Transcriptional regulation of CCSP by interferon-gamma in vitro and in vivo

P. L. Ramsay1,6,*, Z. Luo6,*, S. M. Magdaleno5, S. K. Whitbourne1, X. Cao1, M. S. Park4, S. E. Welty3, L.-Y. Yu-Lee2, and F. J. DeMayo1,6

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


    ABSTRACT
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INTRODUCTION
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Interferon gamma  (IFN-gamma ), 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-gamma concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-gamma 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-gamma stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-gamma -induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-gamma -responsive segment was localized to the proximal 166 bp of the 5'-flanking region. Additionally, IFN-gamma -induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3beta .

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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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-gamma (IFN-gamma ) (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-gamma stimulation may help define the intrinsic mechanisms for modulating pulmonary inflammation in the lung.

The molecular events that regulate IFN-gamma -induced expression of CCSP have not been fully defined. Previous analyses demonstrate that IFN-gamma -induced expressions of CCSP are mediated in part by posttranscriptional mechanisms (31). However, the potential for transcriptional regulation of CCSP by IFN-gamma 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 gamma -interferon activation site (GAS) are localized to this promoter region (13, 17).


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Fig. 1.   Schematic of known cis elements in the Clara cell secretory protein (CCSP) gene promoter localized to distal and proximal segments. The 5'-upstream region of the CCSP promoter includes a proximal segment spanning base pairs +1 to -166 and a distal segment spanning base pairs -166 to -803. The known cis elements for transcription factors previously reported for CCSP regulation in the proximal region include thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3, (HNF-3), activator protein 1 (AP-1), CCAAT enhancer binding protein (C/EBP) binding sites, and gamma -interferon activation site (GAS).

Although the molecular events that regulate IFN-gamma -induced expression of CCSP have not been fully defined, it is known that IFN-gamma 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-gamma 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-gamma could directly effect a change in CCSP gene transcription (13). Alternatively, IFN-gamma 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-gamma -induced expression of the CCSP gene is regulated, at least partially, at the level of transcription. Furthermore, we demonstrate that the IFN-gamma 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-gamma responsiveness of the CCSP gene is mediated through a complex regulatory region in the proximal promoter region and that HNF-3beta binding is a major component of this induction.


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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-3beta (Santa Cruz Biotechnology), C/EBPalpha (Affinity Bioreagents and Santa Cruz Biotechnology), C/EBPbeta (Affinity Bioreagents and Santa Cruz Biotechnology), or C/EBPdelta (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)-beta -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-gamma (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.

beta -Galactosidase assay. As an internal control, all CAT reporters were cotransfected with 1 µg of CMV-beta -galactosidase plasmid, consisting of the CMV promoter driving the expression of the beta -galactosidase gene. The beta -galactosidase activity in each transfection was used to control for variability between transfections. Quantitation of beta -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 beta -mercaptoethanol), 66 µl of 1× ONPG (4 mg/ml of o-nitrophenyl-beta -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-gamma on CCSP gene expression (18). Adult transgenic mice were treated with 0.1 ml of saline or 0.1 ml of IFN-gamma (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-gamma -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-gamma 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-gamma (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-gamma 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-gamma 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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Fig. 2.   Identification of trans-acting elements in mouse transformed Clara cells (mtCC). A: DNase I footprinting of the mouse CCSP (mCCSP) proximal promoter with extracts from mtCC. B: Western blot analyses of whole cell extracts from high- and low-passage mtCC and CV-1 cells utilizing antibodies for TTF-1, HNF-3beta , C/EBP-alpha , C/EBP-beta , and C/EBP-delta proteins. C and D: electrophoretic mobility shift analyses of nuclear protein-DNA interactions in mtCC. C: probe used for mobility shift analyses contained DNA sequence spanning base pairs -110 to -81 of the CCSP promoter, denoted AHC. Bracketed regions represent consensus binding sites for AP-1, HNF-3, and C/EBP. Specific oligonucleotides utilized for localization of key protein-DNA interactions in the proximal CCSP promoter are shown. Mutated nucleotides are shown in lower case. AHC, wild type; mAHC, mutant AP-1 site; AmHC, mutant HNF-3 site; AHmC, mutant C/EBP site; mAmHC, mutant AP-1 and HNF-3 sites; AmHmC, mutant HNF-3 and C/EBP sites; mAHmC, mutant AP-1 and C/EBP sites; mAmHmC, mutant AP-1, HNF-3, and C/EBP sites. D: electrophoretic mobility shift assay (EMSA) of mtCC nuclear extract with 32P-labeled AHC oligonucleotides (lane 2) competed with itself, AHC (lanes 3 and 4) , AHmC (lanes 5 and 6), mAHC (lanes 7 and 8), and AHmC (lanes 9 and 10).


