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Published ahead of print on July 8, 2004, doi:10.1165/rcmb.2004-0050OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 491-500, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0050OC

Characterization of Ciliated Bronchial Epithelium 1, a Ciliated Cell–Associated Gene Induced During Mucociliary Differentiation

Hajime Yoshisue, Sarah M. Puddicombe, Susan J. Wilson, Hans Michael Haitchi, Robert M. Powell, David I. Wilson, Anita Pandit, Ann E. Berger, Donna E. Davies, Stephen T. Holgate and John W. Holloway

Infection, Inflammation, and Repair and Human Genetics Divisions, University of Southampton School of Medicine, Southampton General Hospital, Southampton, United Kingdom; and Pharmacia Corporation, Kalamazoo, Michigan

Address correspondence to: Hajime Yoshisue, Ph.D., Infection, Inflammation, and Repair Division, University of Southampton, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK. E-mail: hyoshisu{at}soton.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lung epithelial structure is altered in asthma; however, the precise mechanisms underlying epithelial repair, including differentiation from basal to columnar epithelial cells, are not well defined. In the course of random sequencing of a cDNA library from human lung biopsies, we have identified a novel gene, ciliated bronchial epithelium 1 (CBE1). Expression of CBE1 was induced during in vitro differentiation of bronchial epithelial cells. Synchronous expression with tektin and hepatocyte nuclear factor 3/forkhead homologue 4, down-regulation by interleukin-13, and its tissue distribution strongly suggested that CBE1 is associated with ciliated cells. Two isoforms of the 0.7-kb full-length cDNA were identified, resulting in open reading frames with different carboxyl termini, with no homology to known proteins. Expression of CBE1 in ciliated epithelial cells was confirmed by immunohistochemistry. Quantitative reverse transcription–polymerase chain reaction analysis using bronchial biopsies showed no difference of expression of CBE1 between normal subjects and subjects with asthma. Expression studies showed that CBE1 is nuclear- or perinuclear-localized, depending on cell type. Regulated expression during differentiation and the subcellular localization of CBE1 suggest that it may play an important role in the differentiation and/or function of ciliated cells in human airways.

Abbreviations: air–liquid interface, ALI • bovine serum albumin, BSA • ciliated bronchial epithelium, CBE • axonemal dynein intermediate-chain gene 1, DNAI1 • enhanced green fluorescent protein, EGFP • fluorescein isothiocyanate, FITC • hepatocyte nuclear factor 3/forkhead homologue 4, FOXJ1 • human bronchial epithelial, HBE • immunohistochemistry, IHC • interleukin, IL • open reading frame, ORF • phosphate-buffered saline, PBS • primary cilia dyskinesia, PCD • polymerase chain reaction, PCR • quantitative PCR, qPCR • rapid amplification of cDNA ends, RACE • reverse transcription PCR, RT-PCR • saline sodium citrate, SSC • T helper type 2, Th2


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mammalian lung epithelium is composed of a variety of cell populations from proximal to distal airways. The individual characteristics of the subtypes of the cells that make up the airway epithelium not only create an effective physical barrier against various noxious substances, but also a highly sophisticated host defense system by producing and releasing a large number of chemical mediators and cytokines (reviewed in Refs. 1 and 2). The epithelium of the upper airways is a continuous layer consisting mainly of three cell types: basal, goblet, and ciliated epithelial cells, the latter two of which make up a suprabasal columnar structure and are necessary for mucociliary clearance. Goblet cells synthesize mucus, providing the surface of the epithelium with a protective cover, and ciliated cells are responsible for propelling the mucus secretions toward the pharynx.

Whereas in the healthy human lung, ciliated cells cover ~ 80% of epithelium, the columnar structure becomes markedly altered in disease states. Epithelial damage that may be accompanied by columnar cell loss is a feature of asthma (3, 4). Columnar cells that are lost are likely to be replaced by goblet cells through differentiation from basal cells, resulting in mucous metaplasia and hypersecretion of mucus. This over-production of mucus is also observed in other chronic airway diseases, including chronic bronchitis, cystic fibrosis, and bronchiectasis. Thus, efficient and adequate epithelial repair, leading to enrichment of ciliated cells, would be of benefit in chronic airway diseases. However, the molecular mechanisms underlining epithelial ciliogenesis remain poorly understood.

Recently, several genes that are induced during ciliogenesis have been cloned and characterized: dynein-related transcript was identified as a testis cDNA, and shown to be upregulated during ciliation of bronchial epithelial cells in culture and the gene product localized to cilia (5). Dynein heavy chain 7 has been identified as a component of the inner dynein arm of cilia, and shown not to be assembled in cilia from patients with primary cilia dyskinesia (PCD) (6). KPL2 was identified by differential display as a gene whose expression was increased during ciliogenesis of rat tracheal epithelial cells under conditions favoring differentiation of ciliated cells, but its cellular localization and function are not known (7). The winged-helix DNA binding domain, containing transcription factor hepatocyte nuclear factor 3/forkhead homolog 4 (FOXJ1), has been shown to be critical in ciliogenesis. Expression of FOXJ1 is temporally regulated during embryogenesis and is localized to the epithelium of lung, testis, choroid plexus, and kidney (8, 9). The targeted deletion of the foxj1 (hfh-4) gene in mouse embryonic stem cells results in a complete absence of airway cilia (10). However, the transcription factor cascade controlling and/or controlled by FOXJ1 remains to be investigated.

Here we report a novel epithelial cell–expressed gene, ciliated bronchial epithelium 1 (CBE1), whose expression parallels ciliated cell differentiation ex vivo. Although its two predicted open reading frames (ORFs) do not show any significant similarity to any known proteins, immunohistochemistry (IHC) has indicated that the protein is expressed in ciliated cells, but does not constitute components of cilia. This suggests that CBE1 is a regulator of ciliogenesis and/or the intracellular function of cilia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of cDNA Libraries from Bronchial Biopsies of Normal Subjects and Subjects with Asthma
cDNA libraries from four subjects with atopy but not asthma and three subjects with atopy and asthma were derived as previously described (11). Bronchial biopsies were obtained from the volunteers by fiber optic bronchoscopy in accordance with standard published guidelines (12).

