Departments of 1 Pediatrics, 2 Medicine, and 6 Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030; 3 Children's Research Institute, Columbus, Ohio 43205; 4 Department of Pediatrics, Ajou University, Suwon, Korea; and 5 Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101
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ABSTRACT |
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Interferon (IFN-
), a potent
cytokine inducing a wide range of immunologic activities, is increased
in the airway secondary to viral infection or during an inflammatory
response. This increase in IFN-
concentration may alter the
expression of specific airway epithelial cell genes that regulate
adaptation of airway inflammatory responses. One protein induced by
IFN-
is Clara cell secretory protein (CCSP), which may contribute to
the attenuation of airway inflammation. This study was done to
investigate the molecular mechanism by which IFN-
stimulates the
expression of the CCSP gene in mouse transformed Clara cells and
transgenic mice. Deletion mapping and linker-scanning mutations
demonstrated that IFN-
-induced expression of CCSP was regulated, in
part, at the level of transcription. In vitro and in vivo studies
verified that the minimal IFN-
-responsive segment was localized to
the proximal 166 bp of the 5'-flanking region. Additionally,
IFN-
-induced expression of CCSP was mediated indirectly through an
interferon regulatory factor-1-mediated increase in hepatocyte nuclear
factor-3
.
hepatocyte nuclear factor-3; CCAAT/enhancer-binding protein; mouse transformed Clara cells; thyroid transcription factor-1; interferon regulatory factor-1; Clara cell secretory protein
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INTRODUCTION |
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PULMONARY INFLAMMATION is a physiological response to a variety of infectious and immune-mediated lung diseases. However, the ability of the lung to limit the inflammatory response to these processes is of critical importance in confining or preventing resultant lung injury. Therefore, the identification of intrinsic pulmonary mechanisms that can restrict or terminate pulmonary inflammation is of extreme importance in maintaining a homeostatic balance within the lung. One protein that may contribute to the lung's modulation of inflammation is the Clara cell secretory protein (CCSP) produced by the nonciliated respiratory bronchiolar epithelial cells (Clara cells) (6).
CCSP is a major component of the surface lining fluid in the lung
(25), and the expression of CCSP is induced by a variety of proinflammatory mediators and cytokines, including interferon- (IFN-
) (5, 13, 31, 32). The physiological function of CCSP in the alveolar lining fluid remains unclear. However, there is
evidence for an anti-inflammatory role of CCSP. This evidence includes
in vitro data that CCSP inhibits the activity of phospholipase A2 (6) and in vivo confirmation that CCSP
deficiency in mouse models, generated by homologous recombination,
increases the susceptibility to pulmonary inflammation secondary to
viral, bacterial, or hyperoxic stimuli (9, 11, 14). These
observations imply that CCSP may have an important role in modulating
intrapulmonary inflammatory events. Therefore, elucidation of the
regulatory mechanisms controlling CCSP gene expression in response to
IFN-
stimulation may help define the intrinsic mechanisms for
modulating pulmonary inflammation in the lung.
The molecular events that regulate IFN--induced expression of CCSP
have not been fully defined. Previous analyses demonstrate that
IFN-
-induced expressions of CCSP are mediated in part by posttranscriptional mechanisms (31). However, the
potential for transcriptional regulation of CCSP by IFN-
remains to
be determined. Many analyses performed to ascertain the regulatory mechanisms responsible for CCSP gene expression have been carried out
in a variety of cell culture models. These analyses have been hampered
by the lack of a cell line in which the endogenous expression of CCSP
and a full complement of the respiratory cell-specific trans-acting factors are present. The known cis
elements identified in the 5'-flanking region of the CCSP gene are
demonstrated in Fig. 1. The CCSP promoter
is divided into proximal and distal promoter regions, with each region
containing multiple cis elements that may function in the
regulation of CCSP gene expression (10, 17, 18, 26). The
proximal 166-bp region is sufficient to maintain cell-specific
expression of reporter genes in vitro and in vivo (16, 18,
22), whereas the distal promoter region is capable of driving
cell-specific expression of a reporter gene to levels comparable to
endogenous CCSP gene expression in vivo (18). The known
cis elements contained in the proximal 166-bp fragment of
the CCSP promoter that may be important in lung-specific gene
expression include two thyroid transcription factor-1 (TTF-1) sites
(17), two CCAAT/enhancer-binding protein (C/EBP) sites (3), an activator protein-1 (AP-1) binding site
(27), and two hepatocyte nuclear factor-3 (HNF-3)
consensus sites (22). Previous analyses of these cis
elements suggest that there may be complex combinatorial
regulation mediated by interactions of the complementary
trans-acting elements (1, 22, 27, 28). The
cis elements in the distal CCSP promoter that regulate the enhanced CCSP gene expression remain to be elucidated. However, it is
known that three TTF-1 sites and a
-interferon activation site (GAS)
are localized to this promoter region (13, 17).
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Although the molecular events that regulate IFN--induced expression
of CCSP have not been fully defined, it is known that IFN-
effects
are mediated by receptor-ligand interactions. These interactions
precipitate the activation of a rapid signal transduction cascade of
events, resulting in the activation and nuclear translocation of the
signal transducer and activator of transcription (STAT1) protein.
Nuclear STAT1 homodimers may affect the transcription of specific
target genes directly by binding to specific DNA regulatory elements or
indirectly through the increased transcription and activity of
interferon regulatory factor-1 (IRF-1) transcription factor. Previous
analysis of the CCSP gene promoter demonstrates at least two regions
through which IFN-
could mediate a change in CCSP gene expression.
