 5B). These results emphasize the importance of
cellular and promoter context, flanking residues such as GT/CAC and
ETS-like motifs, on the regulation of AP-1-dependent gene
expression. Indeed, mutation of these sites significantly
reduced basal-level promoter activity (Fig. 3C). Thus it is
conceivable that both the activation and proximal availability of Fra1
and Fra2 with other proteins such as Jun members and/or cell
type-specific factors might be involved in the regulation of
SPRR1B expression in airway epithelial cells.
It is well established that PMA activates PKC isoenzymes, which in turn
initiate a signaling cascade to stimulate expression of genes involved
in cell growth and differentiation in different cell types. This is
mediated by three well-studied distinct MAPKs, ERK, JNK, and p38
(6). ERK1 and ERK2 are mainly activated in response to
growth stimuli, whereas JNK/SAPK and p38 are activated by cellular
stress and cytokine inflammation and can participate in differentiation
and cell death (6). In turn, these kinases activate
transcription factors such as Elk-1, CCAAT/enhancer binding protein
(C/EBP), C/EBP-homologous protein, c-Jun, and ATF-2 to modulate both
cell type- and stimulus-specific target gene transcription (6). Recently, we demonstrated that a
MAPKK1-ERK-like kinase pathway regulates PMA-stimulated
SPRR1B expression in TBE cells, whereas ERK1 and ERK2,
JNK1/SAPK, and p38 had no effect (31). Consistent with
this, the present study demonstrates an involvement of MAPKK1 in
PMA-stimulated SPRR1B transcription in H441 cells; however,
ERK1/2 do not participate in such a process. Interestingly, dn-JNK1
mutant robustly suppressed PMA-inducible SPRR1B expression in H441 cells (Fig. 9), indicating a regulatory role for JNK1 in the
induction of SCD in distal bronchiolar cells. The fact that both MAPKK1
inhibitors and JNK1 mutant suppressed the PMA-stimulated SPRR1B promoter activation suggests the existence of either
other ERK-related kinase(s) or cross-talk between MAPKK1 and JNK1
pathways that might regulate gene expression. Our data (Fig. 6) clearly support a role for Ras in SPRR1B regulation. Suppression of
PMA-stimulated SPRR1B promoter activity by c-Raf and MAPKKK1
mutants indicates that Ras mediates its effects in part through the
activation of c-Raf and MAPKKK1. However, in S6 cells, PMA-stimulated
SPRR1B promoter regulation by Ras does not require c-Raf but
is mediated by MAPKKK1 (31). Thus activation of
SPRR1B expression, although regulated by a similar set of
AP-1 proteins, may be differentially activated by a unique set(s) of
MAPKs in proximal and distal airway epithelial cells.
 |
ACKNOWLEDGEMENTS |
The authors thank all the scientists who provided us with various
expression vectors used in this study. We thank Reen Wu and Steven
Kleeberger for helpful comments on this study.
 |
FOOTNOTES |
This work was supported in part by National Heart, Lung, and Blood
Institute Grant HL-58122 and a pilot project from National Institute of
Environmental Health Sciences Center Grant ES-03819 to S. P. M. Reddy. We acknowledge the Johns Hopkins Urban Environmental Center
for use of its core facilities.
Address for reprint requests and other correspondence: S. P. M. Reddy, Johns Hopkins Univ., Dept. of Environmental Health Sciences, Div. of Physiology, Rm. W7006, 615 No. Wolfe St., Baltimore, MD 21205 (E-mail: sreddy{at}jhsph.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00125.2001
Received 3 April 2001; accepted in final form 12 September 2001.
 |
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