Isolation, Culture, and In Vitro Differentiation of Primary Human Bronchial Epithelial Cells
Primary bronchial epithelial cells were isolated and cultured as previously described (13). For air–liquid interface (ALI) cultures, passage 2 cells were trypsinized and seeded into 6.5 mm, 0.4-µm pore-size collagen-coated transwell culture inserts at 7.5 x 104 cells/well (Corning Costar, Bucks, UK). The cells were grown submerged in bronchial epithelial growth medium (BEGM) medium for 1–3 d until 90–100% confluent. The apical medium was then removed to expose the cells to the air–liquid interface and 300 µl of basal ALI medium added to bottom wells, comprised of a mixture of an equal volume of Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA), and bronchial epithelial basal medium (BEBM) (Biowhittaker, Wokingham, UK), containing 52 µg/ml bovine pituitary extract, 5 µg/ml insulin, 0.5 ng/ml human epidermal growth factor, 0.5 µg/ml hydrocortisone, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ml triiodothyronine, 1.5 µg/ml bovine serum albumin (BSA; Sigma, Poole, UK), and 50 nM retinoic acid. This basal ALI medium was changed every 1–2 d and differentiation proceeded in a similar manner to that previously described (14). Goblet cell hyperplasia was induced in vitro by chronic stimulation of transwell-cultured epithelial cells with interleukin (IL)-13 (Sigma) at a concentration 20 ng/ml in the basal ALI medium. IL-13 was first added when the cells were exposed to air (start of ALI), and subsequently added whenever ALI medium was changed. At Day 14 or 28 after the start of ALI, RNA was isolated for reverse transcription–polymerase chain reaction (RT-PCR) or cells were fixed for immunostaining, respectively.

General Cell Culture and Transfection
COS-7 and H292 cells were maintained in RPMI1640 medium (Invitrogen) containing 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum. 16HBE 14o(–) (human bronchial epithelial) cells were maintained in minimal essential medium (Invitrogen) containing 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum. Recombinant plasmids were purified by the EndoFree plasmid preparation system (QIAGEN, Crawley, UK) and transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Cloning of Full-Length CBE1 mRNA
Full-length cDNA clones of the CBE1 gene were obtained by the 5' rapid amplification of cDNA ends (RACE) procedure using a SMART RACE cDNA amplification kit (BD Biosciences Clontech, Oxford, UK). First strand synthesis was performed using 0.5 µg of RNA samples from epithelial cells from four volunteers harvested by bronchial brushings as a template and SuperScript II reverse transcriptase (Invitrogen). The gene-specific primer for CBE1 was 5'-GAGTTGTAACAGCACACTGCATTC-3', and the nested, gene-specific primer was 5'-CGAGCAAGCACTTTCGTAACCATG-3'. The DNA fragment amplified by the nested PCR was ligated into a pPCR-Script Amp SK(+) vector (Stratagene, La Jolla, CA). Three independent clones from each subject (12 in total) were subjected to dideoxy-chain termination sequencing reactions using BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, UK), and the reaction products were analyzed using a 377 DNA sequencer (Applied Biosystems).

Isolation of RNA, RT, and PCR
Total RNA was isolated from monolayer- or ALI-cultured bronchial epithelial cells using Trizol (Invitrogen). Human total RNA samples from 20 different tissues were obtained from BD Biosciences Clontech. cDNAs were synthesized from 1 µg of total RNA with SuperScript II reverse transcriptase in a reaction volume of 20 µl according to the manufacturer's instructions; a reaction without reverse transcriptase was performed as a negative control. One-fortieth of each cDNA solution was used for each PCR reaction. The PCR conditions were 94°C, 2 min initial denaturation step followed by a cycle reaction at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min on a DNA Engine (MJ Research, Watertown, MA). The reaction products were subjected to agarose gel electrophoresis. The primers used are shown in Table 1. To specifically amplify the ORF1 and ORF2 isoforms of CBE1, the forward primer (Table 1) and the following isoform-specific reverse primers were used; 5'-CTCGTAGGGAGGTACACATAGTCG-3' (ORF1) and 5'-CTCGTAGGGAGGTACACATCTTAC-3 (ORF2). The specificity of each primer was confirmed by cloning and sequencing of each PCR amplicon.


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  		TABLE 1. Primers used for reverse-transcription polymerase chain reaction

 
Northern Blot Analysis
Six micrograms of total RNA from HBE cells obtained by bronchial brushings, or from H292 cells, were electrophoresed through a 1% agarose gel containing formaldehyde and 3-(N-Morpholino)propanesulfonic acid, and transferred to a positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) by capillary transfer. A CBE1-specific cDNA fragment was amplified by PCR using the same primers as used in RT-PCR, and was 32P-labeled using rediprime II random prime labeling system (Amersham Biosciences, Amersham, UK). The baked membrane was hybridized with the probe at 42°C in a solution of 50% formamide, 5x Denhardt's, 5x saline sodium citrate (SSC), and 0.1% sodium dodecyl sulfate (SDS) for 24 h (1x SSC: 0.15 M NaCl, 15 mM sodium citrate), followed by washing at room temperature twice in 2x SSC/0.1% SDS and at 42°C twice in 0.15x SSC/0.1% SDS. Autoradiography was performed for 24 h with an intensifying screen.

Generation of Polyclonal Antibodies
Anti-CBE1 polyclonal antibodies were generated in rabbits by Abcam (Cambridge, UK). Two synthetic peptides, CPPRPERLNAYERE (amino acids 12–25) and TDRRLRPWCREQPT (amino acids 86–99), both of which are in the common region of the two ORFs, were used for immunization. For IHC, antiserum was purified using an immunoaffinity column that had been coupled with both peptides.

Expression Plasmids
The cDNA fragment containing ORF1 of CBE1, consisting of 127 amino acids, was amplified by Pfu Turbo DNA polymerase (Stratagene) using a sense primer 5'-TAAAGCTTGCCACCATGGAGACAGCAGTTCGAGG-3' (underlined is a HindIII site and double-underlined is a Kozak sequence for efficient initiation of translation) and an antisense primer 5'-TTTCTAGACTAAGGTTCGGATATGGGTAG-3' (underlined is an XbaI site), followed by restriction digestions with HindIII and XbaI and ligation into the HindIII and XbaI sites of pcDNA3.1 (Invitrogen). An expression plasmid for ORF2 was similarly constructed, except that the antisense primer 5'-TTTCTAGACTACCAGCAACGATAGTCGGG-3' (underlined is an XbaI site) was used. A DNA fragment corresponding to the ORF of human FOXJ1 was obtained by PCR using cDNA from well-differentiated primary epithelial cells as a template, a sense primer, 5'-TAAAGCTTGCCACCATGGCGGAGAGCTGGCTGCGCCTC-3' (underlined is a HindIII site), and an antisense primer, 5'-TATCTAGATTACAAGAAGGCCCCCACGCTGGC-3' (underlined is a XbaI site), followed by restriction digestions and ligation into the HindIII and XbaI sites of pcDNA3.1. PCR-amplified fragments were sequence-verified.

