Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Pulmonary emphysema is the abnormal, irreversible enlargement of distal air spaces of the lung, including respiratory bronchioles and alveolar ducts, accompanied by the destruction of their walls without obvious fibrosis. However, the main cause of the dyspnea is a diminished elasticity of the lungs which usually occurs as a consequence of cigarette smoking. Patients who have also a genetic deficiency in the antiprotease 1-antitrypsin (1-AT) have a very high risk of developing emphysema at an early age (1). Emphysema is a major component of chronic obstructive pulmonary disease (COPD) and the main risk factor for the development of this condition is inhalation of cigarette smoke. However, only 15 to 20% of smokers develop COPD, and the underlying genetic and environmental factors that determine its development are not well known (2). With the development of emphysema, changes in gene expression would be expected, but little research has been carried out in this area (although a recent study identified the plasma phospholipid transfer protein as being upregulated in emphysema [3]). To identify changes in gene expression associated with emphysema we used the technique of differential display (4). Such changes may be useful as biomarkers (5) in predicting the rate of loss of lung function, or the responsiveness to different therapies, and the genes themselves may be potential therapeutic targets and therefore help in disease management in the future.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Tissues

Lung samples distal to the hilum were obtained immediately after removal from patients with end-stage respiratory failure secondary to emphysema undergoing lung transplant at Harefield Hospital (Middlesex, UK). Control tissues consisted of similarly sampled pieces of donor lungs not required for the transplant. Tissue was snap-frozen in liquid nitrogen for subsequent RNA extraction. Paraformaldehyde-fixed sections were stained with hematoxylin and eosin. All patient samples had severe panacinar emphysema, and control tissues were found to show no evidence of pathology. The mean age of the emphysema patients (n = 5; three male and two female) was 53 yr (range between 47 and 55 yr) and that of the control donors (n = 5; three male and two female) was 42 yr (range between 18 and 57 yr). All the emphysema patients had forced expiratory volume in 1 s of less than 1 liter. Two of the emphysema patients were 1-AT-deficient. All the emphysema patients were ex-smokers, averaging 10 to 40 cigarettes per day. Four of the five patients were on inhaled steroids before transplantation. No details of smoking history or the use of steroid medications were available for lung donors. However, no evidence of smoking-related deposits was noticed during histologic examinations and RNA extractions.

Cells and Cell Culture

The adenocarcinoma alveolar epithelial cell line A549 (EECACC No. 86012804) was cultured in Dulbecco's modified Eagle's medium and 10% (vol/vol) fetal bovine serum in tissue culture flasks at 37°C and 5% CO₂ and stimulated with 1.0 µM dexamethasone for 24 h. The media for both control and stimulated cells contained 0.0016% (wt/vol) dimethyl sulfoxide as a solvent for dexamethasone. Cells were washed briefly with Hanks' balanced salt solution and were lysed directly into RNA lysis buffer.

Isolation of RNA, Reverse Transcription, and Polymerase Chain Reaction

For differential display and Northern blots, total RNA was isolated from approximately 1 g of frozen tissues and stored at 70°C, using guanidine isothiocyanate (RNeasy method; Qiagen, Crawley, UK). PolyA+RNA was isolated from total RNA using biotinylated oligo(dT) and streptavidin paramagnetic particles (PolyA-Tract messenger RNA [mRNA] isolation kit; Promega, Southampton, UK). For the analysis of human dexamethasone-induced transcript (DEXI) expression in cultured cell isolates and cell lines, total RNA was extracted using guanidine thiocyanate and treated with DNase-I to remove any contaminating genomic DNA (spin or vacuum total RNA isolation system; Promega). The concentration and purity of eluted RNA was determined spectrophotometrically (optical density [OD] 260/280 ratio between 1.8 and 2.0) and the quality of the RNA verified by denaturing agarose gel electrophoresis (28S/18S ratio between 1.5 and 2.5). Total RNA was reverse transcribed with an oligo-dT primer using an AMV RNase H- reverse transcriptase (RT) (ThermoScript; Life Technologies, Paisley, UK). The human DEXI primers were sense, 5'-TCTATGTTCTACGTGGGCCTGTTCTTCGTCA-3', and antisense, 5'-CAACCTCAGCACTCAGTCCCAATCTCTCTTC-3', which gave a 452-base pair (bp) product. The human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers were sense, 5'-CATCACCATCTTCCAGGAGC-3', and antisense, 5'-ATGCCAGTGAGCTTCCCGTC-3', which gave a 474-bp product. As a negative control, RT was omitted from the RT reaction. Polymerase chain reaction (PCR) was carried out on a thermocycler (PE Applied Biosystems 2400) using Taq Gold polymerase (PE Applied Biosystems, Warrington, UK) and complementary DNA (cDNA) from 175 ng of total RNA. Amplification was for 35 cycles for DEXI and 30 cycles for G3PDH. PCR productions were examined by agarose gel electrophoresis and stained with ethidium bromide.