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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-gamma -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-3beta , C/EBP-alpha , C/EBP-beta , C/EBP-delta , and TTF-1 (Fig. 2B). mtCC maintain the expression of these trans-acting factors through >175 passages, although HNF-3beta 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-delta and the low level of expression of C/EBP-alpha are consistent with previous observations in bronchiolar epithelium (3). Our observation of C/EBP-beta expression in mtCC is the first demonstration of this transcription factor in bronchiolar epithelium.

                              
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Table 1.   Known binding sequences protected in DNase I footprinting analysis

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-gamma , 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-gamma , 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-gamma -responsive region of the CCSP promoter is localized to the proximal promoter in vitro. To localize the minimal IFN-gamma -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-gamma (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-gamma 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-gamma treatment. These results demonstrate that IFN-gamma can regulate expression of a CAT reporter gene, mediated by 144 bp of 5'-flanking DNA of the CCSP gene.


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Fig. 3.   Localization of interferon-gamma (IFN-gamma ) responsiveness to the proximal CCSP promoter in vitro and in vivo. A: transfection analysis of mtCC with deletion constructions with 803, 166, 144, 87, and 23 bp of the CCSP 5'-flanking DNA fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Cells were treated with vehicle or IFN-gamma (1,000 IU/ml DMEM). Transfection efficiency was normalized by cotransfection with cytomegalovirus (CMV)-beta -galactosidase (beta -gal). Values (means ± SE for each construct) are representative of several independent transfections and are normalized for beta -galactosidase activity. *P < 0.05. B and C: quantification of RNase protection assay (RPA) of in vivo expression of endogenous CCSP and human growth hormone (hGH) reporter gene activity under direction of the CCSP promoter in transgenic mice after 0, 24, or 48 h of intratracheal instillation of 0.1 ml of IFN-gamma (1,000 IU/ml DMEM). Values (means ± SD) are normalized to cyclophilin for each exposure group. *P < 0.05.

IFN-gamma 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-gamma 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-gamma -treated 166CCSP-hGH transgenic mice. These results demonstrate that the minimal IFN-gamma -responsive elements are contained in the proximal 166 bp of the CCSP promoter.

HNF-3beta binding to the overlapping cis elements in the CCSP proximal promoter is increased by IFN-gamma . Additional EMSAs and antibody inhibition analyses were performed to determine whether exposure to IFN-gamma 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-gamma 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-3alpha (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-3beta (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-3beta is a major component within the protein-DNA complex binding at the complex overlapping the HNF-3 site.


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Fig. 4.   Identification of HNF-3beta interactions in response to IFN-gamma in mtCC. A: EMSA of mtCC nuclear extract of cells exposed for 0, 4, and 24 h to IFN-gamma (1,000 IU/ml DMEM) and probed with 32P-labeled mAHmC oligonucleotide (lanes 2-4) in the presence of 10-fold excess of specific unlabeled mAHmC competitor (lane 5), 10-fold excess of nonspecific competitor (lane 6), antibodies specific for HNF-3alpha (lane 7) and HNF-3beta (lane 8), or nonspecific antibody (lane 9). B: Western blot analysis of whole cell extracts from mtCC after exposure to vehicle or IFN-gamma (1,000 IU/ml DMEM) for 24 h utilizing an antibody for HNF-3beta . C: transient transfection analysis of 166CCSP-Luc construct of mtCC treated with vehicle or IFN-gamma (1,000 IU/ml DMEM) of cotransfected CMV-HNF-3beta . Luciferase activity was normalized by cotransfection with Renilla luciferase under control of the TK promoter. *P < 0.05 vs. vehicle.

Previously, it was shown that HNF-3beta mRNA in mtCC is increased by exposure to IFN-gamma (13). Therefore, we performed Western blot analyses to determine whether the protein content of HNF-3beta was increased as well. The representative Western blot shown in Fig. 4B demonstrates a significant increase in nuclear protein expression of HNF-3beta . To determine whether HNF-3beta was sufficient to mediate an increase in CCSP gene activity, we performed transient cotransfections with HNF-3beta and the 166-bp CCSP reporter gene (Fig. 4C). HNF-3beta expression resulted in a significant increase in CCSP reporter gene expression. Additional transient cotransfections with HNF-3beta and the 166-bp CCSP reporter gene plus IFN-gamma treatment did not show any additional activation above the level of expression induced by HNF-3beta alone (data not shown). These findings clearly demonstrate that HNF-3beta can mediate an induction in the expression of CCSP in mtCC, that HNF-3beta protein content is increased by IFN-gamma , and that HNF-3beta interactions with the CCSP proximal promoter increase with IFN-gamma exposure.

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-gamma 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-gamma . 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-gamma 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-gamma responsiveness.