Western Blot Analysis
A recombinant plasmid encoding full-length ORF1 or ORF2 was transiently introduced by lipofection into COS-7 cells, which had been seeded at a density of 8 x 105 cells per 60 mm dish. Forty-eight hours after transfection, cells were washed by phosphate-buffered saline (PBS) and suspended in 200 µl of SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 5% 2-mercaptoethanol, 1% SDS, and 0.002% bromophenol blue). After heating at 95°C for 5 min, 15 µl of cell lysates were separated using SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose filter (Amersham Biosciences). Western blotting was performed using anti-CBE1 antiserum or pre-immune serum (1:1,000 dilution) and the horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin G (DAKO, Carpinteria, CA) as a secondary antibody. Visualization using ECL Plus reagents was according to the manufacture's instructions (Amersham Biosciences).

IHC
Nasal polyp, bronchial tissue, and bronchial biopsies were fixed in acetone containing protease inhibitors overnight at 4°C and processed into glycol methacrylate resin (15). Two-micron sections were cut and stained by IHC using the streptavidin biotin-peroxidase detection system with diaminobenzidene as the chromagen. Counterstaining was done with Mayer's hematoxylin. The affinity-purified rabbit polyclonal antibody was used for detection of CBE1 at a concentration of 2 µg/ml. For double-staining, to confirm the cellular localization of CBE1, IHC for CBE1 was followed either by IHC for ß-tubulin (Sigma) using the streptavidin biotin-alkaline phosphatase detection system with Fast Red as the chromagen for the identification of ciliated cells, or by the periodic acid Schiff technique for the identification of goblet cells. Specificity of the antibody was confirmed by pre-adsorption with the immunizing peptides.

Preparation of Human Lung Biopsies and Real-Time PCR
Following receipt of approval from the Southampton and South West Hampshire Joint Local Research Ethics Committee, bronchial biopsies were obtained from 10 normal subjects (5:5, male:female; mean age, 37 yr; range, 23–54 yr; FEV1 (% predicted), 107 ± 10%) and 9 subjects with asthma (5:4, male:female; mean age 36 yr; range, 23–50 yr; FEV1 (% predicted), 90 ± 10%). All subjects with asthma were using short-acting ß2-agonists as required, and eight were using inhaled corticosteroids (median dose, 400 [range 200–800] µg/day). Immediately after taking the bronchial biopsies (one to two), the piece of tissue was homogenized in lysing matrix D impact-resistant 2.0 ml tubes containing 1.4 mm ceramic spheres and Trizol reagent in a Hybaid RiboLyser Cell Disrupter (Thermo Life Sciences, Basingstoke, UK). Extracted total RNA samples were treated with DNase (Ambion, Huntingdon, UK). Approximately 300 ng of total RNA was reverse-transcribed using random hexamer primers and 100 U of MMLV reverse-transcriptase (Promega, Southampton, UK) following the manufacturer's protocol. Target gene (CBE1, MUC5AC, and tektin) probes were labeled with a 5'-reporter dye FAM (6-carboxy-fluorescein) and a 3'-quencher dye TAMRA (6-carboxy-N,N,N',N'-tetramethyl-rhodamine). Primer kits directed against 18S rRNA and ß-actin containing a Yakima yellow/dark quencher–labeled probe were used as a normalizing control (Eurogentech, Seraing, Belgium). Specific primers and probes used are shown in Table 2. Validation of target gene primer and probe sets and the method of quantitative PCR (qPCR) were as previously described (16).


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  		TABLE 2. Primers and probes used for real-time polymerase chain reaction

 
Immunofluorescence and Confocal Microscopy
COS-7, 16HBE 14o(–), or primary epithelial cells were seeded and grown on round glass coverslips in a 24-well dish. A recombinant plasmid expressing CBE1 ORF1 or ORF2 was transiently introduced into COS-7, 16HBE 14o(–) or primary epithelial cells by lipofection. Twenty-four hours after transfection, cells were fixed with 2.0% paraformaldehyde in PBS. Background immunofluorescence was quenched with 20 mM NH4Cl in PBS, and then cells were permeabilized with 0.2% Triton X-100 in PBS. After blocking in 1.0% BSA, cells were incubated for 30 min with immunopurified anti-CBE1 antibodies (1:100 dilution), followed by washing with PBS. After re-blocking with 1.0% BSA, cells were incubated for 30 min with fluorescein isothiocyanate (FITC)–conjugated anti-rabbit immunoglobulin G antibody (DAKO) (1:20 dilution). The coverslips were washed with PBS extensively, incubated for 10 min in 2 µM 7-aminoactinomycin D for nuclear staining, and then mounted onto slides in a fluorescent mounting medium (DAKO). Fluorescent images were viewed on a laser scanning confocal microscope (Leica, Bannockburn, IL).

Generation of Mutant CBE1 and Evaluation of Nuclear Localization
The cDNA fragment containing ORF2 of CBE1, consisting of 164 amino acids, was amplified by Pfu Turbo DNA polymerase using a sense primer Eco-S 5'-CTGAATTCTGAGACAGCAGTTCGAGGAATG-3' (underlined is an EcoRI site) and an antisense primer Bam-AS 5'-AAGGATCCTACCAGCAACGATAGTCGGG-3' (underlined is a BamHI site), followed by restriction digestions with EcoRI and BamHI and ligation into the EcoRI and BamHI sites of pEGFP-c1 (BD Biosciences Clontech). As a result, the amino acid sequence downstream from the second residue (glutamic acid) was fused with the carboxyl terminus of enhanced green fluorescent protein (EGFP). To generate a mutant CBE1, amino terminal half portion was amplified using a sense primer Eco-S and an antisense primer 5'-AAGGCCTCAGGGCGGCGTCGGTGCAGGGCGCCCCGC-3' (underlined is a StuI site and double-underlined corresponds to the two amino acids which are substituted), followed by restriction digestions with EcoRI and StuI. Similarly, the carboxyl terminal half of ORF2 was amplified using a sense primer, 5'-AAGGCCTTGGTGCCGGGAGCAAC-3' (underlined is an StuI site), and an antisense primer, Bam-AS, followed by digestions with StuI and BamHI. The two fragments were directionally introduced into the EcoRI and BamHI sites of pEGFP-c1, resulting in an EGFP-fused ORF2 of CBE1 with the two consecutive arginine residues (Figure 2A) changed into two alanines. All clones of PCR products were sequence-verified. Each of these chimeric constructs was transiently introduced into COS-7 cells by lipofection. Twenty-four hours after transfection, the intracellular location of the fused proteins was observed using fluorescent microscope model DM1RB (Leica). The fluorescent images were captured using digital camera model C4742-95 (Hamamatsu Photonics, Welwyn Garden City, UK) and at least 200 EGFP-positive cells were counted for each transfectant. Cells with fluorescence exclusively in their nucleus were defined as nuclear-localized. Four independent transfection experiments were performed to gain reproducibility and statistical significance.