Differential Display

RNA was treated with DNase-I to remove contaminating genomic DNA and the DNase-I heat inactivated. Reverse transcription and ³³P-labeled PCR was carried out using the RNAimage differential display kit-1 according to the manufacturer's instructions (Genhunter Corporation, Nashville, TN). The clone of interest, containing part of the 3' untranslated region (UTR) of the DEXI cDNA, was amplified. Differential display analysis on RNA extracted from emphysematous and control tissue was carried out using the antisense primer 5'-AAGCTTTTTTTTTTTA-3' for both the RT and PCR reactions, and the sense primer 5'-AAGCTTAACGAGG-3' was used in the PCR reaction. PCR products from individual patients were separated on 6% denaturing polyacrylamide sequencing gels (Strategene, Amsterdam, The Netherlands). Bands of interest were excised, reamplified, and cloned into the T-A vector pCR-II-TOPO (Invitrogen, Groningen, The Netherlands), and three clones were sequenced in both directions using the big dye terminator cycle sequencing ready reaction kit and amplitaq DNA polymerase FS, then run on an ABI 373XL stretch sequencer (all from PE Applied Biosystems).

Molecular Cloning of the DEXI cDNAs

Clones encoding the human and murine DEXI cDNA sequences were obtained by 5' rapid amplification of cDNA ends (RACE) from human and murine RACE-ready lung cDNAs, respectively (Clontech, Basingstoke, UK) according to the manufacturer's instructions. The primer used, 5'-ACGAGTAACCTGAAATGAAGGAGCGAGAATC-3', was derived from the sequence of the human DEXI differential display clone; and for the mouse, 5'-TTTTTTTGGAGTTGCTTACATTTTTTTAAT-3', derived from the sequence of a 3' murine expressed sequence tag (EST) (accession AW121786) that had similarity to the human sequence.

Amplification of DEXI Genomic DNA

The human DEXI genomic DNA sequence was obtained from human genomic DNA by PCR amplification using primers designed to the 5' and 3' ends of the cDNA sequence. The primers were: sense, 5'-CCACCCGCTGCATGCT-3'; and antisense, 5'-ATCCAAAGAAGTAAGCCTCCTAAGTATTGC-3'.

RNA Blots

RNA was separated by formaldehyde gel electrophoresis, transferred to Hybond-N membranes (Amersham, Little Chalfont, UK), and hybridized with an [-³²P]deoxyadenosine triphosphate-labeled DEXI cDNA probe or a 453-bp G3PDH probe used as a control. Probes were labeled using the Ambion strip-EZ DNA kit (AMS Biotechnology, Abingdon, UK). Membranes were hybridized overnight in 4× saline sodium citrate (SSC), 5× Denhardt's solution, and 0.5% (vol/vol) sodium dodecyl sulfate (SDS) at 65°C. Stringency washes were in 0.2× SSC and 0.1% SDS at 65°C for 20 min. Membranes were then exposed to Kodak BioMax MS film (Sigma, Poole, UK) at 70°C with intensifying screens and the resultant autoradiograms were scanned and analyzed with Scion Image software (Scion Corp., MD). An analysis of the tissue distribution of the human DEXI cDNA was carried out using a blot of human tissues containing 2 µg of polyA+RNA per lane (mRNA REAL blot; Invitrogen) using DEXI and -actin cDNA probes.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of a Gene Upregulated in Emphysematous Tissue

We identified a band that was upregulated in emphysema by differential display analysis on RNA extracted from four emphysematous and four normal lungs. This band was cloned, sequenced, and found to contain a novel 291-bp gene product. A PCR primer was designed from this sequence and used for 5' RACE on human lung cDNA. The human RACE product was cloned and sequenced. Both clones, taken together, encoded a 1,120-bp cDNA that we renamed DEXI in accordance with the wishes to the human gene nomenclature committee (Genbank accession No. AF108145) (Figure 1A). This gene was previously named MYLE (6). There was a polyadenylation signal at 1091- 1096. The DEXI open reading frame (ORF) encoded for a small, 95-residue acidic protein with a theoretical molecular mass of 10,429 D and an isoelectric point of 3.61 (Figure 1A). There was a 5' UTR in-frame stop codon at 45 before the first initiation codon on an overlapping human genomic DNA shotgun sequence (accession No. GA_x4HGKP3G4BF) (7). Taken together, the shotgun sequence and the DEXI cDNA sequence (up to nucleotide 356) formed a CpG island (69% GC).

