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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 273-285, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0014OC

Regulation of Human Clara Cell 10 kD Protein Expression by Chicken Ovalbumin Upstream Promoter Transcription Factors (COUP-TFs)

Roya Navab, Yimin Wang, Yu-Hua Chow, Anan Wang, Robert P. Jankov, Norio Takamoto, Sophia Y. Tsai, Ming-Jer Tsai, A. Keith Tanswell and Jim Hu

Lung Biology Research Programme and Canadian Institutes of Health Research Group in Lung Development, Hospital for Sick Children; Departments of Laboratory Medicine and Pathobiology, Paediatrics and Physiology, The University of Toronto, Toronto, Ontario, Canada; and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas

Address correspondence to: Jim Hu, Lung Biology Research Programme, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: jhu{at}sickkids.on.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Clara cell 10 kD protein (CC10) is expressed specifically in a portion of nonciliated airway epithelial cells. The molecular mechanisms that determine its high specificity are not clear. Transcription factors implicated in the regulation of CC10 in rodents do not show the same level of cell specificity. We report here that a 3.3 kb human CC10 DNA fragment, containing the 5' flanking region and promoter, directs lacZ reporter expression in a small portion of Clara cells of the airway epithelia of transgenic mice, indicating the requirement of additional regulatory elements for expression. Addition of an intron containing a transcription enhancer from the human cytokeratin 18 gene greatly enhances the level of transgene expression and broadens epithelial specificity. To gain insight into the mechanisms underlying the cell specificity of human CC10 expression, we performed a promoter analysis of the CC10 gene and a yeast one-hybrid screening to identify factors that regulate the promoter. We have found that chicken ovalbumin upstream promoter transcription factors (COUP-TFs) interact with a proximal promoter region and confirmed the interaction by gel-shift assays. Cotransfection analyses with reporter constructs in cultured cells indicated that COUP-TFs inhibit human CC10 expression. These experiments suggest that COUP-TFs may play a pivotal role in cell specificity of the human CC10 gene by inhibiting its expression in nonpermissive cells.

Abbreviations: activation protein-1, AP-1 • 3-amino-1,2,4-triazole, 3-AT • chloramphenical acetyltransferase, CAT • Clara cell secretory protein, CCSP • Clara cell 10 kD protein, CC10 • chicken ovalbumin upstream promoter transcription factors, COUP-TF • Forkhead box A, FoxA • Octamer, Oct • ovalbumin, OVA • secreted alkaline phosphatase, SEAP • Tris-boric acid-EDTA buffer, TBE • thyroid transcription factor, TTF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Clara cells are nonciliated, non–mucus secreting epithelial cells in lung airways. They are believed to be important in metabolism of xenobiotics and regeneration of the airway epithelium (1, 2). Their major secretory product is called Clara cell 10 kD protein (CC10), which is also referred to as uteroglobin, Clara cell secretory protein (CCSP), Clara cell 16 kD protein, human protein 1, urine protein 1, and polychlorinated biphenyl-binding protein (3). In humans, the CC10 gene product is actually 8.5 kD in size (4, 5). CC10 protein forms a dimeric structure linked by disulfide bonds in an antiparallel fashion (4). A property of CC10 is the binding of polychlorinated biphenyls, which can be explained by the presence of a hydrophobic pocket in the dimer of the protein as revealed by X-ray crystallography (4). Although the physiologic role of CC10 is unclear, several lines of evidence suggest that it is involved in regulation of lung inflammation. First, CC10 inhibits phospholipase A2, an enzyme that catalyzes the rate-limiting step in the production of arachidonic acid, the substrate for prostaglandin and leukotriene synthesis, both of which are mediators of inflammation (2, 6). Second, CC10, in combination with transglutaminase, can suppress the antigenicity of foreign proteins (7, 8). In addition, expression of CC10 in rats and rabbits is increased in the lung after administration of corticosteroids (9, 10). Furthermore, the human CC10 gene has been mapped to a chromosomal location, 11p12-q13, linked to atopy (5), an allergic state that is frequently found with asthma. Finally, CC10 knockout mice have an increased susceptibility to hyperoxic injury with an exaggerated inflammatory response (11) and a significantly higher level of pulmonary eosinophilia when challenged with ovalbumin (OVA) (12).

Several groups have reported findings on cis-elements and trans-acting factors involved in regulation of CC10 expression in rat, mouse, and rabbit (3, 5, 13). Stripp and colleagues (14) showed that a 2.3 kb DNA fragment containing the rat CC10 promoter plus the upstream sequence confers cell-specific expression of a reporter gene, chloramphenical acetyltransferase (CAT) in the lungs of transgenic mice, indicating that the DNA regulatory elements required to express CC10 in Clara cells reside in this DNA fragment. In addition, they observed positive as well as negative regulatory elements in the upstream portion of the promoter, and the promoter sequence between -175 and +49 is sufficient to drive reporter gene expression in a Clara cell–like human lung adenocarcinoma cell line, H441 (14). In agreement with the results in rats, a 166 bp fragment of the 5' flanking region of the mouse CC10 gene has been shown to confer cell-specific expression; however, further upstream sequences are needed to achieve higher levels of expression (15). This is consistent with the observation that an expression construct containing an 800 bp promoter fragment of the mouse CC10 gene yielded a higher level of reporter gene expression compared with those containing a shorter fragment from the 5' flanking region (16).