Statistical Analysis
Statistical analysis was undertaken using the Mann-Whitney test implemented in SPSS (SPSS, Inc., Chicago, IL). P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and Identification of CBE1
To investigate differences in gene expression profile between subjects with and without asthma, cDNA libraries were generated from bronchial biopsies. Randomly selected independent clones from the libraries of subjects with and without asthma (4,080 and 4,311, respectively) were sequenced and computer-analyzed, leading to identification of genes with different frequencies of appearance. One of these "putative" differentially expressed genes was CBE1, which was represented by eight independent clones in the library of normal subjects, but none in the library of subjects with asthma, suggesting that expression of the gene is less abundant in the bronchi of subjects with asthma.

Expression of CBE1 Parallels Ciliogenesis Ex Vivo
To identify the pattern of expression of CBE1 in the lung, RT-PCR analysis was performed using cDNA samples derived from different cell types. Significant expression of CBE1 was detected in bronchial brushings, but expression was completely absent in peripheral blood leukocytes, bronchial fibroblasts (data not shown), or undifferentiated primary bronchial epithelial cells. However when primary bronchial epithelial cells were differentiated on an ALI culture model, CBE1 expression was induced (Figure 1A). CBE1 expression is very low in monolayer-cultured cells with a basal phenotype, but is strongly induced 14 d after ALI culture, when ciliated cells are apparent by light microscopy. This temporal expression pattern has greater similarity to that of the ciliated cell markers, tektin (17) and FOXJ1 (9), than to that of the goblet cell–specific MUC5AC and MUC5B (18), which are expressed after 7 d of ALI culture. We did not observe marked upregulation of centrin-2 and -3, which have been reported to show increased transcription during mucociliary differentiation of human nasal epithelial cells in spheroid culture (19). Parallel expression of CBE1 with tektin was also observed in ALI cultures of primary epithelial cells derived from subjects with mild and severe asthma (data not shown). The pattern of CBE1 expression suggests that the gene is temporally regulated during mucociliary differentiation and appears to be associated with ciliogenesis.



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  		Figure 1. Semiquantitative RT-PCR analysis of gene expression during mucociliary differentiation of primary bronchial epithelial cells. (A) Temporal expression pattern. Total RNA samples from monolayer-cultured or ALI-treated primary bronchial epithelial cells at the indicated time points were reverse-transcribed into cDNA, followed by PCR using primers shown in Table 1. (B) Downregulation of CBE1 by IL-13. ALI-cultured cells were treated or untreated with IL-13 for 14 d. PCR cycles were as follows: CBE1 (30), FOXJ1 (35), tektin (30), centrin-2 (30), centrin-3 (30), MUC5AC (27), MUC5B (27), MUC2 (33), and ß-actin (21). PCR products were electrophoresed through a 1.8% agarose gel and stained with ethidium bromide. One representative result from at least two independent experiments is shown.

 
Expression of CBE1 Is Downregulated by IL-13
It has recently been reported that some cytokines, which are produced by type 2 CD4+ T helper (Th2) lymphocytes induce goblet cell hyperplasia and overproduction of mucus in cultured primary lung epithelial cells (20, 21) at the expense of ciliated cells. Thus we treated ALI-cultured epithelial cells with IL-13, a representative Th2 cytokine, and assessed the gene expression profile (Figure 1B). After chronic stimulation with IL-13 for 14 d, expression of MUC5AC, MUC5B, and MUC2 was upregulated, whereas expression of tektin and FOXJ1 was repressed to an undetectable level. Expression of CBE1 was also downregulated by IL-13, strongly suggesting that CBE1 is a ciliated cell–associated gene.

Primary Structure of the CBE1 cDNA and Tissue Distribution
To characterize the entire ORF of the CBE1 cDNA, 5'-RACE analysis was undertaken using total RNA samples from epithelial tissues that had been obtained from bronchial brushings. After determining the 5' end of the mRNA, we amplified the full-length cDNA of CBE1 by PCR. Figure 2A shows the resulting consensus sequence of the full-length cDNA of CBE1. Its length, ~ 0.7 kb, is consistent with the size of the band obtained by Northern blotting (Figure 2B). Within the 0.7-kb sequence, a small ORF consisting of 127 amino acids was identified, designated ORF1. In 6 out of 12 clones that were sequenced, a 5-bp insertion was observed at one of the splicing sites, resulting in a frame shift that, in turn, resulted in a second ORF (ORF2) of 164 amino acids with a different carboxyl terminus (Figure 2A). This was likely to be the result of alternative splicing rather than allelic variation, as RT-PCR analysis using variant-specific primers detected both isoforms of CBE1 in all subjects tested (Figure 2D). Both variants were also expressed in primary epithelial cells undergoing mucociliary differentiation in vitro and their expression kinetics were found to be very similar (data not shown). BLAST (Basic Local Alignment Search Tool) search analysis showed that CBE1 is homologous to NYD-SP22, a testis-derived gene that has three known splice variants, one of which, NYD-SP22 v2 (variant 2) (NM_147168), is identical to ORF1. Two other variants appear to arise from an alternate promoter and/or alternative splicing: NYD-SP22 v1 (NM_032596) and NYD-SP22 v3 (NM_147169) (Figure 2C). This expressed sequence tag (EST) cluster is built on an EST (NYD-SP22, AF367474) that was identified as being overexpressed in adult testis compared with 6-mo–gestation fetal testis (22).



