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Published online before print March 5, 2008, 10.1183/09031936.00172107
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Eur Respir J 2008; 32:121-128
Copyright ©ERS Journals Ltd 2008

Identification of transcripts overexpressed during airway epithelium differentiation

B. Chhin1,2,6, J. T. Pham1,2,6, L. El Zein1,2, K. Kaiser3, O. Merrot4 and P. Bouvagnet1,2,5

1 Laboratory of Cardiogenetics, Laboratory Team 4171, and, 3 Parasitology, University of Lyon 1, 2 Laboratory of Cardiogenetics, Methodology Research Team 0107, National Institute for Health and Medical Research, 4 Ear, Nose and Throat Service, Croix-Rousse Hospital, Hospices Civils de Lyon, 5 Laboratory of Cardiogenetics, East Centre of Biology and Pathology, Groupe Hospitalier Est, Hospices Civils de Lyon, Lyon, France. 6 Both authors contributed equally to this study.

CORRESPONDENCE: P. Bouvagnet, , Laboratoire Cardiogénétique, CBPE, Groupe Hospitalier Est, 59, boulevard Pinel, 69677 Bron, France. Fax: 33 427855900. E-mail: Patrice.Bouvagnet{at}recherche.univ-lyon1.fr

Keywords: Airway epithelium, cilia, human, representational difference analysis, transcriptome

Received: December 19, 2007
Accepted February 8, 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Support statement
 Statement of interest
 ACKNOWLEDGEMENTS
 REFERENCES
 
Human airway epithelium, the defence at the forefront of protecting the respiratory tract, evacuates inhaled particles by a permanent beating of epithelial cell cilia. When deficient, this organelle causes primary ciliary dyskinesia, and, despite numerous studies, data regarding ciliated cell gene expression remain incomplete. The aim of the present study was to identify genes specifically expressed in human ciliated respiratory cells via transcriptional analysis.

The transcriptome of dedifferentiated epithelial cells was subtracted from that of fully redifferentiated cells using complementary DNA representational difference analysis. In order to validate the results, gene overexpression in ciliated cells was confirmed by real-time PCR, and by comparing the present list of genes overexpressed in ciliated cells to lists obtained in previous studies.

A total of 53 known and 12 unknown genes overexpressed in ciliated cells were identified. The majority (66%) of known genes had never previously been reported as being involved in ciliogenesis, and the unknown genes represent hypothetical novel transcript isoforms or new genes not yet reported in databases. Finally, several genes identified here were located in genomic regions involved in primary ciliary dyskinesia by linkage analysis.

In conclusion, the present study revealed sequences of new cilia-related genes, new transcript isoforms and novel genes which should be further characterised to aid understanding of their function(s) and their probable disorder-related involvement.

The airway epithelium is a pseudo-stratified layer, consisting of specialised cell types, including basal cells, goblet/secretory cells and ciliated columnar cells. It plays a critical role in airway defence by protecting the respiratory tract from infections and damage induced by inhaled toxins, pathogens and particles. It constitutes a physical barrier against environmental aggression, through secreted factors that mediate the host immune system and through mucociliary clearance. On respiratory cells, ciliary beat defects cause a disease referred to as primary ciliary dyskinesia (PCD). Cilia are hair-like organelles which can be present on respiratory cells and on many other human cells. Cilia of all types exhibit numerous similarities, but they differ depending on their motility or sensory function. An increasing interest in respiratory epithelia has led researchers to elucidate genes acting in ciliogenesis.

Proteomic analyses have been used to identify components located in the axonemes or centrioles of cilia in humans or flagella in other well-known organisms 1–5. Comparative genomic searches led to the detection of genes conserved in the genome of ciliated organisms versus nonciliated organisms 6, 7. In order to reveal genes specifically expressed during flagellar regeneration or ciliogenesis, several studies have been carried out using various transcriptional strategies 8–12.

Mutations in several genes revealed by these studies turned out to cause diverse human ciliary diseases, such as polycystic kidney disease, retinal dystrophy, neurosensory impairment, Bardet–Biedl syndrome, oral-facial-digital syndrome type 1 and PCD, demonstrating that these genes should be considered in deciphering the aetiology of ciliopathies 13.