Several transcription factors have been shown to play a role in CC10 transcription. Forkhead box A (FoxA, also called hepatocyte nuclear factor 3) (17, 18), activation protein-1 (AP-1), and the transcription factor Octamer (Oct) (19), were shown to bind to two stretches of sequences, designated regions I and II, immediately upstream of the TATA box of the rat CC10 promoter (17–20). Ray and colleagues demonstrated in both cell lines and transgenic mice that TTF1 (thyroid transcription factor 1 or NKX 2.1) also plays a role in expression of the mouse CC10 gene (16). They identified eight sites within the 800 bp promoter region of the mouse CC10 gene interacting with purified TTF1, and these sites were protected in DNase I footprinting analysis, indicating that TTF1 activates transcription of the mouse CC10 gene directly through its binding to the 5' flanking region. Elements further upstream of the CC10 promoter may also play a role in upregulation of CC10 expression through interactions with other factors. Much of the work on rodent CC10 expression has focused on positive regulation. Little is known about how CC10 expression is repressed in other cells that also express the aforementioned factors such as hepatocyte nuclear factor 3, AP-1, and Oct. It is likely that negative regulatory elements are involved in repressing nonspecific expression of CC10 in other tissues or cells.

DNA control elements of the human CC10 gene as well as the regulatory factors are less well studied. The human CC10 mRNA expression in freshly isolated lung epithelial cells has been shown to occur (5) in Clara cells as well as in non-Clara cells. The expression of human CC10 in other types of nonciliated epithelial cells other than Clara cells (5, 21) has been documented, indicating a distinction of the human CC10 gene from the rodent CC10 gene. In this article, we report the results of reporter gene expression driven by human CC10 DNA regulatory elements in cell lines as well as in transgenic mice. We also report the identification of chicken OVA upstream promoter transcription factors, COUP-TFI and COUP-TFII, as putative regulators of the human CC10 gene. COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily (22). They have been shown to regulate negatively the activation role of vitamin D, thyroid hormone, retinoic acid, retinoid X, and the peroxisome proliferator-activator receptors (22). COUP-TF genes have been identified in many species and they are highly conserved through evolution. COUP-TFII expression is detected in the mesenchyme but not in the terminally differentiated epithelium (23). Our results suggest that COUP-TFs act as repressors for inhibition of CC10 expression in nonpermissive cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid Construction
A 3.3 kb DNA sequence corresponding to the promoter and the upstream region of the human CC10 gene was isolated by polymerase chain reaction-cloning using human genomic DNA as templates. To facilitate the cloning process, restriction sites Kpn I and Mlu I were included in the 5' (CAG GTA CCA TTG GCC ACA GCT TTC TT) and 3' (CAA CGC GTG AGA CAT GCC CAG CAA GG) primers, respectively. The amplified DNA fragment was then cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and pSEAP (secreted alkaline phosphatase) II, which contains a reporter gene encoding secreted placental alkaline phosphatase (ClonTech, Palo Alto, CA). The resultant plasmids, pCR2.1CC10 and pCC10SEAP II, were confirmed by restriction mapping and DNA sequencing. A series of SEAP II CC10 deletion reporter constructs (BglIIMluI CC10SEAPII,EcoRVMluICC10SEAPII, SmaIMluICC10SEAPII, EcoRIMluICC10SEAPII,MunIMluICC10SEAPII, SacIMluICC10 SEAPII) was built by taking DNA fragments with various lengths from the 3.3 kb human CC10 5' flanking sequence and inserting them between the Kpn I (polished) and Mlu I sites of pSEAP II.

Cell Culture and Reporter Gene Assays
A549 cells were maintained as recommended by the American Type Culture Collection. For transient transfection assays, cells were grown to 60–80% confluency and transfected with a mixture of 1 µg DNA:12 µg Lipofectamine (GIBCO BRL, Burlington, ON, Canada) at a density of 2.5 x 105 per 35-mm dish as recommended by the manufacturer. Quantification of SEAP activity was performed using the Phospha-Light chemiluminescent system (Tropix, Bedford, MA). Chemiluminescence in a solution equivalent to 6.25 µL of the conditioned media was measured on a microplate luminometer (E.G. & G. Berthold LB96V, Bad Wildbad, Germany). ß-galactosidase activity was measured in cell lysates using a chemiluminescent assay; light emission was measured with a luminometer after the addition of an accelerator which terminates the ß-galactosidase activity and enhances light emission. For cotransfection assays using the eukaryotic expression vectors mCOUP-TFI and mCOUP-TFII, the cells were transfected with a mixture of 0.25 µg COUP-TFs DNA, 0.75 µg deletion constructs DNA, and 12 µg lipofectamine. As a control in cotransfection studies, we used pcDNA3, which carries the cytomegalovirus promoter as do the eukaryotic expression vectors for COUP-TFs.