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  		Figure 2. Primary structure of the CBE1 cDNA. (A) Nucleotide and deduced amino acid sequences of the full-length cDNA of CBE1, which was revealed by 5'-RACE. There is a 5-bp insertion in one of the splicing sites within the cDNA, leading to an alternative ORF with a longer and different carboxyl terminus. Putative protein kinase C–phosphorylation sites are underlined. Two arginine residues that could provide a nuclear localization signal are shown by asterisks. (B) Expression of CBE1 in bronchial epithelium analyzed by Northern blotting. Six micrograms of total RNA from human pulmonary mucoepidermoid cell line H292 cells (lane 1) or brushed bronchial epithelial tissues (lane 2) were electrophoresed through a 1% denaturing agarose gel, transferred onto a nylon membrane, and subjected to hybridization with a 32P-labeled DNA probe corresponding to bp 121–515 of A. A band of 0.7 kb in length is indicated by an arrow. The integrity of RNA samples used is shown by ethidium bromide staining before blotting. (C) Schematic representation of the intron/exon organization and comparison of amino acid sequences of splicing variants of CBE1. Blank represents untranslated regions, but the 5'-untranslated portion of NYD-SP22 v1 is different from that of the other three variants due to an alternate promoter. (D) RT-PCR analysis of the mRNA expression of ORF1 and ORF2 isoforms of CBE1 in human lung tissues. Total RNA was prepared from brushed lung epithelial tissues from four individuals (lanes 1 and 2, subjects with asthma; lanes 3 and 4, subjects without asthma). The isoform-specific primers were used for RT-PCR. PCR cycles were 30 for ORF1 and ORF2 of CBE1, and 20 for ß-actin. No amplification was observed without RT (data not shown). (E) Tissue distribution of CBE1 analyzed by RT-PCR. Lanes: 1, adrenal gland; 2, bone marrow; 3, cerebellum; 4, whole brain; 5, fetal brain; 6, fetal liver; 7, heart; 8, kidney; 9, whole lung; 10, placenta; 11, prostate; 12, salivary gland; 13, skeletal muscle; 14, spleen; 15, testis; 16, thymus; 17, uterus; 18, colon; 19, small intestine; and 20, stomach. Primers shown in Table 1 were used to detect expression of both variants of CBE1. PCR cycles were 35 for CBE1 and tektin, and 23 for ß-actin. PCR products were analyzed as in Figure 1.

 
The predicted amino acid sequences of all of these isoforms of CBE1 have no significant similarity to known protein families or domains, although cDNA sequences that can be assumed to be mammalian orthologs are found in the databases. Protein sequence analysis using PSORTII (http://psort.nibb.ac.jp/) resulted in the identification of no subcellular localization signals in ORF1 or ORF2, but both ORFs were predicted to have the highest probability of being nuclear-located using the k-nearest neighbor algorithm for assessing the probability of localizing at each subcellular location (23). Furthermore, both ORFs contain consensus sequences for recognition by protein kinase C. Although two other splicing variants in addition to CBE1 seem to be expressed in testis, the 0.7-kb forms appear to be predominantly expressed in bronchial epithelium when assessed by Northern blotting using a probe that could detect all splicing forms reported (Figure 2B). To assess the tissue distribution of CBE1, RT-PCR was performed on human tissue RNA samples using primers detecting both isoforms (Figure 2E). Although not quantitative, the result clearly shows that CBE1 is highly expressed in brain and testis in addition to lung; all tissues containing cilia. This was also the case with tektin, strongly suggesting that CBE1 is a cilia-associated gene. Interestingly, substantial expression of CBE1 was also observed in heart, kidney, and salivary gland, where expression of tektin was absent.

Expression in Adult Human Tissues by IHC
To analyze the protein products of CBE1 in human tissues, we raised polyclonal antibodies for CBE1 by immunization of rabbits with synthetic peptides. Because the two synthetic peptides are in the common region of ORF1 and ORF2, the polyclonal antibodies reacted with both isoforms of CBE1 that were produced in COS-7 cells by Western blotting (Figure 3). Although we also tried to immunoblot lysates of bronchial brushings and ALI-differentiated primary cells, we could not detect any specific signals, suggesting that the polyclonal antibody is not sufficiently sensitive to detect small quantities of endogenous CBE1 by Western blotting (data not shown). Using IHC, strong staining for CBE1 was observed in the epithelial cells of both nasal polyp and in intact epithelium of bronchial biopsies from subjects with and without asthma (Figure 4). Staining was strong in columnar cells in both bronchial and nasal polyp tissues, whereas little or no staining was observed in basal epithelial cells along the basement membrane. Careful observation of the stained sections revealed that not every columnar cell was equally positive; staining was much less in columnar cells in which obvious secretory granules are seen (i.e., goblet cells) (Figure 4D). To further characterize the epithelial cell type that expressed CBE1, sections were costained with either anti–ß-tubulin IV to identify ciliary structures (Figure 4F) or periodic acid-Schiff to identify mucus-secreting cells (Figure 4G). These costained sections clearly demonstrate that CBE1 is strongly expressed in ciliated epithelial cells but much less in goblet cells. Staining for CBE1 was mostly in the perinuclear areas of positive cells, but some signal may overlap with the hematoxylin-stained nucleus (Figure 4C). Pre-adsorption of the purified antibody with the immunizing peptides abolished epithelial staining (Figure 4H).



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  		Figure 3. Western blotting using anti-CBE1 antibodies. A recombinant plasmid encoding full-length ORF1 or ORF2, or vector alone (pcDNA3.1), was transiently introduced into COS-7 cells by lipofection. After 48 h after transfection, cellular extracts were analyzed by immunoblotting using anti-CBE1 antiserum or pre-immune serum. Bands corresponding to ORF1 and ORF2 are indicated by arrows. Lane 1, vector alone; lane 2, CBE1 ORF1; lane 3, CBE1 ORF2.

 


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  		Figure 4. Endogenous expression of CBE1 analyzed by IHC. Anti-CBE1 antibodies were used for detection of in vivo expression of CBE1 in adult human bronchi (A–D) and nasal polyp (E–H). Bronchial sections were obtained from normal subjects (A–C) and subjects with asthma (D). In both bronchi and nasal polyp, positive signals with brown staining are observed in columnar epithelial cells. No or little staining is observed in the basal epithelium, and staining is completely absent in mesenchymal and submucosal tissues underneath. Note that staining in goblet cells with evident secretory granules is less (arrows in D). The specificity of the antibody was tested by pre-absorption of the antibody with a molar excess of the immunizing peptide (H). Localization of CBE1 expression to ciliated epithelial cells is shown by costaining with ß-tubulin IV (red; F) and periodic acid-Schiff (purple; E, G). Note that most immunoreactive signals are in the perinucleus, but some staining inside the nucleus may be seen (arrows in C). ALI-differentiated primary epithelial cells, which were treated (J, L, N) or untreated (I, K, M) with IL-13 for 28 d, were also subjected to an immunostaining of CBE1 (I, J), ß-tubulin IV (K, L), or CBE1 plus ß-tubulin IV (M, N). Note strong signals of CBE1 in columnar cells with visible cilia on their apical surface without obvious staining in basal epithelium (I, M), and drastic reduction of staining by IL-13 treatment (J, N). Scale bars = 20 µm, except in panels A and E, which are 60 µm.