Using a different approach, to discover genes specifically expressed in human ciliated respiratory cells that could be responsible for human disorders, a method referred to as representational difference analysis (RDA) was used. This method, first described by Lisitsyn et al. 14, represents a process of subtraction coupled to amplification and was initially applied to the detection of differences between two genomes. Subsequently, Hubank and Schatz 15 adapted RDA for use with complementary DNA (cDNA) in order to isolate genes expressed differentially in two cell populations.

In the present study, the sequential culture system described by Jorissen et al. 16, in which epithelial cells covering the turbinates of the nasal cavity are dediffentiated in flat nonciliated cells and then redifferentiated in ciliated cells, was utilised. The transcriptome of flat nonciliated cells was subtracted from that of re-ciliated cells in order to characterise transcripts specific to ciliated cells. Differentially expressed genes were cloned and sequenced, resulting in the identification of bona fide and predicted genes. Moreover, genomic fragments that lay in intergenic intervals were cloned, suggesting the existence of new putative genes. The increased expression of some known and predicted genes during ciliogenesis was confirmed by real-time PCR validation studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Support statement
 Statement of interest
 ACKNOWLEDGEMENTS
 REFERENCES
 
Cell culture
Human respiratory cells from normal subjects were obtained from nasal turbinates, which were removed and discarded, thereby providing access to the ethmoidal sinus (at the Ear Nose and Throat Service, Croix-Rousse Hospital, Lyon, France). Patients were operated on for tumours located in the ethmoidal region and showed no respiratory disease. Cells were grown using the immerged cell culture method described by Jorissen et al. 16. Briefly, ciliated cells were isolated by pronase digestion and expanded in collagen-coated 25-cm2 flasks to dedifferentiate in nonciliated cells at 37°C under 5% CO2. When they reached 80–90% confluence, collagen was digested and cells were suspended in flasks with rotation (80 revolutions·min–1) at 37°C to redifferentiate in the form of ciliated vesicles. Nonciliated cells were collected at 80–90% confluence, when they stopped proliferating, and vesicles were collected when they were fully covered by cilia.

Isolation of mRNA and complementary DNA synthesis
RNA was extracted from nonciliated and ciliated cells using Extract-All® (Eurobio, Courtaboeuf, France), following the manufacturer’s instructions. Poly(A)+ mRNA was separated from total RNA using the Dynabeads Oligo(dT)25 purification kit (Dynal Biotech, Oslo, Norway) and its quality was assessed on an agarose gel. cDNA was synthesised from 2.85 µg poly(A) mRNA by oligo-deoxythymidine (dT) priming using SuperScript II Reverse Transcriptase as recommended by the manufacturer (Invitrogen, Grand Island, NY, USA). Double-stranded cDNA was prepared in an 80-µL total volume, containing 20 µL cDNA template, 400 µM deoxyribonucleoside triphosphates, 5 U DNA ligase (New England Biolabs, Ipswich, MA, USA), 24 U DNA polymerase (Invitrogen) and 1 U RNase H (Invitrogen). The reaction was performed for 2 h at 16°C and then supplemented by 6 U T4 DNA polymerase (Invitrogen) for an additional 30-min incubation.

The absence of genomic DNA contamination was confirmed by PCR using {alpha}-tubulin primers, which could amplify either a 320-bp cDNA fragment or a 468-bp genomic DNA fragment (protocol available on request).

Generation of difference products
cDNA RDA was performed on the basis of the protocol described by Hubank and Schatz 15 with slight modifications. Double-stranded cDNA (2 µg) from the two cell populations was digested with DpnII (New England Biolabs) to generate tester (ciliated cells) and driver (nonciliated cells) cDNA representations. In order to facilitate purification of the digested representations, primers pair sets were biotinylated and removed using a Streptavidin M-280 kit (Dynal Biotech), following the manufacturer’s recommendations. The first subtractive hybridisation tester:driver cDNA ratio was 1:50. In the second and third rounds of subtractive hybridisation, the ratio was increased to 1:500 and 1:250,000, and mung bean nuclease digestion of PCR products was omitted (detailed protocol available on request).