Generation of Transgenic Mice and Detection of LacZ Gene Expression
To examine the gene expression activity of the 3.3 kb human CC10 5' flanking region/promoter sequence in vivo, a reporter expression plasmid, pCC10lacZ, was constructed by replacing the SEAP coding sequence of pCC10SEAP II with a Bgl II–Sal I fragment containing the lacZ coding region, from pK18 mTElacZ (24). A 7.6 kb DNA fragment containing the lacZ reporter gene driven by the CC10 promoter and upstream regulatory region was isolated from pCC10lacZ and injected into pronuclei of SJL/B6 mouse fertilized eggs. To test the effect of a heterologous intronic enhancer from the human K18 gene on lacZ reporter expression under the control of the CC10 regulatory elements, pCC10K18iTElacZ was constructed by replacing SEAP coding sequence of pCC10SEAP with an Mlu I–Sal I fragment containing the K18 intron 1 and lacZ coding sequence from pK18 mTElacZ. For generating CC10K18 mlacZ transgenic mice, a 8.3 kb DNA fragment, containing the lacZ reporter gene under the control of the CC10 promoter/upstream regulatory region and the K18 intron 1, was isolated from pCC10K18iTElacZ and injected into pronuclei of SJL/B6 mouse fertilized eggs. Embryos at the two-cell stage were implanted into pseudopregnant CD1 surrogate mothers by standard procedures. The mice were genotyped by PCR analysis of the tail DNAs 2 wk after birth and reporter gene expression in lung was assessed by X-gal staining at various stages of development.

Analysis of COUP-TFII Expression in Murine Tracheal Epithelial Cells
Nuclear-lacZ knock-in mice of COUP-TFII were generated by homologous recombination, and lacZ expression was analyzed to recapitulate the endogenous COUP-TFII expression pattern. Mouse embryos heterozygous for COUP-TFII–lacZ knock-in were dissected at E10.5 or E12.5 and fixed in 2% paraformaldehyde for 30 min at room temperature. After washing in phosphate-buffered saline, embryos were cryopreserved in 30% sucrose, then embedded in OCT compound (Sakura Finetek USA, Torrence, CA). Cryostat sections (20 µm) were incubated with staining solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal. Sections were counterstained with Eosin Y (Sigma, St. Louis, MO).

Yeast One-Hybrid System
The yeast one-hybrid system is a genetic selection method for isolating genes encoding transcription regulatory factors that bind to a target cis-element (25, 26). The one-hybrid assay is based on the finding that many eukaryotic transcriptional activators are composed of physically and functionally independent DNA-binding domains and activation domains. This allows the construction of various gene fusions that, when expressed as fusion proteins in yeast, can simultaneously bind to a target sequence and activate transcription. Theoretically, in the one-hybrid assay, any target element can be used to trap a protein with a binding domain specific for that element.

We cloned an Sma I–Eco RI (337 bp) DNA fragment of the human CC10 gene 5' region into placZ and pHISi (both from ClonTech) to generate reporter constructs placZCC10SE and pHISiCC10SE, respectively. A double reporter yeast strain was constructed by sequential integration of placZCC10SE linearized with Nco I and pHISiCC10SE linearized with Xho I into the genome of YM4271 (ClonTech). Because of the integration events, the double reporter strain becomes Ura+ and is able to produce a minimal level of histidine for survival on plates with synthetic media lacking histidine. However, addition of 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of the HIS3 gene product (His3p), at 45 mM is sufficient to inhibit cell growth, and this very property is used to screen for fusion proteins that can bind to the CC10 sequence to enhance the HIS-3 expression to allow cell survival in the presence of 3-AT.

For isolation of factors in human lung cells that interact with the CC10 target sequence, plasmid DNA was prepared from a Matchmaker human lung cDNA library (ClonTech) and introduced into the double reporter yeast strain. The transformants were selected on plates with a synthetic medium containing 45 mM 3-AT free of histidine and leucine (plasmids in the cDNA library contain the Leu-2 gene so that the transformants do not require leucine in the media). A very small portion of transformed cells were plated on plates containing the same selective medium, but without 3-AT, to estimate the total number of transformed cells. The colonies selected were further tested for activation of lacZ reporter expression by the filter assay as described in the Yeast Protocols Handbook provided by ClonTech. Fresh yeast colonies were lifted to 125-mm filters (#28321–113; VWR, Mississauga, ON, Canada). To permeabilize the cells, the filters were completely submerged (colonies facing up) in a pool of liquid nitrogen for 10 s and then allowed to thaw at room temperature. Each of the filters was then placed, colony side up, on top of another filter presoaked in Z buffer/X-gal solution (60 mM Na2PO4, 40 mM Na2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0, 50 mM ß-mercaptoethanol, 1 mg/ml X-gal). Blue colonies were evident within 15 min of incubating the filters at room temperature.