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Fig. 5.   Intact HNF-3beta cis elements are mandatory for IFN-gamma -induced expression of the CCSP gene. Wild-type (WT) 803 bp of the CCSP promoter as well as linker-scanning mutations of the 803-bp promoter with mutations in the GAS (mGAS), distal HNF-3 (mdH) binding site, and proximal HNF-3 (mpH) binding site fused to the CAT reporter gene were transiently cotransfected with an internal control, CMV-beta -gal, into mtCC. At 24 h after transient transfection, mtCC were treated with vehicle or IFN-gamma (1,000 IU/ml DMEM) in the absence of fetal calf serum. Data represent independent transfections, which are normalized for beta -galactosidase activity, and have been repeated >3 times with similar results. Values are means ± SD for each construct. *P < 0.05.

IFN-gamma -stimulated expression of mCCSP expression in vivo is mediated through IRF-1. IFN-gamma signaling is known to involve cytokine binding to the IFN-gamma 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-3beta is induced by IFN-gamma in hepatocytes through mechanisms involving STAT1 and IRF-1 (19). Therefore, to determine whether the underlying mechanism for IFN-gamma 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-gamma . Figure 6A is a representative RPA demonstrating the CCSP mRNA expression after intratracheal administration of saline or IFN-gamma . We observed no change in CCSP expression in the IRF-1-deficient mice after administration of IFN-gamma , 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-gamma to IRF-1-deficient mice, i.e., 129 and FVB wild-type mice, is shown in Fig. 6B. To further clarify whether IFN-gamma responsiveness was mediated through IRF-1-mediated induction of HNF-3beta , Western blot analyses were done to determine the level of HNF-3beta 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-gamma (1,000 IU/ml DMEM). There was no detectable HNF-3beta in the whole lung homogenates from the IRF-1-/- mice with saline or IFN-gamma administration or from the 129 wild-type mice with saline administration. However, there was a significant increase in detectable HNF-3beta protein in whole lung homogenates from the 129 wild-type mice after IFN-gamma administration (Fig. 6C).


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Fig. 6.   IFN-gamma induction of mCCSP expression is mediated through the interferon regulatory factor (IRF-1) pathway. A: representative RPA of CCSP mRNA in IRF-1-deficient, 129 wild-type, and FVB wild-type mice (4 animals per group) treated intratracheally with saline or 0.1 ml of IFN-gamma (1,000 IU/ml DMEM). B: quantification of relative mCCSP mRNA from mouse lungs in A. Values are means ± SD for each mouse strain and/or genotype normalized to cyclophilin. *P < 0.05. C: representative Western blot analysis for HNF-3beta protein in whole lung extracts from IRF-1-deficient and 129 wild-type mice treated intratracheally with 0.1 ml of saline or 0.1 ml of IFN-gamma (1,000 IU/ml DMEM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-gamma 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-gamma by mononuclear cells further supports the speculation that IFN-gamma 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-gamma . The present investigations utilized a variety of in vitro and in vivo techniques to determine that IFN-gamma -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-gamma 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-gamma , 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-gamma 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-gamma 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-gamma 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-3beta protein-DNA interactions mediate the IFN-gamma -induced transcriptional increase in CCSP gene expression. Moreover, the observation that both HNF-3 sites are critical to the basal and IFN-gamma -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-gamma stimulation of mtCC results in an increase in HNF-3beta levels and an increase in functional protein-DNA interactions at the HNF-3 binding site. We identified HNF-3beta as the major protein contained within two of the protein-DNA complexes with a minor contribution by HNF-3alpha . Interestingly, HNF-3 proteins have been shown to mediate the IFN-gamma 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-3beta 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-alpha and C/EBP-delta 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-gamma 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-3beta in the IFN-gamma -induced expression of CCSP is shown in Fig. 7. The proposed mechanism for IFN-gamma induction of CCSP expression mediated by the proximal mCCSP promoter was suggested by previous studies, which demonstrated that IFN-gamma induced the expression of IRF-1 (15), which then induced an increase in HNF-3beta expression and functional DNA interactions. Our findings clearly demonstrate that IFN-gamma responsiveness of mCCSP is lost in the absence of IRF-1 in association with a lack of induction of HNF-3beta .


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Fig. 7.   Model of IFN-gamma induction of CCSP expression in mtCC. Binding of IFN-gamma to its cell surface receptor leads to phosphorylation of signal transducer and activator of transcription (STAT1), which forms a homodimeric complex and moves into the nucleus. STAT1 dimer can activate transcription of IRF-1. Transcription factor, IRF-1, then activates transcription of HNF-3beta , which in turn activates induction of the CCSP gene through increased HNF-3beta binding with the proximal CCSP promoter.

In summary, the present study demonstrates that CCSP gene regulation by IFN-gamma is, in part, regulated at the level of transcription. Furthermore, it shows that the IFN-gamma responsiveness of the CCSP gene is mediated through a complex regulatory region in the proximal promoter and that HNF-3beta binding to this region is a major component of the induction of CCSP transcription. This report proposes a mechanism by which IFN-gamma 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-gamma 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-3beta and HNF-3alpha 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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