Cloning and DNA sequencing
The products of the third round of the PCR were digested with DpnII, and, to facilitate their identification, bands of 200, 300, 400 and 600 bp were separately gel-purified using a QIAquick gel extraction kit (Qiagen, Germantown, MD, USA). Purified products were shotgun cloned into a BamHI-digested dephosphorylated pBlueScript® II KS+ vector (Stratagene, San Diego, CA, USA) and used to transform DH5{alpha} One Shot competent cells (Invitrogen), according to the manufacturer’s protocol. Bacteria were plated on Luria–Bertani medium/ampicillin plates and colonies screened for inserts by SacII and XhoI double digestion, following conventional plasmid extraction.

Cloned products were sequenced using the M13–20 primer. The sequencing reaction was set up using the plasmid asa template and the Big Dye® Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. Sequence analysis was performed on a 3100 automated ABI sequencing apparatus (Applied Biosystems), and sequences were aligned using Staden 1.7.0 for Windows from the Staden Package 17 after extraction of primer and vector sequences.

Sequence analysis
Sequences were formatted using the FAST-All program 18 and compared to the public human genomic databases, Ensembl 19 and National Center for Biotechnology Information 20, with the nucleotide–nucleotide basic local alignment search tool (BLASTN).

Real-time PCR
Nonciliated and ciliated cells were collected, centrifuged to remove cell medium and washed in PBS (pH 7.4). The cell pellet was stored at -80°C until processing. Purified mRNA was prepared using the Chemagic mRNA direct kit (Chemagen, Baesweiler, Germany) following the manufacturer’s recommendations. DNA contamination was removed with a DNase I treatment (Invitrogen), and mRNA was quantified using an ND-1000 spectrophometer (NanoDrop, Wilmington, DE, USA). mRNA (10 µg) was reverse transcribed to generate cDNA using the Transcriptor First Strand cDNA synthesis kit (Roche Applied Science, Rotkreuz, Switzerland) and anchored oligo(dT)18, according to the manufacturer’s recommendations.

Real-time PCR was carried out in a LightCycler System® using the FastStart DNA Master SYBR Green I kit (Roche Applied Science). Reference and target gene primers were obtained from QuantiTect Primer Assays (Qiagen), which contained validated primers sets for the reduced glyceraldehyde-3-phosphate dehydrogenase (GAPDH), axonemal dynein intermediate chain 1 (DNAI1), glycoprotein nmb (GPNMB), retinitis pigmentosa GTPase regulator (RPGR), chromosome 7 open reading frame 49 (C7orf49) and chromosome 14 open reading frame 166 (C14orf166) genes. PCR reactions were set up in a total volume of 20 µL, containing 2 µL SYBR Green FastStart reaction mix, 2.4 mM MgCl2, 2 µL 10x primers mix and 2 µL cDNA. The temperature cycling profiles were as follows: 95°C for 10 min, 40 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 20 s. Melting curve analysis was carried out in the range 65–95°C to confirm the specificity of the PCR products.

Gene expression level was determined with the comparative threshold method, using the level of the housekeeping gene GAPDH as a reference value 21. The threshold cycle of PCR at which amplified product was first detected (Ct) was determined for the real-time PCR. The corrected Ct ({Delta}Ct) for each determination was then used to calculate the relative n-fold differential expression of a specific gene in a ciliated cell compared with a nonciliated cell sample and expressed as the ratio of the 2-{Delta}Ct values.

Statistical analysis
Data from triplicate experiments are presented as mean±SD. For each target gene, the 2-{Delta}Ct values of ciliated and nonciliated cells were analysed using an unpaired t-test with the significance set at a p-value of <0.05 for a one-tailed test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Support statement
 Statement of interest
 ACKNOWLEDGEMENTS
 REFERENCES
 
The goal of the present study was to identify genes specifically expressed in human ciliated respiratory cells. To this end, the transcriptome of dedifferentiated epithelial cells was subtracted from that of fully redifferentiated cells. The cDNA RDA procedure used in the present study was closely based on the protocol described by Hubank and Schatz 15, which permits the enrichment of transcripts specifically expressed in a cell type through iterative cycles of amplification/subtraction.