Gel-Shift Assays
mCOUP-TFI and mCOUP-TFII proteins were produced from pCR3.1 mCOUPTF-I and pCR3.1 mCOUPTF-II, respectively, using T7 RNA polymerase and the TnT reticulocyte lysate system (Promega, Madison, WI). The proteins produced were confirmed by immunostaining with antibodies against COUP TF-I or COUP TF-II (Santa Cruz Biotechnology, Santa Cruz, CA). As a probe, oligonucleotides corresponding to the sequences 5'-GTC AAG GCC ATT GGG AGG TCA AGT GAG-3' and 5'-CTC ACT TGA CCT CCC AAT GG-3' containing a putative binding site for COUP-TFs were hybridized using 1 µg of each pair of oligonucleotides. The complementary oligonucleotides were mixed, heated to 90°C for 5 min and then cooled to room temperature, and the DNA was subsequently labeled with [{alpha}-32P] dCTP using the large Klenow fragment DNA polymerase. The gel-shift assays were performed by incubating 10 ng of a labeled probe in a binding buffer containing 5% glycerol, 10 mM Hepes, 100 mM KCl and 1 µg poly (dI.dC) (Sigma), with proteins translated in the in vitro rabbit reticulocyte lysate system. For competition, 100 ng and 300 ng of the same unlabeled probe was used. For supershift, affinity-purified goat polyclonal antibodies against COUP-TFI and ARP-1/COUP-TFII (Santa Cruz Biotechnology) were added to the binding reaction 30 min before addition of the probe whenever indicated. The protein–DNA complexes formed during the band shift reactions were separated on precooled, preelectrophoresed (2 h, 200 V) 5% polyacrylamide gels with 0.5 x Tris-boric acid-EDTA buffer (TBE) (1 x TBE: 90 mM Tris borate and 1 mM ethylenediamine-tetraacetic acid) as the running buffer at 4°C at 200 V. In addition to the double-stranded oligonucleotide as a probe, a 337 bp Sma I-Eco RI fragment, containing nucleotides 335 to 672, was taken out from pBpuMluICC 10SEAPII and used as a probe in gel shift assays. The fragment was labeled with [{alpha}-32P] dATP by filling in the Eco RI end with Klenow polymerase.

Immunohistochemistry
The lungs from CC101acZ and CC10K18 mlacZ transgenic mice, already stained for X-gal, were dehydrated, embedded in paraffin, and cut into 5-µm sections. Sections were mounted on aminopropyl-triethoxysilane-coated slides. Lung sections were examined for CC10-positive cells with goat polyclonal antibodies against murine CC10 protein (S-20; Santa Cruz Biotechnology); dilutions of the primary and secondary antisera (Donkey anti-goat immunoglobulin G biotin; Santa Cruz Biotechnology) were 1:1,000 (0.2 µg/ml) and 1:250, respectively. Antibody specificity was verified by omitting the primary antiserum. After completion of immunohistochemical studies, using an avidin–biotin–peroxidase complex method (27), slides were lightly counterstained with Carazzi hematoxylin, dehydrated, cleared in xylene, and mounted. Images were digitally captured using a Leica DC200 camera and Leica DC Viewer software (Leica Microsystems AG, Wetzlar, Germany).

Statistical Analysis
All numeric values are presented as means ± SD. Statistical significance was determined by analysis of variance followed by assessment of differences using Duncan's multiple range test (28) and the Dunnet two-sided test (28). Values were considered significantly different if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reporter Expression from the Human CC10 Promoter in Transgenic Mice
To examine whether the 3.3 kb CC10 promoter/5' regulatory region is sufficient to confer cell specificity of reporter expression in vivo, we used the DNA fragment to drive lacZ reporter expression in transgenic mice. Transgenic mice, as well as age-matched control mice, were killed and their lungs and other organs such as brain, gall bladder, and kidney were taken for the analysis of the reporter expression at different stages of development. As shown in Figures 1A–1C , the lacZ expression in the F1 generation of CC10 transgenic mice was predominantly localized in the bronchioles and no expression was seen in the brain, gall bladder, kidney (Figure 1D), or trachea (Figure 1C). Other organs, including the esophagus, liver, intestine, heart, blood vessels, spleen, skeletal muscle, and skin, were examined and they were all negative for lacZ expression. Interestingly, airway expression appeared to have decreased in adult animals (Figure 1C) compared with the 17-d fetuses (Figure 1A). To determine whether the cells expressing the reporter gene were Clara cells, we performed immunostaining with antibodies against the murine CC10 protein. As shown in Figure 2 , these reporter-expressing cells in lung sections from the transgenic mice are CC10-positive cells, but they only account for a small portion of the CC10-positive cells.



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  		Figure 1. Epithelium-specific expression of the lacZ reporter gene in CC10lacZ transgenic mice. Mouse tissues were stained with X-gal and lung sections were prepared after staining. (A) Lungs (left panel) and a lung section (right panel) from a 17.5-d prenatal transgenic mouse. (B) Lungs and a lung section from a Day-8 postnatal mouse. (C) Lung (left and middle panels) and trachea (right panel) sections of an adult CC10lacZ trangenic mouse. (D) X-gal staining of different organs from an adult CC10lacZ transgenic mouse: brain (left), liver and gall bladder (middle), and kidney (right).

 


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  		Figure 2. Immunohistochemical detection of CC10 positive cells in the lungs of a CC10lacZ transgenic mouse. (A) X-gal staining showing the expression of the lacZ reporter in lung airway cells of a Day-8 postnatal CC10lacZ transgenic mouse. Arrow points to positive X-gal staining cells. (B) CC10 immunostaining (brown stain using 3-diaminobenzidine (DAB) and light blue counterstain using Carazzi hematoxylin) of the same lung tissue. CC10 immunoreactive cells (arrows) were distributed throughout the lung airways. (C) Negative control where immunostaining was performed as in B, but without CC10 primary antibody. Panels D, E, and F are high power views of areas enclosed by boxes in A, B, and C respectively. Bar length = 1,000 µm. Note that only in A and D is the blue color indicative of lacZ expression.