DNase I footprinting demonstrates an interferon activation site (GAS)
spanning nucleotides
314 to
284, through which IFN-
could
directly effect a change in CCSP gene transcription (13).
Alternatively, IFN-
could indirectly affect CCSP gene expression
through IRF-1-mediated interactions with the HNF-3 sites in the
proximal promoter region, as observed for hepatic expression of the
transthyretin gene (20).
In the present study, we demonstrate that IFN--induced expression of
the CCSP gene is regulated, at least partially, at the level of
transcription. Furthermore, we demonstrate that the IFN-
responsiveness of the CCSP gene is localized to the proximal 166-bp region of the 5'-flanking region of the CCSP gene in vivo and in
vitro. Moreover, we demonstrate that the IFN-
responsiveness of the CCSP gene is mediated through a complex
regulatory region in the proximal promoter region and that HNF-3
binding is a major component of this induction.
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MATERIALS AND METHODS |
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Cell culture. Mouse transformed Clara cells (mtCC) were cultured at 37°C in a humidified atmosphere with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (0.1 mg/ml).
Footprinting.
The murine CCSP (mCCSP) promoter sequence previously reported
(26), spanning base pairs 291 to
11, was amplified by
PCR. The PCR product was subcloned into the pCR2.1-TOPO vector
(Invitrogen), and the fragment was isolated by digestion with
HindIII and XbaI. The ends were then labeled with
Klenow and [32P]dATP and [32P]dCTP.
Unincorporated nucleotides were removed by passing the reaction mixture
through a Sephadex G-25 quick-spin column (Boehringer Mannheim). The
DNA was cut with BamHI, extracted with
phenol-chloroform-isoamyl alcohol, and precipitated with 100% ethanol.
The DNase I reaction was performed according to the manufacturer's
recommendation using the Promega Core Footprinting system. Briefly,
nuclear extracts obtained from mtCC were incubated in binding buffer
(12 mM HEPES, 12% glycerol, 50 mM KCl, 0.12 mM EDTA, 3 mM
MgCl2, and 1.2 mM dithiothreitol) with 40 ng of
poly(dI-dC) for 10 min on ice and then for 1 min at room temperature.
Pancreatic DNase I (0.375 U) was incubated with increasing amounts of
nuclear proteins (10 or 30 ng). After 1.5 min, the reaction was stopped
by addition of 100 µl of stop buffer (10 mM EDTA, 0.1% SDS, and 50 ng/ml proteinase K). The samples were incubated at 37°C for 15 min
and then subjected to phenol-chloroform-isoamyl alcohol extraction and
ethanol precipitation. The reaction products were analyzed on a
polyacrylamide-8 M urea sequencing gel. A nucleotide reaction for the
region being footprinted was run in parallel in the same gel for
accurate identification of the sizes and nucleotide sequences of the
footprinted regions. After electrophoresis, the gels were fixed, dried,
and exposed to X-ray film at
80°C.
Western blot analyses.
For Western blot analyses, we utilized samples obtained from whole lung
homogenates or mtCC whole cell extracts. Samples were supplemented with
SDS loading buffer and boiled. The proteins were separated by 12%
SDS-polyacrylamide gels and electroblotted onto a polyvinylidene
difluoride membrane (Millipore) at 4°C. Western blot analysis was
done using a number of primary antibodies: TTF-1 at a 1:6,000 dilution
(Santa Cruz Biotechnology and the laboratory of F. J. DeMayo),
HNF-3 (Santa Cruz Biotechnology), C/EBP
(Affinity Bioreagents and
Santa Cruz Biotechnology), C/EBP
(Affinity Bioreagents and Santa
Cruz Biotechnology), or C/EBP
(Santa Cruz Biotechnology) at a
1:3,000 dilution. This was followed by luminescent detection according
to the protocol of the manufacturer (Amersham Life Technologies).
Transient transfection analyses.
At 1 day before transfection, mtCC were plated at a density of 3 × 106/100-mm plate. Cells at 50-70% confluency were
transfected on 100-mm dishes by using 30 µl of the Superfect
transfection reagent (Qiagen) with 10 µg of reporter plasmid and 0.5 µg of cytomegalovirus (CMV)--galactosidase plasmid as an internal
control. Transfected cells were incubated for 3 h and then washed
with DMEM to remove the transfecting agent. Cells were then fed DMEM
without fetal calf serum and treated with vehicle or recombinant mouse
IFN-
(Invitrogen) at 1,000 IU/ml DMEM. The cells were harvested,
centrifuged for 5 min, and resuspended in 100 µl of 250 mM Tris (pH
7.5). The cells were lysed by three cycles of freezing in liquid
N2 and thawing at 37°C. The cell debris was cleared by
centrifugation, and protein concentration was measured using Bradford
reagent (Bio-Rad). Alternatively, for RNA analyses, the cells were
harvested with TRIzol reagent (Invitrogen), and total RNA was
quantified by measuring the absorbency at 260 nm using a spectrophotometer.
Transfection analysis of CCSP-chloramphenicol acetyltransferase
constructions.
The generation of the CCSP-chloramphenicol acetyltransferase (CAT)
plasmids containing various lengths of the CCSP promoter ligated to the
bacterial CAT gene has been reported previously (18). The
linker-scanning mutation of the GAS site and the two HNF-3 binding
sites were introduced into the 803-bp promoter fragment of the
wild-type CCSP gene by modifications of the procedure previously described (12). Mutagenic oligonucleotides were generated
to replace the GAS site or the proximal or distal HNF-3 site with the
sequences that compose a BglII-BamHI ligation
(replaced sequence 5'-CGGGATCTTC-3'). The mutagenic oligonucleotides
contained 30 nt: 20 nt were complementary to the CCSP promoter sequence
surrounding the targeted site, and 10 nt contained a BglII
restriction endonuclease sequence (forward primer) or a
BamHI restriction endonuclease sequence (reverse primer).