Determination of differentially expressed genes
Of the bacterial clones, 25% contained an insert. All clones with an insert (n = 78) were sequenced, including four chimeric clones that each contained two cDNA fragments. Altogether, 82 individual cDNA fragments were identified. Four of these cDNA fragments were recovered several times, and corresponded to the following genes: GPNMB (nine clones), zinc finger protein 236 (ZNF236; six clones), ribosomal protein large P0 (RPLP0; four clones) and ribosomal protein L14 (RPL14; two clones). Finally, 65 unique cDNA fragments were identified and mapped to the genomic human sequence by BLASTN. Of these, 53 (82%) cDNA fragments mapped to exonic sequences of known genes, seven (11%) to intronic regions or the boundary of an intron–exon junction and five (7%) to intergenic regions.

A list of the 53 cDNA fragments corresponding to exonic regions is presented in table 1Go. Genes are clustered by their described functions. It is notable that nine ribosomal proteins and an initiation factor involved in translation were identified. Genes related to immunity, inflammation and defence were also detected. The mitochondrial cluster includes seven genes encoding mitochondrial components. Among these, six are nuclear genes and one is mitochondrial (the mitochondrially encoded cytochrome b gene (MT-CYB)). Components of channels, transporters or related proteins are listed in one group, which includes two solute carrier family genes. Genes for cell signalling and signal transduction, such as those encoding spermatogenesis associated 13 (SPATA13) and testis enhanced gene transcript (TEGT; Bax inhibitor 1), are present. Several cytoskeletal genes, such as the actin gamma 1 (ACTG1), keratin and tropomyosin genes were evidenced. Despite the fact that ciliated cells do not replicate, several cell proliferation genes were found, including the non-metastatic cells 2, protein (NM23B) expressed in gene (NME2). Among the remaining genes of table 1Go, only one, the beta-tubulin gene (TUBB), had been previously demonstrated to be implicated in cilia. Finally, two genes corresponding to predicted open reading frames were identified, C7orf49 and C14orf166.


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  		Table 1— Genes overexpressed in human ciliated respiratory cells: complementary DNA fragments mapping to exonic regions

 
The cDNA fragments with sequences aligning to introns are reported in table 2Go. These cDNA fragments are presumably representative of new isoforms of mRNA. Only the cDNA fragment mapping to the exportin 6 gene (XPO6) includes intronic and exonic sequences. The six other fragments come from intronic regions. One fragment maps to a genomic region in which two genes overlap on opposite strands (family with sequence similarity 82 (FAM82B) and copine III (CPNE3)). Since the present cloning strategy was not oriented, it is not known whether this fragment is derived from one or the other gene.


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  		Table 2— Genes overexpressed in human ciliated respiratory cells: complentary DNA fragments mapping to intronic regions

 
The five cDNA fragments that mapped to intergenic regions are presented in table 3Go. The first two cDNA fragments (A and B) could represent a new 5’ or 3’ exon since the distance from the closest gene is at most 16 kb. By contrast, the last two cDNA fragments (D and E) are so isolated (minimum distance of 75 kb from the nearest gene) that they can only be segments of new genes.


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  		Table 3— Complementary DNA fragments mapping to intergenic regions

 
Real-time PCR validation studies
First, in order to validate the present culture system, expression of DNAI1 and RPGR, two genes which are known to be overexpressed in ciliated cells, was analysed. DNAI1 encodes a component of outer dynein arms in ciliary axonemes and is essential for ciliary function, whereas RPGR is specifically expressed in tissues containing cells with cilia or cilia-like organelles, such as retina, lung, cochlea and epithelial cells lining bronchi and sinuses 22. For data analysis, relative quantification of target gene transcripts was performed for each sample, with normalisation to GAPDH expression, since it is an endogenous unregulated gene transcript. By real-time PCR, it was found that these two genes exhibit a 3,696- and 83-fold relative increase, respectively (table 4Go).