 
Because the reporter expression driven by the human CC10 regulatory elements was localized in a small portion of Clara cells in the bronchioles, it is likely that other enhancer elements required for CC10 expression were missing in our construct. Currently, it is not known whether these elements are present in introns or the 3' region of the CC10 gene. Increasing the number of epithelial cells in the whole airway expressing the transgene through the CC10 promoter/regulatory region would be valuable for specifically targeting transgene expression to airway epithelial cells. We hypothesized that addition of enhancers from other epithelium-specific genes may increase the number of epithelial cells that express the reporter gene. To test this hypothesis, we added intron 1 of the human cytokeratin 18 gene to the upstream of the SEAPII and lacZ reporter genes in our CC10 expression constructs (Figures 3A and 3B) . The K18 intron contains an enhancer which is highly active in terminally differentiated epithelial cells of many internal organs, including the lung (24). It has been shown that for lacZ expression, the modified K18 intron (K18 m) is much more efficient (29). We tested the new construct CC10K18 mlacZ in transient transfection assays and showed that the modified K18 intron can significantly (P < 0.05) enhance lacZ expression from the human CC10 promoter (Figure 3D). We further confirmed with a different reporter construct, CC10K18iSEAPII (Figure 3A), that the K18 intron can also significantly (P < 0.05) enhance the SEAP reporter expression from the human CC10 promoter (Figure 3C).



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  		Figure 3. Enhancement of gene expression activity from the human CC10 promoter by intron 1 of the cytokeratin 18 gene. (A) Schematic diagram showing CC10 reporter constructs with or without the K18 intron. Both of these constructs contain the same SEAPII reporter gene. (B) CC10 reporter constructs as in A but with the lacZ gene and with or without the modified K18 intron (K18 m). (C) The effect of K18 intron 1 on the human CC10 promoter in SEAP expression. A549 cells were transfected with the constructs in A and the SEAP reporter assays were performed 48 h post transfection. (D) The effect of K18 m on the human CC10 promoter in lacZ expression. A549 cells were transfected with the constructs in B and chemiluminescent ß-galactosidease activity assays were performed with cell lysates of the transfected cells 48 h post transfection. Results are from two independent experiments, each as average of triplicates and presented as mean ± standard deviation. CC10, the human Clara cell 10 kD protein gene promoter; K18i, cytokeratin 18 intron 1; K18 m, modified K18 intron 1. * = P < 0.05 versus values without K18i or K18 m.

 
To examine whether the K18 intron can enhance the expression activity of the human CC10 promoter in vivo, we generated transgenic mice with the CC10K18 mlacZ construct and analyzed the reporter expression in airway epithelia. As shown in Figure 4 , K18 m appeared to significantly enhance the reporter expression in transgenic mice based on the X-gal staining of Day 8 postnatal lungs. The CC10 promoter in combination with K18 m was also able to drive reporter expression in a much larger portion of airway epithelial cells compared with the CC10lacZ mice (Figure 1). In these transgenic mice, expression in the tracheal epithelium is sporadic (Figure 4), and this expression pattern, which differs from that conferred by the K18 promoter (29), is consistent with the lower proportion of Clara cells present in this region. Apparently, there was no lacZ reporter expression in the trachea of the CC10 transgenic mice (Figure 1C). Expression was not observed in alveolar epithelial cells in either CC10lacZ or CC10K18 mlacZ transgenic mice. Strong expression in CC10K18 mlacZ mice was seen in the bronchiolar epithelial cells (Figure 4). To examine whether the enhanced reporter expression from the combination of the CC10 promoter and K18 m was confined to CC10-positive cells, we performed immunostaining of lung sections with antibodies against the murine CC10 protein. As shown in Figure 5 , the reporter lacZ and murine CC10 were coexpressed in the airway epithelia of the transgenic mice. These results reinforce our speculation that other regulatory elements are needed for the 3.3 kb human CC10 promoter fragment to drive gene expression in CC10-expressing cells.



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  		Figure 4. Expression of the lacZ reporter gene by the human CC10 promoter and the modified K18 intron 1 in a transgenic mouse at postnatal Day 8. Top panels: lungs from transgenic (left) and age-matched control (right) mice were stained with X-gal. The reporter expression is largely confined to the lung airway cells (left) while a lung from the age-matched control is negative for X-gal staining (right). Bottom panels: Expression of the lacZ reporter in lung airway cells. The bottom left panel represents a tissue section showing strong reporter expression in bronchi (B), but sporadic expression in tracheal (T) epithelial cells. The bottom middle panel represents a tissue section at a higher magnification showing reporter expression in tracheal epithelial cells. The bottom right panel shows a section of lung airway at a higher magnification.

 


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  		Figure 5. Immunohistochemical detection of CC10 positive cells in the lungs of CC10K18 mlacZ transgenic mice. (A) X-gal staining showing the expression of the lacZ reporter gene by the human CC10 promoter and K18 m in a Day-8 postnatal transgenic mouse (arrow points to positive X-gal staining cells). (B) CC10 immunostaining of a tissue section of the same lung. CC10 immunoreactive cells were widely distributed throughout the lung airways (arrows). (C) Negative control where primary antibodies were omitted in the immunostaining as in B. D, E, and F are high power views of areas enclosed by boxes in A, B, and C, respectively. Bar length = 1,000 µm.