PCR amplification was performed using the 803-bp CCSP-CAT plasmid as a
template. For each PCR, one of two anchor primers was used along with
the specific mutagenic primer. A 5' anchor, 1M, contains a
HindIII overhang and anneals at position 803 of the CCSP
promoter. A 3'-anchor primer, pBLCAT3-BalI, anneals a
BalI restriction site within the CAT reporter gene. The
amplified fragments were cloned into the pCRII vector (Invitrogen) and
sequenced to verify the specific mutation. The appropriate pairs of
fragments were then cloned into the HindIII-BamHI
site of the pBLCAT3 vector. Plasmid DNA for transfection was isolated using Maxi-Prep plasmid preparations (Qiagen).
Liquid CAT assay. The CAT activity of the lysate was assayed as described by Seed and Sheen (23). The activity of 50 µg of lysate was assayed at 37°C for 5 h with a mixture of 10 µl of butyryl CoA (2.5 mg/ml) and 20 µl of [3H]chloramphenicol (0.01 µCi/µl). The reaction was then extracted with 200 µl of tetramethylpentadecane-xylenes (2:1), and 150 µl of the top aqueous phase were counted in a liquid scintillation counter with 4 ml of scintillation fluid.
-Galactosidase assay.
As an internal control, all CAT reporters were cotransfected with 1 µg of CMV-
-galactosidase plasmid, consisting of the CMV promoter
driving the expression of the
-galactosidase gene. The
-galactosidase activity in each transfection was used to control for
variability between transfections. Quantitation of
-galactosidase activity was achieved essentially as described by Sambrook et al.
(21). In summary, equal amounts of protein in 30 µl from each transfection plate were mixed with the following
reagents: 3 µl of 100× MgCl2 (0.1 M MgCl2
and 4.5 M
-mercaptoethanol), 66 µl of 1× ONPG (4 mg/ml of
o-nitrophenyl-
-D-galactopyranoside dissolved
in 0.1 M sodium phosphate, pH 7.5), and 201 µl of 0.1 M sodium
phosphate, pH 7.5, and incubated for 30 min at 37°C. The reactions
were stopped by the addition of 500 µl of
Na2CO3, and the optical density of the
reactions was read at a wavelength of 420 nm.
Luciferase activity. The CCSP-luciferase plasmid containing the 800 bp of the CCSP promoter ligated to the firefly luciferase reporter gene was generated by PCR using the 2.1-kb CCSP-CAT plasmid as a template and oligonucleotides with synthesized KpnI and HindIII sites 5' and 3', respectively, and TOPO TA cloned into the pCR2.1 vector (Invitrogen). The 800-bp promoter was then cut out with KpnI and HindIII and directionally ligated into the pGL3-basic luciferase reporter gene (Promega). The 166-bp CCSP-luciferase plasmid was generated by cutting the 800-bp CCSP-luciferase plasmid with SacI, blunt ended with T4 polymerase, and ligated with T4 DNA ligase (Boehringer Mannheim). Transient transfection analyses were then done utilizing the luciferase reporter genes in mtCC and harvested in 1× reporter lysis buffer (Promega). The cell lysate was subjected to a freeze-thaw cycle and centrifuged at 12,000 g in a microcentrifuge for 15 s at room temperature to pellet the cell debris. The luminescent signals generated by the firefly and Renilla luciferase reporter genes were measured using the dual-luciferase reporter assay (Promega) and a Monolight Luminometer (Pharmingen).
Transgenic mouse analysis.
Previously generated transgenic mice that express a human growth
hormone (hGH) reporter gene driven by a 166-bp segment of the CCSP
promoter were utilized for the in vivo assessment of the effects of
IFN- on CCSP gene expression (18). Adult transgenic mice were treated with 0.1 ml of saline or 0.1 ml of IFN-
(1,000 IU/ml DMEM) via tracheotomy and allowed to recover on a warming pad for
30 min. The mice were then killed by lethal injection of
tribromoethanol (Avertin) at 24 or 48 h after treatment. Lung samples were harvested for RNA and protein analyses.
IRF-1 mouse analysis.
Previously generated IRF-1-deficient mice (29) were
utilized for in vivo assessment of whether the IRF-1 pathway was
involved in the IFN--induced expression of mCCSP, in addition to two
wild-type controls: 129, which was the strain background of the
IRF-1-deficient mice, and FVB, which is known to respond to IFN-
with an increase in CCSP expression (13). Adult null mice
and wild-type controls were treated with 0.1 ml of saline or 0.1 ml of
IFN-
(1,000 IU/ml DMEM) via tracheotomy and allowed to recover on a
warming pad for 30 min. Mice were killed by lethal injection of
tribromoethanol 48 h after IFN-
treatment. Lung samples were
harvested for RNA and protein analyses.
RNase protection assay. Total RNA was extracted from mouse lungs by using TRIzol reagent (Invitrogen). Expression of the mRNAs for the CCSP and hGH was accomplished by RNase protection with a [32P]UTP (ICN)-labeled probe using an RNase protection assay (RPA) kit (Ambion). The template for the CCSP mRNA was generated by insertion of a 327-bp BamHI and NotI fragment of the mouse CCSP cDNA into pCRII (Invitrogen). An antisense riboprobe for RPA was generated by digesting the plasmid with BamHI, and T7 RNA polymerase was used for in vitro RNA synthesis. A template for hGH mRNA was generated by insertion of a 2.0-kb EcoRI fragment of the hGH cDNA into pBluescript (Stratagene). An antisense riboprobe for RPA was generated by digesting this plasmid with BglII and by using T7 RNA polymerase for in vitro RNA synthesis. A cyclophilin probe was used as an internal control for all RPA analyses.