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  		Table 4— Relative changes in expression of selected genes

 
Secondly, in order to validate the list of genes, the expression of one gene (GPNMB) and two open reading frames, namely C7orf49 and C14orf166, were evaluated by real-time PCR in nonciliated and ciliated cells. The three selected genes exhibited an expression in ciliated cells that was significantly higher than in nonciliated cells (fig. 1Go). GPNMB displayed a relative 7.89-fold increase, a change consistent with the 3.01–12.17-fold change reported by Ross et al. 12. C7orf49 and C14orf166 showed increased expression in human respiratory ciliated cells, with 2.94- and 4.92-fold relative changes, respectively (table 4Go).


Figure 1
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  		Fig. 1— Validation of the expression data for a number of the identified human ciliated cell genes: expression in nonciliated ({square}), and ciliated cells (blk34). Data are presented as mean±SEM. AU: arbitrary unit; DNAI1: axonemal dynein intermediate chain 1 gene; GPNMB: glycoprotein nmb gene; RPGR: retinitis pigmentosa GTPase regulator gene; C7orf49: chromosome 7 open reading frame 49 gene; C14orf166: chromosome 14 open reading frame 166. *: p<0.05 versus nonciliated cells.

 
Comparison with other studies on cilia
In order to confirm the present data, the genes list was compared to other lists obtained by various methods from the ciliated cells of various organisms (table 5Go). Ten out of 53 genes showing increased transcript synthesis in the present study had been reported once in other studies: annexin A1 (ANXA1); eukaryotic translation initiation factor 1 (EIF1); GPNMB; nebulette (NEBL); NME2; RPL14; secretoglobin family 1A member 1 (uteroglobin) (SCGB1A1); SPATA13; thymosin beta 4, X-linked (TMSB4X); and ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52) 1, 3, 11, 12. Two additional genes were mentioned in the studies of both Ostrowski et al. 1 and Pazour et al. 3: ribosomal protein SA (RPSA); and ACTG1. TUBB was also detected twice 4, 8. Finally, a computer-predicted gene, referred to as C14orf166, was mentioned three times 6–8.


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  		Table 5— Genes overexpressed in human ciliated respiratory cells: genes reported in previous studies

 
In agreement with the study of Pazour et al. 3, it was found that FK506 binding protein 1A, 12kDa gene (FKBP1A) showed increased expression during ciliogenesis 3. By contrast, Ross et al. 12 noted decreased expression (-2.33-fold) of this gene. Three genes that showed increased expression during ciliogenesis in the present study showed a reversed pattern in the study of Ross et al. 12: serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2; -2.82-fold), tropomyosin 1 (TPM1; -3.31/-3.73-fold) and TPM3 (-3.49/-3.21-fold) 12.

The identification of causal genes in PCD and situs inversus by positional cloning is difficult because of the potentially numerous genes involved in these diseases. As a consequence, it is important to note that 22 genes from the present series map to chromosomal regions which may contain a causal gene as determined by a positional cloning approach 23–26. These 22 genes are presented in table 6Go. Half of these have never previously been reported in studies aimed at characterising components specific to cilia/flagella. Among these 22 genes, four are located in chromosomal regions showing definite linkage in familial PCD: chemokine (CXC) ligand 17 (CXCL17), sulphide quinone reductase-like (yeast) (SQRDL), solute carrier family 6 (amino acid transporter), member 14 gene and TMSB4X 24–26.