 
Deletion Analysis of the 5' Regulatory Region of the Human CC10 Gene
To determine the regulatory properties of protein-binding motifs within the CC10 promoter region, a series of deletion constructs of the CC10 promoter/5' flanking region was made (Figure 6A) using SEAP as the reporter gene; activity in reporter gene expression in these constructs was monitored in transient expression assays. These human CC10 reporter constructs were introduced into A549 cells (from the lung epithelium) as liposome–DNA complexes, and reporter gene expression was measured by chemiluminescent assay (30). Deletion up to 2.4 kb from the 5' end of the 3.3 kb CC10 promoter/5' flanking sequence led to a significant (P < 0.05) increase in reporter gene expression (Figure 6B); however, further deletion led to a significant (P < 0.05) decrease in reporter expression, suggesting there are regulatory elements present in the 5' region of the CC10 promoter fragment. In general, all these deletion promoter fragments as well as the entire 3.3 kb sequence showed relatively low levels of expression activity in transient transfection assays. These results together with those from the transgenic analysis in mice indicate that other enhancer elements required for CC10 expression may be present elsewhere.




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  		Figure 6. Deletion analysis of the human CC10 promoter. (A) Schematic representation of the CC10 deletions. Deletion constructs were made by inserting the human CC10 promoter fragments of various lengths between the KpnI and MluI sites of the vector pSEAPII. The positions of the unique restriction sites are indicated on top of the 3,319 bp DNA fragment. (B) Reporter expression analysis of deletion constructs of the human CC10 5' flanking region. A549 cells were transfected with deletion promoter constructs indicated on the left side of the figure. The expression levels of the various deletion constructs after being normalized to total protein are shown as relative light units measured with the luminometer. The results are presented as means of two experiments, each in triplicate, and are presented as means ± SD. * = P < 0.05 versus the value of transfection with CC10SEAPII construct.

 
Identification of Regulatory Factors Involved in Human CC10 Expression
To understand the regulation of human CC10 expression, we used the yeast one-hybrid system to identify trans-acting factors that bind to a region that upregulates the CC10 expression in transient transfection analysis. For the strategy of the one-hybrid system, see MATERIALS AND METHODS. We selected, as shown in Figure 7 , the Sma I–Eco RI fragment as the target sequence because it resides near the CC10 promoter and does not contain any of the three Alu repeats present in the 5' region of the human CC10 gene (31). The size of this fragment is potentially big enough to contain binding sites for many transcription factors, therefore maximizing our chance of isolating putative transcription factors. We introduced these target-reporter constructs into yeast cells and, by marker gene selection, obtained recombinants with genomically integrated reporters. To isolate genes encoding DNA-binding-proteins of interest, we transformed the target-reporter yeast cells with a library of human lung cDNAs fused with a sequence for the target-independent activation domain. Transformants were selected on growth medium without histidine and in the presence of 3-AT for colonies that enhanced expression of the HIS3 reporter gene. Thirty-four colonies growing on the selective medium were then assayed for ß-galactosidase activity to verify the DNA–protein interaction. Plasmid DNA was recovered from the colonies positive in ß-galactosidase assays (data not shown) and were subsequently subject to DNA sequence analysis. As shown in Table 1, four interacting factors, COUP-TF I (32), COUP-TF II (33), TR4 (34), and RAR{gamma} (35), were identified following screening of more than 106 transformants.



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  		Figure 7. Schematic representation of the yeast one-hybrid reporter constructs with a CC10 5' fragment as target sequence. A Sma I-Eco RI (337 bp) DNA fragment of the human CC10 gene 5' region was cloned into the pHIsi, and placZ, respectively. These target-reporter constructs were introduced into yeast cells, and recombinants with genomically integrated reporters were obtained by marker gene selection.

 

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  		TABLE 1 Putative CC10 regulators identified by the yeast one-hybrid system

 
Interaction of COUP-TFs with the Target DNA Sequence
Since two of the four factors selected by the one-hybrid system were COUP-TFs (Table 1), we decided to identify the COUP-TF binding site in the Sma I–Eco RI fragment of the CC10 5' region used as the target sequence for the yeast one-hybrid system. Following examination of the DNA sequence of the fragment, one perfect half site (aggtca) and an imperfect half site (aggcca) for COUP-TFs were found to be separated by five base pairs present in the Sma I–Eco RI fragment. To test whether COUP-TFs really bind to the putative sequence, we designed a double stranded oligonucleotide probe (see MATERIALS AND METHODS) and labeled it with [{alpha}-32P]-dCTP. COUP-TFI and II were produced using an in vitro reticulocyte lysate-translation system. As shown in Figure 8 , protein–DNA complexes formed with COUP-TFI or COUP-TFII were observed and they were supershifted when an anti–COUP-TFI antibody or anti–COUP-TFII antibody was included in the binding reaction. These results suggested that both COUP-TFI and II proteins can bind specifically to the DNA probe. We have also confirmed that both COUP-TF I and II bind to the 337 bp Sma I–Eco RI fragment labeled with [{alpha}-32P]-dATP (data not shown).