 
To verify the association of CBE1 expression with ciliated cells, we also analyzed ex vivo expression of CBE1 using well-differentiated primary bronchial epithelial cells by ALI culture. Strong staining was seen in the perinuclear region of columnar cells with visible cilia on their apical surface, whereas no staining was observed in basal cells (Figure 4I), resembling in vivo expression. Efficient ciliogenesis of the ALI culture was verified by strong expression of ß-tubulin IV (Figure 4K), and expression of CBE1 in ciliated cells was confirmed by costaining with ß-tubulin IV (Figure 4M). Chronic treatment of the ALI culture with IL-13, which is known to cause goblet cell hyperplasia at the expense of ciliated cells in an ALI culture model (21), drastically reduced expression of the CBE1 protein (Figure 4J) with concomitant downregulation of ß-tubulin IV (Figures 4L and 4N), reflecting downregulation of the mRNA of CBE1 and ciliated cell–associated marker genes (tektin and FOXJ1) by IL-13 (Figure 1B).

Expression of CBE1 in Asthma
As cDNA clones of CBE1 were identified as a result of overrepresentation in cDNA libraries derived from bronchial biopsies of normal subjects compared with subjects with asthma, we sought to confirm whether expression of CBE1 is downregulated in patients with asthma using RNA extracted from a separate collection of bronchial biopsies obtained from normal subjects (n = 10) or from those with asthma (n = 9). Quantitative RT-PCR analysis showed that expression levels of CBE1 were not different between normal subjects and subjects with asthma when normalized using ß-actin and 18S rRNA (Figure 5A). This was also the case for tektin (Figure 5B). On the other hand, the expression of MUC5AC was 3.0 times higher in subjects with asthma compared with normal individuals, although this was not statistically significant (Figure 5C). This is consistent with a previous report showing that goblet cell numbers are significantly increased in subjects with asthma (24). These results suggest that expression of CBE1 is not related to asthma.



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  		Figure 5. In vivo expression of CBE1 in bronchial biopsies from subjects with (solid bars) and without (open bars) asthma, analyzed by qPCR. The expression of CBE1 (A), tektin (B), or MUC5AC (C) in subjects with and without asthma is expressed relative to the geometric mean of 18S ribosomal RNA and ß-actin. Error bars represent 1 SD and P values were calculated using a Mann-Whitney U test for nonparametric data.

 
CBE1 Is Localized to the Nucleus or Perinucleus
To begin to explore CBE1 function, indirect immunofluorescence was performed using cells that were transiently transfected with a CBE1 expression vector. Figure 6 shows that in COS-7 cells transfected with ORF1 of CBE1, the majority of FITC-positive signals overlap with nuclei that were stained with 7-aminoactinomycin D, although the perinuclear region was stained in some cells (Figure 6B). No FITC signals were detected when the pre-immune serum was used instead of anti-CBE1 antibodies (Figure 6A). Immunofluorescence was also detected mainly in the nucleus when ORF2 was introduced (Figures 6G–6I). Similar nuclear localization was seen in bronchial epithelial–derived 16HBE 14o(–) cells (Figures 6J–6O). We also transfected monolayer-cultured primary bronchial epithelial cells with CBE1. Although transfection efficiency was very low (only 1–2% of cells were FITC-positive) due to the difficulty of transfecting primary cells, FITC signals were mainly observed in the cytoplasm, especially in the perinucleus (Figures 6P, 6R–6T, and 6X–6Z). Intranuclear staining was also observed in a small population of cells (Figure 6U–6W). Immunofluorescence in primary cells seems to reflect the subcellular localization observed using IHC, in that the staining is perinuclear in most cells, with nuclear staining observed in only a small population of cells (Figure 4).



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  		Figure 6. Intracellular localization of CBE1 analyzed by immuofluorescence. A recombinant plasmid expressing CBE1 ORF1 (A–F, J–L, P–W) or ORF2 (G–I, M–O, X–Z) was transiently introduced into COS-7 (A–I), 16HBE 14o(–) (J–O), or monolayer-cultured primary lung epithelial cells (P–Z) by lipofection. Twenty-four hours after transfection, cells were fixed and indirectly immunostained with anti-CBE1 antibodies or pre-immune serum (A). Nuclei were stained with 7-aminoactinomycin D. C and Q are interference contrast images of B and P, respectively. F, I, L, O, T, W, and Z represent merged images of D and E, G and H, J and K, M and N, R and S, U and V, and X and Y, respectively. No fluorescent FITC signals were detected without either primary or secondary antibodies (data not shown). In COS-7 and 16HBE 14o(–) cells, most FITC signals overlap with nuclear staining, but some are outside the nucleus (arrowhead). In contrast, most signals are perinuclear when expressed in primary epithelial cells, although intranuclear staining is also observed in some of the cells (U–W). Nuclear localization in primary cells was also observed for ORF2 (data not shown). Representative results from at least three independent transfection experiments are shown. Scale bars = 40 µm.

 
Identification of Nuclear Localization Signal by Mutational Analysis
Although no obvious nuclear localization signal is found in the predicted amino acid sequences of ORF1 and ORF2 of CBE1, both isoforms are relatively rich in arginine residues (16 in ORF1 and 19 in ORF2), and we hypothesized that this basic amino acid could be responsible for nuclear location. Thus, we substituted the two consecutive arginine residues (amino acids 88 and 89), which are in the common region of ORF1 and ORF2 (Figure 2A), for alanine. As the substitution occurred in one of the peptide sequences used to raise the anti-CBE1 polyclonal antibody, we fused the mutant or wild-type CBE1 with EGFP and transiently introduced the chimeric constructs into COS-7 cells to examine nuclear location. Figure 7 shows that 72.8 ± 9.1% of wild-type CBE1–EGFP transfected cells showed complete nuclear localization, compared with 18.8 ± 8.6% transfected with mutant CBE1–EGFP, which mostly showed a rather diffused fluorescence throughout cytoplasm. This indicates that the two consecutive arginine residues could serve as at least one nuclear localization signal for CBE1.