EMSAs.
A synthetic oligonucleotide was generated that contained 30 bp of the
CCSP promoter from 110 to
81, which includes the overlapping binding sites for AP-1, HNF-3, and C/EBP. Subsequent oligonucleotides were generated with mutations in the AP-1, HNF-3, or C/EBP binding sites and followed by oligonucleotides, which contained double mutations in the AP-1/HNF-3, AP-1/C/EBP, or HNF-3/C/EBP binding sites.
Also, a triple-mutant synthetic oligonucleotide that contained mutations in all three binding sites was generated to be utilized as a
nonspecific competitor (Fig.
2C). In addition, the
oligonucleotides contained a 5' overhang and were end-labeled with
[32P]dATP and [32P]dCTP using a Sequenase
reaction kit (US Biochemicals). EMSA was performed by incubating 5 × 104 cpm of labeled oligonucleotide with 5-10 µg
of nuclear extract from the mtCC in gel shift binding buffer (Promega)
treated with vehicle or IFN-
for 4-24 h. The formation of
complexes was performed at room temperature for 15 min. The complexes
were separated by electrophoresis through a 6% nondenaturing
polyacrylamide gel, dried on filter paper, and exposed to
autoradiographic film.
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RESULTS |
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mtCC express a full complement of respiratory epithelial
trans-acting factors.
Previous analysis of the mCCSP promoter was conducted in H441 cells,
which do not express CCSP or recognize the distal promoter elements
required for in vivo expression of CCSP (18). With the use
of tumor cells derived from mice expressing the simian virus 40 large T
antigen gene under the control of the CCSP promoter, an mtCC line was
generated. This cell line expresses IFN--regulatable CCSP, albeit at
low levels (13). Before these cells were used in
transfection analysis, a survey was conducted to determine the nuclear
trans-acting proteins expressed in these cells; these proteins would bind to the endogenous mCCSP promoter. DNase I footprinting analyses demonstrate three distinct regions, RI, RII, and
RIII, within the proximal CCSP promoter that contain the DNA binding
sequences for the known respiratory epithelial transcription factors,
HNF-3, C/EBP, and TTF-1 (Fig. 2A, Table 1). Western blot analyses were done to
establish whether the mtCC lines contained these endogenous
trans-acting factors expressed in native respiratory
epithelium. mtCC do express the transcription factors HNF-3
,
C/EBP-
, C/EBP-
, C/EBP-
, and TTF-1 (Fig. 2B). mtCC
maintain the expression of these trans-acting factors
through >175 passages, although HNF-3
protein content diminishes
with increasing passage number (Fig. 2B). The passage number
does not appear to influence expression of TTF-1 or the three C/EBP
isoforms. The high level of expression of C/EBP-
and the low level
of expression of C/EBP-
are consistent with previous observations in
bronchiolar epithelium (3). Our observation of C/EBP-
expression in mtCC is the first demonstration of this transcription
factor in bronchiolar epithelium.
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mtCC nuclear proteins interact with the HNF-3 binding site
contained within a region of overlapping cis elements in the CCSP
proximal promoter.
There are two cis elements for each of the
trans-acting factors TTF-1, HNF-3, and C/EBP proteins in the
proximal 144 bp of the CCSP promoter. To determine whether these
proteins were functionally interactive with the cis elements
in the CCSP proximal promoter in mtCC and whether the protein-DNA
interactions were altered by IFN-, EMSAs were performed. The region
of the CCSP promoter investigated was a complex sequence of nucleotides
spanning base pairs
110 to
81, which contains overlapping cis
elements for AP-1, HNF-3, and C/EBP. To determine whether the
potential protein-DNA interactions at this region of the CCSP promoter
were affected by IFN-
, we utilized the double-stranded
oligonucleotides shown in Fig. 2C. The representative EMSA
shown in Fig. 2D demonstrates three specific protein-DNA
complexes (B1, B2, and B3) after incubation of nuclear extracts from
mtCC with the labeled intact AHC probe (lane 2). These three
specific protein-DNA complexes are competed away by the addition of
increasing amounts (10× and 50×) of intact cold AHC probe
(lanes 3 and 4). To identify whether the
trans-acting factors were binding specifically to the AP-1
(22), HNF-3 (22), or C/EBP (4)
binding sites, we generated additional mutations to abolish the
individual binding sites for AP-1, HNF-3, and C/EBP. These mutations
were termed mAHC, AmHC, and AHmC, respectively. Increasing amounts of
the unlabeled mutant oligonucleotides were used in an EMSA competition
assay to interact with the mtCC nuclear extract (lanes
5-10). Increasing amounts of unlabeled AHmC oligonucleotides, which contain a mutation in the C/EBP binding site but retain the
capacity to bind AP-1 and HNF-3, showed competition with bands B2 and
B3 (lanes 5 and 6). This indicates that B1
interacts with the C/EBP binding site. Competition with mAHC, a
mutation in the AP-1 binding site (lanes 7 and
8), demonstrated that there is very little, if any,
difference in competition between the wild-type AHC oligonucleotides
(lane 4) and the mutant mAHC oligonucleotides (lane
8). Thus interaction of the AP-1 site in the AHC probe with mtCC
nuclear extract could not be demonstrated. Competition with unlabeled
AmHC, which contains a mutation in the HNF-3 binding site, showed that
complexes B2 and B3 remained, indicating that these complexes interact
with the HNF-3 binding sites (lanes 9 and 10).