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  		Table 6— Genes overexpressed in human ciliated respiratory cells: complementary DNA fragments mapping to loci related to known ciliary structure defects and diseases

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Support statement
 Statement of interest
 ACKNOWLEDGEMENTS
 REFERENCES
 
In the present study, RDA was used to identify genes differentially expressed in human ciliated respiratory cells. Subtraction of the nonciliated cell representation from that of ciliated cells resulted in the detection of 53 genes and 12 new coding sequences of known genes (n = 7) or putative new genes (n = 5). These 65 cDNA fragments are truly overexpressed in ciliated cells since a subset of three cDNAs were tested using real-time PCR analysis and all three were expressed at a significantly higher level in ciliated than nonciliated cells. In addition, 18 out of the 53 cDNAs had already been reported in other studies aimed at deciphering transcripts or proteins specific to ciliated cells. The remaining 35 cDNAs were newly identified genes overexpressed in ciliated cells. Of the sequences, 22 were located at loci related to known ciliary structure defects and diseases.

RDA is a method that is long and difficult to set up, but which is recognised as providing no false positives. Consistent with this observation, all of the cDNAs tested by real-time PCR had their overexpression confirmed, and 15 of the cDNAs of the present study had previously been reported in other studies as being overexpressed in ciliated cells. One limitation of this method is that there is selection of short cDNA fragments, meaning that there is no hope of obtaining a complete list of all overexpressed transcripts. Although microarrays provide a systematic view of transcript expression and can only detect preselected sequences, RDA has the power to detect additional expressed sequences either as novel transcript variants of known genes or even as new genes.

In table 1Go, a set of genes that could be linked to cells with cilia/flagella are reported. Indeed, seven genes encoding proteins of the mitochondrial respiratory chain and oxidative phosphorylation system were identified. In ciliated cells, increased ATP production is presumably necessary for intracellular and intraflagellar transport and ciliary beating. Mutations in ACTG1, a major component of sensory ciliated cells of the cochlea, have been described as causing dominant deafness 27. Finally, NME2 was demonstrated to be involved in spermiogenesis and flagellar movement 28, and TMSB4X was found to be highly represented in lung parenchyma and unrelated tissue types relative to the bronchial epithelium in a previous study 11.

For the real-time PCR analysis, two positive controls that have previously been described as showing increased expression in ciliated tissues, DNAI1 and RPGR, were used. Two other genes, GPNMB and C14orf166, have been reported, in others studies, to show increased expression in ciliated cells, but their function in ciliogenesis remains to be elucidated. GPNMB, a transmembrane glycoprotein, was hypothesised to be involved in growth delay and reduction of metastatic potential 29, but its role in ciliogenesis remains elusive. The C14orf166 and C7orf49 genes were predicted by bioinformatic searches of the human genome. C14orf166 is frequently detected during ciliogenesis since it has been reported by three other studies. This gene encodes protein involved in the functional regulation of human ninein in the centrosome structure 6–8, 30. It would be interesting to obtain complete data on C14orf166 and elucidate the biological function of C7orf49.

Finally, five totally new genes (~10% of the whole set) were detected. Further work is warranted to characterise in detail these putative new genes, in particular sequence B, which is located in a chromosomal region implicated in situs inversus, a disturbance of lateralisation which can be secondary of ciliary dysfunction in the early embryo.


   Support statement
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Support statement
Statement of interest
 ACKNOWLEDGEMENTS
 REFERENCES
 
B. Chhin was supported by a stipend from the French Association against Myopathies (Evry, France) between 2004 and 2007. The Laboratory of Cardiogenetics (Hospices Civils de Lyon, Lyon, France) is supported by grants from Scientific Interest Group – Rare Diseases 2003 and Hospital Project of Clinical Research regional 2003 (Paris, France), and the Laboratory of Cardiogenetics (University of Lyon 1, Lyon, France) by grants from the ProKartagener Foundation 2005 (Geneva, Switzerland) and Renaud Febvre Foundation (Les Etards, France).


   Statement of interest
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Support statement
 Statement of interest
ACKNOWLEDGEMENTS
 REFERENCES
 
None declared.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Support statement
 Statement of interest
 ACKNOWLEDGEMENTS
REFERENCES
 
The authors warmly thank S. Picot for the contribution of the Parasitology Laboratory (University of Lyon 1, Lyon, France) to the real-time PCR experiments.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Support statement
 Statement of interest
 ACKNOWLEDGEMENTS
 REFERENCES
 

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