View larger version (61K): [in this window] [in a new window]   View larger version (60K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 1.   The cDNA and encoded amino-acid sequences of the human (Hs) (A) and mouse (Mm) (B) DEXI cDNAs. * represents a stop codon. The in-frame stop codon tga and the polyadenylation signals are underlined and bolded (attaaa). (C) Alignment of the human and mouse DEXI proteins. The DEXI protein is predicted to contain a central transmembrane domain (underlined), a CT leucine zipper motif with four leucines (L) that contain a potential casein kinase II phosphorylation site (S) and a negatively charged CT (underlined residues). Identical residues are indicated by *, strongly similar residues by a :, and weakly similar residues by a period (.).

Because the first initiation codon of the ORF was in a relatively weak sequence context for an initiation consensus sequence (8), the murine DEXI cDNA was cloned for comparison. The human cDNA was searched against the murine EST database. A tentative murine electronic contig was derived and a 5' RACE primer designed from the 3' end of the contig. This primer was used in 5' RACE on mouse lung cDNA and a 1,095-bp cDNA was obtained (Genbank accession No. AF152470) (Figure 1B). There was an in-frame stop codon at 48 before the first initiation codon on the mouse sequence. There were three well-conserved regions between the cDNAs from two species that had greater than 82% identity at the nucleotide level. A 5' region, corresponding to nucleotides 8-429 on the human sequence, contained the ORFs. In the 3' UTR, regions 652-732 and 1049-1120 were also well conserved, suggesting that these regions of the mRNA may be important in regulating its stability and polyadenylation. The mouse ORF encoded a 95-residue protein (Figure 1B) with a theoretical molecular mass of 10,402 D and an isoelectric point of 3.61. The mouse and human predicted proteins were 94% identical (Figure 1C). Taken together, the identical length and high degree of homology of the human and mouse ORFs and the presence of 5' UTR in-frame stop codons suggest that the ORFs are correct. The DEXI proteins were predicted to contain a central transmembrane domain, a carboxy-terminal (CT) leucine zipper motif containing a predicted casein kinase II phosphorylation site and a negatively charged CT (Figure 1C).

Searches of the EST database with the cDNA and protein sequences revealed the presence of homologues only in mammals (human, mouse, cow, pig, and rat), but not in the genomes of Drosophila melanogaster and Caenorhabditis elegans. No other related genes were identified, suggesting the DEXI is the sole member of a novel gene family.

In view of the small size of the human DEXI cDNA sequence and predicted protein, the genomic DNA was examined for the presence of introns. Genomic DNA and cDNA isolated from A549 cells were amplified by PCR using primers designed to the 5' and 3' ends of the cDNA sequence. The amplicons from cDNA and genomic DNA were the same size, showing that the gene is intronless (Figure 2). Direct sequencing of these PCR products identified only one transcribed sequence, but the genomic sequence contained many pseudoheterozygous sequence differences, indicating that there are two genes in the genome (data not shown). The human DEXI gene has been localized to 15q11-q13, a region of chromosomal duplication (9). A search of the human genomic sequence showed that the complete DEXI genomic DNA sequence has not been finished. However, it identified a DEXI pseudogene on chromosome 15 (clone RP11-578F21, Genbank accession No. AC055876) (Figure 3A). The ORF of the DEXI pseudogene has 98% identity to the human DEXI ORF (Figure 3B), but it does not appear to be expressed inasmuch as there were no EST matches. However, the nucleotide sequence of the ORF is more conserved than the 3' UTR (Chi-squared with Yate's correction, P < 0.01), suggesting that either it has only recently lost its transcriptional activity or that it is expressed at low levels in rarely investigated tissues.

View larger version (53K): [in this window] [in a new window]   Figure 2.   The human DEXI gene has one exon. Agarose gel electrophoresis of PCR products amplified using primers designed to the 5' and 3' ends of the cDNA sequence. The lanes are: lane M, phiX174 DNA/HaeIII markers showing location of the 1,078-bp band; lane 1, RT-PCR from RNA treated with DNase-I isolated from A549 cells; lane 2, same as lane 1, but without RT; lane 3, human genomic DNA; lane 4, negative control with no DNA input. A 1,092-bp amplicon was obtained from both cDNA and genomic DNA.