These results demonstrate that mtCC contain multiple nuclear proteins
that bind specifically to the CCSP proximal promoter region spanning
nucleotides
110 to
81 upstream from the start of transcription.
The minimal IFN--responsive region of the CCSP promoter is
localized to the proximal promoter in vitro.
To localize the minimal IFN-
-responsive region of the CCSP promoter,
a variety of CCSP deletion reporter genes were transiently transfected
into mtCC and then treated with vehicle (DMEM without fetal calf serum)
or IFN-
(1,000 IU/ml DMEM) without fetal calf serum. As shown in
Fig. 3A, basal expression of
the CAT reporter gene under the control of 803 bp of the CCSP
5'-flanking region is greater than that for the 166 bp of 5'-flanking
DNA. This demonstrates that, unlike H441 cells, mtCC recognize the
elements in the distal region of the mCCSP promoter. Figure
3A also shows IFN-
responsiveness in the 803-, 166-, and
144-bp CCSP promoter reporter constructs. However, when the CCSP
promoter was deleted to
87 bp, the baseline level of expression of
the CCSP reporter gene was greatly reduced and failed to demonstrate a
response to IFN-
treatment. These results demonstrate that IFN-
can regulate expression of a CAT reporter gene, mediated by 144 bp of
5'-flanking DNA of the CCSP gene.
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IFN- activates the CCSP gene in vivo via the proximal 166-bp
segment of the CCSP promoter.
The ability of 166 bp of the 5'-flanking region of the CCSP promoter to
mediate the effects of IFN-
on CCSP gene expression in vivo was
confirmed using previously generated adult transgenic mice with the
166-bp segment (166CCSP-hGH) of the CCSP promoter ligated to the hGH
coding region (18). Quantification of representative RPA
results are shown in Fig. 3, B and C, which
demonstrates a time-dependent increase in endogenous CCSP and hGH RNA
transcripts in the IFN-
-treated 166CCSP-hGH transgenic mice. These
results demonstrate that the minimal IFN-
-responsive elements are
contained in the proximal 166 bp of the CCSP promoter.
HNF-3 binding to the overlapping cis elements in the CCSP
proximal promoter is increased by IFN-
.
Additional EMSAs and antibody inhibition analyses were performed to
determine whether exposure to IFN-
would increase the protein-DNA
interactions within the proximal
110 to
81 bp of the CCSP promoter
and, if so, to identify the proteins interacting with the specific
binding site. For these analyses, we utilized nuclear extracts from
mtCC treated for 4-24 h with vehicle or IFN-
and a radiolabeled
mAHmC probe spanning base pairs
110 to
81, which contains an intact
HNF-3 binding site but mutant AP-1 and C/EBP binding sites. We observed
a time-dependent increase in protein-DNA interactions at the HNF-3
binding site (Fig. 4A, lanes
2-4). Competition with the cold
probe (lane 5) demonstrates the formation of three specific
protein-DNA complexes, H1, H2, and H3, with H1 and H2 running as a
doublet. Antibodies were added during the incubation of hot probe and
nuclear proteins for the samples in lanes 7-9. The
addition of an antibody specific for HNF-3
(lane 7) shows
some minor inhibition of the upper bands (H1 and H2) in the nuclear
protein-DNA complex. The addition of an antibody for HNF-3
(lane 8) shows a complete inhibition of complex H1 and
partial inhibition of complexes H2 and H3. The addition of a
nonspecific antibody (lane 9) does not show any inhibition
or supershift. These results suggest that HNF-3
is a major component
within the protein-DNA complex binding at the complex overlapping the
HNF-3 site.
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Proximal and distal HNF-3 cis-acting elements are required for CCSP
gene expression.
To determine the contribution of the GAS site in the distal promoter
region and the two HNF-3 binding sites in the proximal promoter to
IFN- induction of CCSP gene expression, transient transfections were
conducted utilizing specific linker-scanning mutations to the GAS,
distal HNF-3, and proximal HNF-3 sites. Figure
5 demonstrates that mutation of the
distal GAS site resulted in an attenuation of the baseline expression
of the CCSP reporter gene but that this construct remained responsive
to IFN-
. However, mutation of the distal or proximal HNF-3
cis-acting element reduced the expression of the CCSP
reporter gene constructs to background levels and further eliminated
the IFN-
responsiveness of the CCSP promoter. These results
demonstrate that the distal and proximal HNF-3 cis elements
in the CCSP promoter are required for CCSP gene expression, as well as
IFN-
responsiveness.
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IFN--stimulated expression of mCCSP expression in vivo is
mediated through IRF-1.
IFN-
signaling is known to involve cytokine binding to the IFN-
receptor on the cell surface, which leads to activation of the Janus
kinases and phosphorylation and homodimerization of STAT1 proteins.