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  		Figure 8. Electrophoretic mobility shift assay of COUP-TFI and COUP-TFII binding sites. Gel shift experiments were performed with an [{alpha}-32p]-dCTP labeled double-stranded oligonucleotide (10 ng) and COUP-TFI and COUP-TFII produced with an in vitro transcription/translation system. The control proteins were obtained by in vitro transcription/translation with vector DNA (pCDNA3.1). For competition, 100 and 300 ng of the same unlabeled double-stranded oligonucleotide were used (see MATERIALS AND METHODS). Four µl (0.8 µg) antibody to COUP-TFI and COUP-TFII were added to the binding buffer prior to addition of the labeled oligonucleotide.

 
Effect of COUP-TFs on Human CC10 Expression
To investigate whether COUP-TFs affect the human CC10 promoter activity in cultured cells, we performed cotransfection assays in A549 cells using reporter constructs with (SmMCC10SEAPII) or without (EMCC10SEAPII) the Sma I–Eco RI DNA fragment used in the screening with the one-hybrid system. Both COUP-TFs showed suppression activity on the SmMCC10SEAPII in cotransfection assays. As shown in Figure 9 , both COUP-TFI and COUP-TFII caused a significant (P < 0.05) reduction in the SEAP reporter expression. Interestingly, they showed significant (P < 0.05) stimulation of SEAP expression when the cotransfection was done using the EMCC10SEAPII deletion construct. This transactivation is likely an indirect effect of COUP-TFs because they can regulate a variety of genes (22), some of which may upregulate CC10 expression. These results confirm that the COUP-TFs regulate CC10 expression through the DNA fragment used for one-hybrid screening.



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  		Figure 9. Regulation of human CC10 promoter by COUP-TFI and COUP-TFII in transfected cells. The effect of COUP-TFs on reporter gene expression was studied in A549 cells using chemiluminescent assay of SEAP. A549 cells were cotransfected with various deletion constructs of human CC10 gene 5' flanking region in combination with COUP-TFI, COUP-TFII expression plasmid, or pcDNA vector (as a negative control). Expression levels are shown as relative light units measured with luminometer after being normalized to total protein. Results are presented as means of two experiments, each in triplicates, and are presented as means ± SD. * = P < 0.05 versus value of cotransfection with control pcDNA vector.

 
Expression of COUP-TFII in Tracheal Epithelial Cells of Mouse Embryos
COUP-TFs are expressed in all three germ layers during mouse development. COUP-TFI is highly expressed in the nervous system and COUP-TF II is highly expressed in the messenchyme of internal organs (22, 23). To further analyze the temporal and spatial expression patterns of COUP-TFII, we generated COUP-TFII–lacZ knock-in mice and examined the lacZ reporter expression in tracheae of developing mouse embryos. Although in general COUP-TFII is not expressed in terminally differentiated epithelial cells, it was consistently found to be expressed in tracheal epithelial cells. Figure 10 represents COUP-TFII expression in fetal tracheal epithelial cells capitulated by lacZ reporter expression.



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  		Figure 10. COUP-TFII expression in tracheal epithelial cells recapitulated by lacZ reporter expression in COUP-TFII–lacZ knock-in fetal mice. Shown in the first two panels are cross-sections of tracheae at the low end from a normal mouse embryo (A) and a COUP-TFII–lacZ knock-in mouse embryo (B) at E10.5. (C) represents a sagittal section of a trachea from a COUP- TFII–lacZ knock-in mouse embryo at E 12.5. Cryostat sections (20 µm) of the embryos were stained for lacZ activity and counter-stained with Eosin Y. Intense staining was observed in tracheal epithelial cells as well as nonepithelial cells of the COUP-TFII–lacZ knock-in mice. Arrows indicate the tracheal epithelial cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The airway specificity of CC10 expression is a logical characteristic to use for targeting transgene expression in the airway. Our transgenic analysis demonstrated that the 3.3 kb DNA sequence of the human CC10 gene can target transgene expression to a small portion of Clara cells, indicating that it contains sufficient DNA elements needed for silencing its expression in nontarget cells. We have observed this type of lung expression pattern in three independent transgenic lines. Because the expression from this fragment is confined to a small portion of Clara cells, it is likely that other enhancer elements are required for its expression in all Clara cells. It would be useful if the epithelial specificity of our construct could be expanded to all the Clara cells or other types of epithelial cells in the lung. As in many other mammalian genes, the additional enhancer elements could be present in introns or the 3' downstream region. Because there is no information available about additional regulatory elements of the CC10 gene, we have tested the possibility of broadening the epithelial specificity by the addition of a heterologous intron from another epithelium-specific gene, intron 1 of the human cytokeratin 18 gene, which contains a strong enhancer. Indeed, the K18 intron greatly enhanced the level of expression as well as the number of epithelial cells that expressed the reporter gene (Figures 3 and 4), with no expression in nonepithelial cells. However, the addition of the intron also resulted in expression of the reporter gene in other organs (data not shown) such as the brain, kidney, trachea, and gall bladder. Therefore, it is important to identify putative enhancer elements of the CC10 gene to maintain the organ and cell-specificity for transgene expression.