View larger version (54K): [in this window] [in a new window]   Figure 3.   (A) The human DEXI pseudogene sequence and its potential ORF translated from the sequence of chromosome 15 clone RP11-578F21, Genbank accession No. AC055876 (nucleotides 62835-63951). (B) Alignment of the human DEXI protein with the potential protein sequence of the pseudogene. The pseudogene has 96% identity at the nucleotide level and 98% identity at the protein level with the DEXI cDNA.

Expression of DEXI in Tissues and Emphysema

On RNA blots the human DEXI cDNA had a single 1.3-kb transcript (Figure 4). The tissue distribution of the human DEXI cDNA was examined in heart, brain, liver, pancreas, placenta, and lung using a blot on which equal amounts of poly+RNA were loaded. It was present in all tissues examined. Expression levels were highest in heart and more than 3-fold lower in pancreas. However, when compared with the expression of the housekeeping gene -actin, the expression of DEXI was greatest in liver and lowest in placenta.

View larger version (69K): [in this window] [in a new window]   Figure 4.   DEXI expression in human tissues. A Northern blot with 2 µg of mRNA per tissue (equal amounts of RNA loaded) was probed with the human DEXI cDNA. The DEXI mRNA has a single transcript of 1.3 kb. For comparison, the expression of a housekeeping gene was examined using an 1.8-kb -actin cDNA probe. Size markers are in kb. Lane 1, heart; lane 2, brain; lane 3, liver; lane 4, pancreas; lane 5, placenta; lane 6, lung.

To examine the differential expression of DEXI in emphysema, an RNA blot of total RNA from human distal lung samples was probed with the human DEXI cDNA (Figure 5A). The level of expression of DEXI relative to G3PDH was 147% greater in the emphysematous lungs when compared with the control lungs (0.47 ± 0.02 standard error of the mean [SEM] versus 0.32 ± 0.06, arbitrary OD units; P < 0.05 using a two-tailed unpaired Student's t test) (Figure 5b), and relative to 28S ribosomal RNA, was 158% greater in the emphysematous lungs when compared with the control lungs (0.71 ± 0.02 SEM versus 0.45 ± 0.07; P < 0.01). There was no obvious difference in the level of DEXI expression between patients who were 1-AT-deficient and those who were not deficient.

View larger version (53K): [in this window] [in a new window]   View larger version (9K): [in this window] [in a new window]   Figure 5.   Human DEXI expression in emphysema and control lung tissues. (A) A Northern blot loaded with 15 µg total RNA was probed with the human DEXI cDNA. Samples were from five control lungs (lanes 1-5) and five emphysematous lungs (lanes 6-10). Samples in lanes 7 and 8 were taken from patients who have 1-AT deficiency. (B) Ratio of DEXI to G3PDH expression in control and emphysematous lungs. The horizontal lines represent the means.

Expression of DEXI in Cell Types

Overall, in human tissue libraries the DEXI mRNA is of low abundance, being expressed at a level of approximately 1.7% of that of the housekeeping gene G3PDH. This was ascertained by comparing the number of blast matches for the two sequences in the Genbank EST database. To determine which cell types in the lung may express DEXI mRNA and therefore may play a part in the upregulation of DEXI in emphysema, we examined its expression in various cell types by RT-PCR (Figure 6). DNase-1-treated RNA was used because the DEXI gene was found to be intronless. DEXI was expressed in cells derived from endothelium (12), epithelium, fibroblasts, and lymphocytes, and in the erythroleukemia cell line HEL, but not in the promyelocytic leukaemia cell line HL60. DEXI mRNA was not detected by RT-PCR in some, but not all, isolates of pulmonary artery smooth-muscle cells.

View larger version (43K): [in this window] [in a new window]   Figure 6.   Expression of DEXI in human cells. RT-PCR for DEXI (top panel) and G3PDH (bottom panel). The cell types examined were: lane 1, Daudi (Burkitt's lymphoma); lane 2, BGRL-169 (EB-transformed lymphocytes); lane 3, HL60 (promyelocytic leukemia); lane 4, HEL (erythroleukemia); lane 5, A549 (adenocarcinoma alveolar epithelial); lane 6, H322 (adenocarcinoma bronchial epithelial); lane 7, placental microvascular endothelial; lane 8, umbilical vein endothelial; lane 9, HFL-1 (pulmonary fetal fibroblasts); lane 10, pulmonary adult fibroblasts; lane 11, pulmonary artery smooth muscle (patient 1); lane 12, pulmonary artery smooth muscle (patient 2); lane 13, bronchial smooth muscle; lane 14, negative control; and lane M, phiX174 DNA/HaeIII markers.