Previously, it was shown that HNF-3
is induced by IFN-
in
hepatocytes through mechanisms involving STAT1 and IRF-1
(19). Therefore, to determine whether the underlying
mechanism for IFN-
responsiveness, localized to the proximal mCCSP
promoter, was mediated through a similar mechanism involving IRF-1, we
analyzed CCSP expression in IRF-1-deficient mice exposed to
intratracheal administration of IFN-
. Figure
6A is a representative RPA
demonstrating the CCSP mRNA expression after intratracheal
administration of saline or IFN-
. We observed no change in CCSP
expression in the IRF-1-deficient mice after administration of IFN-
,
in contrast to the significant increase in CCSP expression in the 129 and FVB wild-type mice. Quantification of CCSP mRNA expression after administration of saline or IFN-
to IRF-1-deficient mice, i.e., 129 and FVB wild-type mice, is shown in Fig. 6B. To further
clarify whether IFN-
responsiveness was mediated through
IRF-1-mediated induction of HNF-3
, Western blot analyses were done
to determine the level of HNF-3
in whole lung homogenates obtained
from the IRF-1
/
and 129 wild-type mice after
administration of 0.1 ml of saline or 0.1 ml of IFN-
(1,000 IU/ml
DMEM). There was no detectable HNF-3
in the whole lung homogenates
from the IRF-1
/
mice with saline or IFN-
administration or from the 129 wild-type mice with saline
administration. However, there was a significant increase in detectable
HNF-3
protein in whole lung homogenates from the 129 wild-type mice
after IFN-
administration (Fig. 6C).
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DISCUSSION |
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The cell-specific expression of respiratory epithelial cell genes
is not regulated by lung-specific transcription factors. This
lung-specific gene regulation is, in fact, conferred by complex interactions of general transcription factors with distinct cis elements in the 5'-flanking regions of the respiratory epithelial cell genes (2, 8, 30). The cell-specific gene expression of the CCSP gene represents an interesting model to decipher the molecular mechanisms involved in cell-specific transcriptional control
of pulmonary genes. However, these analyses have been hampered by the
lack of a faithful cell line to mirror the in vivo Clara cell
environment and function. In this study, we utilized CCSP reporter
genes in transgenic mice and mtCC to elucidate the transcriptional
control of CCSP in response to IFN- stimulation. In addition, we
have further clarified the utility of the mtCC line as a faithful model
for analysis of CCSP expression mediated by the proximal and distal
promoter regions with greater fidelity than present models for CCSP
gene expression.
The importance of elucidating the molecular mechanisms that regulate
IFN- responsiveness of the CCSP gene may exemplify a cell-specific
scheme for the control of host defenses and modulation of pulmonary
inflammation. The observation that CCSP inhibits the production and
biological activity of IFN-
by mononuclear cells further supports
the speculation that IFN-
induction of CCSP expression in vivo may
function to restrict intrapulmonary inflammation (33).
Extensive investigations have failed to clearly characterize the
molecular mechanisms that mediate the increase in CCSP expression
secondary to exposure to IFN-
. The present investigations utilized a
variety of in vitro and in vivo techniques to determine that
IFN-
-induced expression of CCSP is mediated, in part, at the level
of transcription. Our findings extend the findings of Yao et
al. (31), who showed that IFN-
treatment of
human BEAS-2B cells increased CCSP protein production in a dose- and
time-dependent manner. They found the greatest stimulation in CCSP
expression at 1,000 IU/ml IFN-
, the same dose used in this study; at
a lower dose (300 IU/ml), however, these same investigators found that
the increase in CCSP protein expression was mediated by an increase in
CCSP mRNA stability, with the half-life of CCSP mRNA increased from 15 to 40 h. The transcriptional regulation of the CCSP promoter in
mtCC in the present study highlights the utility of this cell line to
respond to stimuli in a manner similar to endogenous Clara cells. This
is the first report of an in vitro tool that mirrors the responsiveness
of the in vivo Clara cells.
In addition, there is the potential that IFN- treatment affects
specific gene expression by multiple mechanisms, resulting in an
alteration in the rate of transcription and/or an alteration in message
stability (24). These results, in combination with those
of Yao et al. (31), would suggest that IFN-
treatment regulates CCSP gene expression through multiple mechanisms,
including an increase in gene transcription, message stability, and
protein production. The use of multiple mechanisms to ensure an
increase in CCSP expression may signify the importance of limiting
intrapulmonary inflammatory events.
Our promoter deletion and linker-scanning analyses demonstrate that the
proximal 144 bp of the CCSP promoter were sufficient to maintain
IFN- responsiveness of the CCSP gene. The mutational analyses were
critical in verifying our conclusions from the promoter deletions, the
results of which may be misleading because of the potential for
disruption of specific protein-protein or protein-DNA interactions of
enhancing elements in the 5'-flanking region of the gene. Further
verification of our results, in addition to recognition of the
physiological importance of the cis elements in vivo, was
obtained from our analyses in the transgenic mouse model. The
consistency in the results from these different models further
strengthens our hypothesis that increased HNF-3
protein-DNA interactions mediate the IFN-
-induced transcriptional increase in
CCSP gene expression. Moreover, the observation that both HNF-3 sites
are critical to the basal and IFN-
-induced CCSP gene expression is
consistent with emerging evidence that combinatorial action of
transcription factors may provide a method of stimulus- and cell-specific gene regulation through complex protein-DNA and protein-protein interactions (7).
The complex regulatory region of the CCSP proximal promoter between
nucleotides 110 and
81, represented by R2 on the DNase footprinting
analysis, includes the overlapping consensus sites for HNF-3 and C/EBP
proteins. We have shown that IFN-
stimulation of mtCC results in an
increase in HNF-3
levels and an increase in functional protein-DNA
interactions at the HNF-3 binding site. We identified HNF-3
as the
major protein contained within two of the protein-DNA complexes with a
minor contribution by HNF-3
. Interestingly, HNF-3 proteins have been
shown to mediate the IFN-
regulation of the transthyretin gene in
the liver (19). In the cytokine regulation of
transcription, similarities can be drawn between the liver-specific
gene and respiratory epithelial-specific genes. Analysis of cytokine
regulation of transcription of transthyretin showed that HNF-3
and C/EBP binding sites are involved in the cytokine regulation of this
gene. Thus the use of these transcription factors to mediate cytokine
regulation of lung and liver genes may be conserved.