CC10 expression is highly regulated and the mechanisms underlying the regulation are very intriguing. Several transcription factors, such as Nkx2.1, FoxA, AP-1, and Oct, have been implicated in involvement in the regulation (16–19), but none of them is considered Clara cell-specific. Much of the work on rodent CC10 expression has focused on positive regulation and not much is known about how CC10 expression is repressed in other cells that also express the transcription factors such as FoxA, AP-1, and Oct. It is likely that some negative regulatory elements are involved in repressing the expression of CC10 in the nonpermissive tissues or cells. We have performed transient transfection assays to identify putative promoter elements involved in the regulation of the human CC10 promoter. We noticed that reporter constructs with different lengths of 5' region of the human CC10 promoter display different gene expression activity. We also observed that the human CC10 promoter expresses in epithelial cell lines of non-Clara cell origin, such as A549 (Figure 3) or HeLa cells (data not shown), whereas the rat CC10 promoter was reported to be highly Clara-cell specific and not expressed in these cells (14). It is possible that the human CC10 gene may be regulated differently from that of rodents. Eoers and colleagues showed that a significant portion of human airway goblet cells express CC10 (21).

The one-hybrid system can theoretically be used to isolate putative factors that interact with a target DNA sequence or to identify DNA sequences for a particular protein factor. However, its application to protein factor isolation is relatively more successful, probably due to the fact that this approach can use a cDNA library prepared from a particular organ, tissue, or cell type, and therefore minimize the pool size for screening. False positives are expected in any type of genetic screening procedure, but there are ways to eliminate the false positives in the yeast one-hybrid system. First, addition of various concentrations of 3-AT can distinguish the clones expressing strongly interacting factors from those expressing false positives or weakly interacting factors. Second, a reporter system, in this case the lacZ reporter system, can be used to confirm the interaction. In addition, genes encoding the identified putative factors can be used to analyze their functions through cotransfection assays. This method is designed to isolate putative factors that interact with a target DNA sequence, but does not discriminate between positive and negative regulators, since a piece of DNA encoding an activation domain from VP16 is included in the cloning vector to generate the fusion cDNA library. This feature offers the possibility of identifying negative factors that are not expressed in the cells where the targeting gene is expressed. These negative factors can then be tested in cotransfection assays in cultured cells.

Based on the one-hybrid system, we identified four putative factors that interact with the Sma I–Eco RI (337 bp) DNA fragment of the human CC10 gene 5' region (Table 1). Among these factors are transcription factors COUP-TFI (EAR-3) and COUP-TFII (ARP-1). The interactions of these factors with the target DNA sequence have been confirmed by gel-shift assays using either the same fragment or the double-stranded oligonucleotide containing a putative binding site for COUP-TFs (Figure 8). Furthermore, the specificity of the interactions was demonstrated by the supershift assay using antibodies to the COUP-TFI and COUP-TFII (Figure 8). Interestingly, the target DNA sequence has been shown to contain positive DNA regulatory elements based on deletion analysis in cultured lung epithelial cells (Figure 6B). Yet the majority of factors identified with the yeast one-hybrid system, such as COUP-TFs, are negative regulators. This observation suggests that the target DNA sequence contains both positive and negative elements. No positive regulators were identified in our screening, probably because the cDNAs encoding positive factors in epithelial cells are under-represented in the cDNA library (the number of epithelial cells is relatively low compared with mesenchymal cells). It is also possible that the positive regulators may have a low binding affinity and the stringency of our screening may be too high to allow the identification of weak binding.

The identification of COUP-TFs as negative regulators for CC10 expression sheds new light on the mechanisms of the regulation. COUP-TFs form dimers in solution and bind to an imperfect direct repeat separated by a variable number of nucleotides (36). Because of their promiscuous DNA binding properties and the ability to compete for binding sites with other nuclear factors, COUP-TFs negatively regulate a large number of genes (22, 37–39). In epithelial cell lines, such as A549, there is no expression of COUP-TFs (40). When a COUP-TF is co-expressed in A549 cells with a CC10 reporter construct with the COUP-TF binding site present, the level of reporter gene expression is decreased (Figure 9), suggesting COUP-TFs negatively regulate CC10 expression. Interestingly, COUP-TFII is expressed in mesenchymal cells, but not in the epithelial cells of the lung (23), except those in the upper end of the conducting airway, such as tracheal epithelial cells (Figure 10). This expression pattern is in inverse correlation with that of CC10. The results of these studies suggest that COUP-TFs repress CC10 expression in non-Clara cells in vivo.


   Acknowledgments
 
The authors thank M. Kuliszewski, L. Ye, and J. Plumb for technical assistance, and M. Liu for helpful suggestions during preparation of this manuscript. This work was supported by Operating Grants from the Canadian Institutes of Health Research to J.H. and A.K.T. and Operating Grants from the Canadian Cystic Fibrosis Foundation to J.H. R.N. and R.P.J. hold Fellowships from the Canadian Lung Association/Canadian Institutes of Health Research and J.H. is a CCFF Scholar. A.K.T. holds the Women's Auxiliary Chair in Neonatology at the Hospital for Sick Children.

Received in original form February 1, 2002

Received in final form April 3, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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