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  		Figure 7. Identification of a nuclear localization signal. COS-7 cells were transiently transfected with wild-type (A–C) or mutant (D–F) EGFP-CBE1. Twenty-four hours after transfection, the intracellular location of the fused proteins in live cells was observed using a fluorescent microscope (A, D) and nuclear localization was evaluated. Nuclei were stained with Hoechst 33,342 (B, E) and phase-contrast images were also taken (C, F). Statistical analysis from four independent experiments is shown in G. Scale bars = 40 µm.

 
Interrelationship between CBE1 and FOXJ1
As FOXJ1 is an important regulator of ciliogenesis, we tested whether CBE1 is under the control of FOXJ1. 16HBE 14o(–) cells were transiently transfected with an expression vector containing a full-length cDNA of human FOXJ1, and total RNA was isolated 24 h after transfection, followed by semiquantitative RT-PCR analysis. Transfection of FOXJ1 clearly induced tektin expression (Figure 8A), verifying our experimental system. However, expression of CBE1 was not detected in FOXJ1-transfected cells, suggesting that FOXJ1 alone is not sufficient to transactivate the promoter of CBE1. Given its nuclear localization, we postulate that the CBE1 protein may function as a transcription factor. However, introduction of an expression vector containing ORF1, ORF2, or both into 16HBE 14o(–) cells failed to induce the expression of tektin and FOXJ1 (Figure 8B). The same results were obtained when H292 cells were used (data not shown).



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  		Figure 8. (A and B) Forced expression of CBE1 does not induce tektin and FOXJ1, whereas FOXJ1 induces tektin in 16HBE 14o(–). Cells were transiently transfected by lipofection with a FOXJ1 (F), CBE1 ORF1 (1), CBE1 ORF2 (2), both ORFs of CBE1 (1 + 2)–expressing plasmid or pcDNA3.1 (vec). Total RNA was isolated 24 h after transfection, followed by semiquantitative RT-PCR analysis using primers for tektin, CBE1, FOXJ1, or ß-actin (Table 1). PCR cycles are 35 for tektin, CBE1, and FOXJ1, and 20 for ß-actin. Reaction products along with the positive control (ALI-differentiated primary cells at Day 21: ALI-21) were electrophoresed on a 1.8% agarose gel and ethidium bromide–stained. (C) Expression of CBE1 in 16HBE 14o(–) cells at 24 h after transfection was confirmed by Western blotting. One representative result from at least three independent transfection experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The critical role of the ciliated cell in mucociliary clearance is clearly demonstrated by the severe bronchitis and sinusitis that occurs in patients with the inherited disease, PCD (Online Mendelian Inheritance in Man [OMIM] #242650). PCD is an autosomal recessive and highly heterogenous disorder that is characterized by bronchiectasis and chronic sinusitis due to the long-term results of recurrent respiratory tract infections. In addition, half of the affected patients have situs inversus (reversed organs) (25). The chronic respiratory dysfunction and, in approximately half of the cases, male infertility can be explained by the abnormality of cilia structure and its resultant immotility. As expected, given the heterogeneity of the disease phenotype, many genetic loci have been suggested for PCD (26). Recently, mutations in several genes, especially those encoding components of dynein arms essential for ciliary beating, have been reported to be associated with PCD, including the axonemal dynein intermediate-chain gene 1 (DNAI1) (27) and the heavy dynein chain 5 (28). FOXJ1 is also a candidate gene for PCD as mice with targeted disruption of foxj1 show phenotypes similar to those of PCD, namely, defective cilia formation and situs inversus (10). However, mutation screening of the FOXJ1 gene in patients with PCD has failed to identify any mutations (29). DPCD, a recently identified gene whose knockout mice cause a PCD phenotype, could be a novel candidate for PCD, although no disease-associated mutations have been identified yet (30). The CBE1 gene identified in this study may also be a candidate gene for PCD, not only because of its spatial and temporal expression, but also its genetic locus on 9p12, adjacent to the DNAI1 gene (noted above). Given the juxtaposition of these two genes in a head-to-head orientation, it is possible that both genes may be under a common transcriptional regulation, although the temporal and spatial expression pattern of DNAI1 during ciliogenesis has not yet been investigated.

Using an ex vivo differentiation system of primary HBE cells, we have analyzed the expression profile of CBE1. Its expression during differentiation from basal to columnar phenotypes induced by ALI culture paralleled that of the ciliated cell markers tektin and FOXJ1 rather than goblet cell markers. FOXJ1 is a transcription factor localized to cells with motile cilia and necessary for ciliogenesis (10). Tektins are evolutionally conserved proteins that are associated with microtubules of cilia and flagella. Recently, human TEKTIN1 has been cloned and characterized and shown to be expressed in the cilia of HBE cells (31).

Although little is known about factors affecting differentiation of lung epithelial cells, several recent reports have shown that some Th2 cytokines induce goblet cell hyperplasia in vitro (20, 21). Laoukili and colleagues used human nasal epithelial cells to show that both IL-4 and IL-13 increase the population of goblet cells and decrease that of ciliated cells, accompanied by a decrease in ciliary beat frequency (20). We thus stimulated human lung epithelial cells on ALI culture with IL-13, and observed a marked upregulation of mucin genes with a corresponding downregulation of ciliated cell markers. Furthermore, we also observed a dramatic downregulation of immunostaining of the CBE1 protein in ALI-differentiated cells, accompanied by reduced staining of ß-tubulin. The dramatic reduction of CBE1 expression by IL-13 at both mRNA and protein levels provides further evidence that CBE1 is associated with ciliated cells.

Tissue distribution of CBE1 was analyzed by RT-PCR, which clearly suggested that it is expressed at higher levels in tissues that contain motile cilia (lung, brain, and testis) and is not expressed or is expressed at lower levels in other tissues examined. The expression pattern of CBE1 was similar to that of tektin, confirming that CBE1 is associated with ciliated cells. However, expression of CBE1 was also observed in salivary gland, kidney, and heart. Tektin is reported not to be expressed in kidney and heart (31, 32). Salivary gland is reported to have ciliated cells; an ultrastructural analysis of electron micrographs showed the 9 + 2 microtubule pairs typical for motile cilia (33). On the other hand, there are nonmotile sensory cilia on epithelial cells of the renal tubules, suggesting that CBE1 is expressed in not only motile but also sensory ciliated cells. Although substantial expression in the heart tissue was unexpected, primary cilia are present in both embryonic and adult heart (34); during development, motile and sensory monocilia are involved in the directional flow of extraembryonic fluid surrounding the node providing signals for left–right asymmetry and normal cardiac development (35).