C/EBP- and C/EBP-
proteins have been shown to be involved in the
regulation of the CCSP gene through complex protein-DNA interactions at
the overlapping C/EBP binding site in conjunction with a second C/EBP
binding site in close proximity (3). This temporal
arrangement of cis elements in the CCSP proximal promoter for the C/EBP binding sites is strikingly similar to the dual binding
sites for the HNF-3 proteins. Our findings that both HNF-3 binding
sites are critical for CCSP gene regulation in mtCC, in conjunction
with the previous reports of required interactions at both C/EBP
binding sites, suggest that such compound regulatory units may serve as
a more universal molecular mechanism for organ- or cell-specific gene expression.
Our results support the hypothesis that CCSP induction by IFN- is
mediated by the interaction of multiple trans-acting factors interacting with a compound regulatory unit in the proximal promoter. One potential molecular mechanism that illustrates the observed interactions of HNF-3
in the IFN-
-induced expression of CCSP is
shown in Fig. 7. The proposed mechanism
for IFN-
induction of CCSP expression mediated by the proximal mCCSP
promoter was suggested by previous studies, which demonstrated that
IFN-
induced the expression of IRF-1 (15), which then
induced an increase in HNF-3
expression and functional DNA
interactions. Our findings clearly demonstrate that IFN-
responsiveness of mCCSP is lost in the absence of IRF-1 in association
with a lack of induction of HNF-3
.
|
In summary, the present study demonstrates that CCSP gene
regulation by IFN- is, in part, regulated at the level of
transcription. Furthermore, it shows that the IFN-
responsiveness of
the CCSP gene is mediated through a complex regulatory region in the
proximal promoter and that HNF-3
binding to this region is a major
component of the induction of CCSP transcription. This report proposes
a mechanism by which IFN-
mediates an increase in mCCSP expression. Moreover, this report reveals the utility of the mtCC line as a model
for further investigation of the mechanistic features of cell-specific
transcriptional regulation of gene expression in respiratory
epithelium. Although the present study was focused on the determination
of the minimal cis elements responsive to IFN-
regulation
for the CCSP gene, we also noted that the distal CCSP promoter was
important in CCSP gene expression to a level >10 times the expression
mediated by the proximal promoter alone. These findings are important
for future investigations of CCSP gene regulation and the
identification of enhancing elements in this distal regulatory region.
![]() |
ACKNOWLEDGEMENTS |
---|
Dr. R. Costa (Dept. of Molecular Genetics, University of Illinois,
Chicago, IL) generously provided the antibodies for HNF-3 and
HNF-3
as well as the HNF-3 expression vector. J. Wang, M. Gu, and J. DeMayo provided technical assistance. J. Ellsworth aided in preparation
of the manuscript.
![]() |
FOOTNOTES |
---|
* P. L. Ramsay and Z. Luo contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-61406.
Address for reprint requests and other correspondence: F. J. DeMayo, Dept. of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (E-mail: fdemayo{at}bcm.tmc.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.
August 16, 2002;10.1152/ajplung.00186.2002
Received 12 June 2002; accepted in final form 8 August 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bingle, CD,
Hackett BP,
Moxley M,
Longmore W,
and
Gitlin JD.
Role of hepatocyte nuclear factor-3 and hepatocyte nuclear factor-3
in Clara cell secretory protein gene expression in the bronchiolar epithelium.
Biochem J
308:
197-202,
1995[ISI][Medline].
2.
Bohinski, R,
Di Lauro R,
and
Whitsett JA.
The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the forgut axis.
Mol Cell Biol
14:
5671-5681,
1994[Abstract].
3.
Cassel, TN,
Nordlund-Moller L,
Andersson O,
Gustafsson JA,
and
Nord M.
C/EBP and C/EBP
activate the Clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites.
Am J Respir Cell Mol Biol
22:
469-80,
2000
4.
Cassel, TN,
Suske G,
and
Nord M.
C/EBP and TTF-1 synergistically transactivate the Clara cell secretory protein gene.
Ann NY Acad Sci
923:
300-302,
2000
5.
Cowan, MJ,
Huang X,
Yao XL,
and
Shelhamer JH.
Tumor necrosis factor- stimulation of human Clara cell secretory protein production by human airway epithelial cells.
Ann NY Acad Sci
923:
193-201,
2000
6.
Facchiano, A,
Cordella-Miele E,
Miele L,
and
Mukherjee AB.
Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action.
Science
48:
453-464,
1991.
7.
Granger, RL,
Hughes TR,
and
Ramji DP.
Stimulus- and cell-type-specific regulation of CCAAT-enhancer binding protein isoforms in glomerular mesangial cells by lipopolysaccharide and cytokines.
Biochim Biophys Acta
1501:
171-179,
2000[ISI][Medline].
8.
Hackett, BP,
Bingle CD,
and
Gitlin JD.
Mechanisms of gene expression and cell fate determination in the developing pulmonary epithelium.
Annu Rev Physiol
58:
51-71,
1996[ISI][Medline].
9.
Hayashida, S,
Harrod KS,
and
Whitsett JA.
Regulation and function of CCSP during pulmonary Pseudomonas aeruginosa infection in vivo.
Am J Physiol Lung Cell Mol Physiol
279:
L452-L459,
2000
10.
Ikeda, K,
Shaw-White JR,
Wert SE,
and
Whitsett JA.
Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells.
Mol Cell Biol
16:
3626-3636,
1996[Abstract].
11.
Johnston, CJ,
Mango GW,
Finkelstein JN,
and
Stripp BR.
Altered pulmonary response to hyperoxia in Clara cell secretory protein-deficient mice.
Am J Respir Cell Mol Biol
17:
147-155,
1997
12.
Kunkel, TA,
Roberts JD,
and
Zakour RA.
Rapid and efficient site-specific mutagenesis without phenotypic selection.
Methods Enzymol
154:
367-382,
1987[ISI][Medline].
13.
Magdaleno, SM,
Wang G,
Jackson KJ,
Ray MK,
Welty S,
Costa RH,
and
DeMayo FJ.
Interferon- regulation of Clara cell gene expression: in vivo and in vitro.
Am J Physiol Lung Cell Mol Physiol
272:
L1142-L1151,
1997
14.
Mango, GW,
Johnston CJ,
Reynolds SD,
Finkelstein JN,
Plopper CG,
and
Stripp BR.
Clara cell secretory protein deficiency increases oxidant stress response in conducting airways.
Am J Physiol Lung Cell Mol Physiol
275:
L348-L356,
1998
15.
Mori, K,
Stone S,
Khaodhiar L,
Braverman LE,
and
DeVito WJ.
Induction of transcription factor interferon regulatory factor-1 by interferon- (INF
) and tumor necrosis factor-
(TNF
) in FRTL-5 cells.
J Cell Biochem
74:
211-219,
1999[ISI][Medline].
16.
Nord, M,
Cassel TN,
Braun H,
and
Suske G.
Regulation of the Clara cell secretory protein/uteroglobin promoter in lung.
Ann NY Acad Sci
923:
154-165,
2000
17.
Ray, MK,
Chen CY,
Schwartz RJ,
and
DeMayo FJ.
Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family members thyroid transcription factor 1 and cardiac muscle-specific homeobox protein (CSX).
Mol Cell Biol
16:
2056-2064,
1996[Abstract].
18.
Ray, MK,
Magdaleno SW,
Finegold MJ,
and
DeMayo FJ.
Cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis.
J Biol Chem
270:
2689-2694,
1995
19.
Samadani, U,
Porcella A,
Pani L,
Johnson PF,
Burch JB,
Pine R,
and
Costa RH.
Cytokine regulation of the liver transcription factor hepatocyte nuclear factor-3 is mediated by the C/EBP family and interferon regulatory factor 1.
Cell Growth Differ
6:
879-890,
1995[Abstract].
20.
Samadani, U,
Qian X,
and
Costa RH.
Identification of a transthyretin enhancer site that selectively binds the hepatocyte nuclear factor-3 isoform.
Gene Expr
6:
23-33,
1996[ISI][Medline].
21.
Sambrook, J,
Fritsch E,
and
Maniatis T.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press, 1989.
22.
Sawaya, PL,
Stripp BR,
Whitsett JA,
and
Luse DS.
The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families.
Mol Cell Biol
13:
3860-3871,
1993[Abstract].
23.
Seed, B,
and
Sheen JY.
A simple phase-extraction assay for chloramphenicol acyltransferase activity.
Gene
67:
271-277,
1988[ISI][Medline].
24.
Sen, GC,
and
Lengyel P.
The interferon system. A bird's eye view of its biochemistry.
J Biol Chem
267:
5017-5020,
1992
25.
Singh, G,
Singh J,
Katyal SL,
Brown WE,
Kramps JA,
Paradis IL,
Dauber JH,
MacPherson TA,
and
Squeglia N.
Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 kD protein.
J Histochem Cytochem
36:
73-80,
1988[Abstract].
26.
Stripp, BR,
Huffman JA,
and
Bohinski RJ.
Structure and regulation of the murine Clara cell secretory protein gene.
Genomics
20:
27-35,
1994[ISI][Medline].
27.
Stripp, BR,
Sawaya PL,
Luse DS,
Wikenheiser KA,
Wert SE,
Huffman JA,
Lattier DL,
Singh G,
Katyal SL,
and
Whitsett JA.
Cis-acting elements that confer lung epithelial cell expression of the CC10 gene.
J Biol Chem
267:
14703-14712,
1992
28.
Toonen, RF,
Gowan S,
and
Bingle CD.
The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene.
Biochem J
316:
467-473,
1996[ISI][Medline].
29.
White, LC,
Wright KL,
Felix NJ,
Ruffner H,
Reis LF,
Pine R,
and
Ting JP.
Regulation of LMP2 and TAP1 genes by IRF-1 explains the paucity of CD8+ T cells in IRF-1/
mice.
Immunity
5:
365-376,
1996[ISI][Medline].
30.
Whitsett, JA.
A lungful of transcription factors.
Nat Genet
20:
7-8,
1998[ISI][Medline].
31.
Yao, XL,
Ikezono T,
Cowan M,
Logun C,
Angus CW,
and
Shelhamer JH.
Interferon- stimulates human Clara cell secretory protein production by human airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
274:
L864-L869,
1998
32.
Yao, XL,
Levine SJ,
Cowan MJ,
Logun C,
and
Shelhamer JH.
Tumor necrosis factor- stimulates human Clara cell secretory protein production by human airway epithelial cells.
Am J Respir Cell Mol Biol
19:
629-635,
1998
33.
Zhang, L,
Whitsett JA,
and
Stripp BR.
Regulation of Clara cell secretory protein gene transcription by thyroid transcription factor-1.
Biochim Biophys Acta
1350:
359-367,
1997[ISI][Medline].