Because the alveolar epithelial cell line A549 expressed DEXI, together with the evidence that airway epithelial cells respond to noxious stimuli by producing a variety of inflammatory mediators and antioxidants that are glucocorticoid-regulated (13), we sought to determine whether DEXI was glucocorticoid-regulated in this cell line by RNA blot analysis. Using dexamethasone as a reference steroid we found that DEXI mRNA was upregulated 230% relative to the expression of G3PDH by 1 µM dexamethasone treatment for 24 h (Figure 7).

View larger version (42K): [in this window] [in a new window]   Figure 7.   Human DEXI expression was upregulated by 1.0 µM dexamethasone treatment for 24 h in A549 human lung epithelial cell line. A Northern blot loaded with 2 µg polyA+RNA was probed with the human DEXI cDNA. The blot was stripped and reprobed with a control G3PDH cDNA probe. This result is representative of two similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We used the differential display technique to examine differential gene expression in lung samples taken from patients with emphysema. The novel DEXI gene that we identified was upregulated in emphysema, suggesting that it may play a role in this airway disease. We cannot rule out the possibility that the increase in expression of DEXI found in patient tissues was iatrogenic, because dexamethasone treatment of an airway epithelial cell line upregulated the expression of DEXI and most of the emphysema patients examined in this study were treated with inhaled corticosteroids.

The human DEXI gene has been localized to 15q11-q13, a region of chromosomal duplication (9). In this region many genes are imprinted, and abnormalities in imprinting led to the development of the Prader-Willi and Angelman syndromes. These syndromes are associated with significant developmental, behavioral, and mental problems (16). However, the active DEXI gene has been shown recently not to be imprinted (17).

The DEXI protein has little similarity to any known protein and therefore, on the basis of homology, no definite function or intracellular localization for this protein can be forecast. However, analysis of the protein sequence suggests the presence of a central transmembrane domain, a CT leucine zipper motif containing a predicted casein kinase II phosphorylation site and a negatively charged CT. A leucine zipper is a specialized coiled-coil motif in which the leucine side chains extending from one -helix interact and interdigitate with those from a similar -helix of a second polypeptide, facilitating dimerization, and the resulting structure formed by cooperation of these two regions forms a coiled coil (18, 19). The leucine zipper motif is present in many transcription factors and gene regulatory proteins (20). However, the presence of a predicted central transmembrane domain suggests that DEXI is not a nuclear protein. Because leucine zippers have also been implicated in oligomeric assemblies on membranes (21), DEXI is likely to be a membrane protein. The CT leucine zipper contains a predicted casein kinase II phosphorylation site which suggests that the DEXI protein's interactions, either with itself, by dimerization, or another protein, may be regulated by phosphorylation.

Acute exacerbations of underlying COPD are a common cause of respiratory deterioration, and patients have been shown to benefit from systemic corticosteroids that may act by decreasing airway inflammation (22). Further, inhaled and intranasal corticosteroids are some of the most efficacious treatments for airway inflammatory diseases (23), and about 15% of patients with COPD will respond to inhaled corticosteroids (24). The upregulation of human DEXI mRNA in the A549 lung epithelial cell line by the glucocorticoid analogue dexamethasone indicates that the DEXI promoter may contain functional glucocorticoid response elements and that DEXI is a potential marker for glucocorticoid responsiveness in treated patients and may play a role in modulating the function of inflammatory proteins. Recently, dexamethasone treatment of A549 epithelial cells has also been shown to increase the p65 subunit of the nuclear factor B, a transcription factor with a pivotal role in orchestrating immune and inflammatory processes, although the significance of this effect is not known (25). Both asthma and, to some extent, COPD are characterized by the presence of airway inflammation (26). Studies have shown that treatment of asthmatic patients with inhaled glucocorticoids inhibits the bronchial inflammation and improves their lung function. However, the inflammatory process in COPD appears to be resistant to the anti-inflammatory effects of glucocorticoids (30). Glucocorticoids upregulate the transcription of hormone-inducible genes through binding of the activated glucocorticoid receptor to glucocorticoid response elements located in the promoter region of the target genes (31). The mechanisms by which glucocorticoids reduce inflammation are many and varied, but the reduction in the expression of proinflammatory cytokines is thought to play a major role (14, 32, 33) although some anti-inflammatory mediators are upregulated by glucocorticoids (34, 35).

Future studies on the DEXI protein, both in vivo and in vitro, and its interactions with other proteins will lead hopefully to the identification of the function of this unique protein and its role in emphysema and inflammatory airway disease.