Indirect immunofluorescence showed that, although exogenously expressed CBE1 resided mainly in the nucleus in COS-7 and 16HBE 14o(–), it was mostly cytoplasmic when expressed in primary epithelial cells. Given that endogenous expression was mostly in the perinucleus when assessed by IHC using tissue sections and ALI culture, it would be reasonable to conclude that CBE1 is mainly perinuclear-localized, at least in bronchial epithelial cells. On the other hand, its clear nuclear localization in cell lines and in a small percentage of primary cells suggests that localization of CBE1 is cell-type–dependent and that CBE1 might be a nucleocytoplasmic shuttling protein. We have previously found that expression of some well known nucleocytoplasmic factors, nuclear factor {kappa}B (36, 37), signal transducer and activator of transcription 6 (38), and p21waf (cyclin-dependent kinase inhibitor) (39), all showed predominantly perinuclear staining in epithelium using this immunohistochemical method in human bronchial biopsies. p21waf is reported to be translocated in the nucleus when transfected in cell lines (NIH3T3 and COS-7) (40). The possibility of CBE1 acting as a nucleocytoplasmic factor, and the stimuli controlling its shuttling, remain to be elucidated.

Despite the differential representation of CBE1 cDNA clones in random sequencing of biopsy libraries, we did not observe any difference in expression of CBE1 between bronchial biopsies from subjects with and without asthma. This may reflect the fact that the qPCR analysis was undertaken using biopsies from different volunteers from whom the initial cDNA libraries were constructed. On the other hand, expression of MUC5AC was higher in subjects with asthma compared with normal subjects, reflecting goblet cell metaplasia, a characteristic of asthmatic lung epithelium (24). The results of qPCR analysis suggest that CBE1 is not associated with goblet cells in vivo and that expression of CBE1 is not correlated with asthma, in contrast to our initial expectations.

Given that FOXJ1 is expressed in ciliated cells, that its temporal expression pattern precedes the appearance of cilia during embryogenesis, and that foxj1-null mice have defects in ciliogenesis (10), FOXJ1 is likely to induce the expression of ciliated cell–specific genes. Although the importance of FOXJ1 in ciliogenesis is undoubted, its in vivo molecular targets have not yet been well defined. An in vitro study using electrophoretic mobility shift assays and cotransfection reporter assays has established that FOXJ1 binds to and transactivates the promoters of bronchial epithelial–expressed genes, such as hepatocyte nuclear factor (HNF)-3ß and Clara cell secretory protein genes, but the expression of these two genes is not restricted to ciliated cells, and they seem not to be involved in the differentiation and/or function of ciliated cells (41). Thus, we examined whether FOXJ1 induced expression of CBE1 by transfection of 16HBE 14o(–) cells with an expression plasmid of human FOXJ1. However, CBE1 mRNA was not induced upon expression of FOXJ1 24 h after transfection, implying that another transcription factor(s), expressed only in well-differentiated cells, may be required, or both the factor(s) and FOXJ1 may be required for the expression of CBE1. On the other hand, endogenous expression of tektin was induced upon transfection with FOXJ1, indicating that tektin is a direct target of FOXJ1.

Due to its nuclear localization, CBE1 may be acting as a transcription factor. To investigate whether CBE1 might regulate the expression of ciliated cell–expressed genes, we transfected 16HBE 14o(–) cells with CBE1-cloned into a mammalian expression vector. However, this did not result in the induction of transcription of any ciliated cell marker genes tested. This does not exclude the possibility that CBE1 works as a nuclear factor, because we used 16HBE 14o(–) cells that do not develop cilia on ALI culture. Forced expression of CBE1 in primary lung epithelial cells under differentiation using a viral expression system would be of great interest.

Deduced amino acid sequences of both ORFs of CBE1 and those of other splicing variants of NYD-SP22 from testis have neither similarity to proteins that are deposited in public databases nor motifs that would be helpful in predicting the function of these proteins. Although there are orthologs of CBE1 in the mouse, rat, and monkey, similar proteins have not been identified in invertebrates or microbes, contrasting with components of cilia, such as tektin, which is conserved from sea urchin to mammals. Given this, and the IHC costaining using anti–ß-tubulin IV, it is clear that CBE1 does not constitute a component of cilia. Rather, judging from the presence of putative protein kinase C–phosphorylation sites, CBE1 might play a role in differentiation and/or maintenance of ciliated cells not only in the lung, but also in other motile and/or nonmotile cilia-containing tissues. The possible role of CBE1 in ciliogenesis and/or embryogenesis remains to be experimentally proven by loss-of-function analysis.


   Acknowledgments
 
The authors thank Dr. M. Quinlan for the method of immunofluorescence, and they acknowledge generous support from the Asthma, Allergy, and Inflammation Research (AAIR) and Hope Charities. H.Y. was funded by a fellowship from Kyowa Hakko Kogyo Co. Ltd, Japan; J.W.H. is a Medical Research Council (UK) Research Training Fellow.


   Footnotes
 
Conflict of Interest Statement: H.Y. was an employee of Kyowa Hakko Kogyo Co. Ltd. (Japan) and received a salary from 2001 to May 2002; S.M.P. has no declared conflicts of interest; S.J.W. has no declared conflicts of interest; H.M.H. has no declared conflicts of interest; R.M.P. has no declared conflicts of interest; D.I.W. has no declared conflicts of interest; A.P. has no declared conflicts of interest; A.E.B. was an employee of Pharmacia/Pfizer, Inc. from 1978 to September 2003 and received salary from Pharmacia/Pfizer, Inc., which originally contracted with University of Southampton for collection of samples from which the current study was derived. Pharmacia performed microarray experiments on the samples, and analysis of the resulting data, followed by Pharmacia exiting Inflammation as an area of research, and data from the studies was given to University of Southampton under an agreement which involved no monetary transfer; D.E.D. received £686,371 as a research grant from AstraZeneca, £167,000 as a research grant from Novartis, and $60,000 as a research grant from Aventis, and also consults for Synairgen; S.T.H. has no declared conflicts of interest; and J.W.H. has no declared conflicts of interest.

Received in original form February 9, 2004

Received in final form June 29, 2004


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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