From the Center for Comparative Respiratory Biology
and Medicine, Division of Pulmonary & Critical Care Medicine,
School of Medicine and the ¶ Department of Pathology, Medical
Center of the University of California, Davis, California 95616
Received for publication, October 15, 2002
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ABSTRACT |
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Retinoids, such as all-trans-retinoic
acid, play an essential role in the regulation of airway epithelial
cell growth, differentiation, and gene expression. Using cDNA
microarray, we identified a clone, DD4, that contains the cDNA of a
novel gene, spurt (secretory protein in upper respiratory
tracts) that was significantly induced by
all-trans-retinoic acid in primary cultured human
tracheobroncheal epithelia. Two alternatively spliced spurt
transcripts of 1090 and 1035 base pairs exist that contain the same
open reading frame expressing a 256-amino acid peptide. The full-length
spurt cDNA sequence spans a genomic DNA fragment of
7,313 bp, and the gene is located on chromosome 20q11.21.
spurt mRNA is notably expressed at high levels in human
nasal, tracheal, and lung tissues. In situ hybridization
demonstrated that spurt message is often present in
secretory cell types. The human spurt gene product is a
secretory protein that contains a distinct signal peptide sequence in
its first 19 amino acids. Mono-specific antibodies were generated to
characterize spurt expression. Our data demonstrate that
spurt is secreted onto the apical side of primary human
airway epithelial cultures and is present in clinical sputum samples.
spurt gene expression is higher in sputum and tissue
samples obtained from patients with chronic obstructive lung disease.
Our results provide the cloning and characterization of this
tissue-specific novel gene and its possible relationship with airway diseases.
The mucociliary functions of conducting airway epithelia play an
essential role in pulmonary defense against various inhaled pollutants.
It has been reported that basal, ciliated, and secretory cells form a
coordinated airway defense mechanism against inhaled air pollutants
(1). Goblet cells in the airway epithelium and secretory cells in the
submucosal glands are the major contributors to mucus secretion (2, 3).
The secretory products contributed by each cell type are currently
unknown. Therefore, identifying secretory products for each cell type
provides a marker to either assess the functional role of a specific
cell type in maintaining homeostasis of mucociliary function or
evaluate its role in the pathogenesis of various airway diseases.
Airway epithelial secretory proteins are divided into two major
classes: mucins and non-mucins. Mucins are major secretory components
that play an essential role in protecting and lubricating the airway
epithelial surface. Mucins constitute a very large molecular weight
glycoprotein family that are poly-dispersed and heavily
O-glycosylated (4, 5). Currently, there are at least 12 mucin genes identified. MUC1, MUC2,
MUC4, MUC5AC, and MUC5B are expressed
at relatively high levels in the human respiratory tracts compared with
other mucin genes (6-10). Non-mucin secretory proteins include
lysozyme, lactoferrin, peroxidase, and antileukoprotease. These
proteins contribute directly or indirectly to the pulmonary defense
mechanism against infectious pathogens (12, 13). The cell of origin for
most of these non-mucin secretory proteins is the serous cell contained
within submucosal glands (11-13). However, there is still a large gap
of information about the nature of these secretory proteins and their
role in pulmonary defense.
Vitamin A and its metabolites, retinoids, are critical to the
maintenance of mucociliary functions and secretions in the airway epithelium. In vitamin A-depleted animals, the airway epithelial surface changes from a mucociliary cell layer to a protective, squamous
cell surface. This alteration can be reversed upon the addition of
retinoids, such as all-trans-retinoic acid
(ATRA)1 (14). Similar changes
can be demonstrated in vitro in primary cultures of airway
epithelial cells derived from humans and other species (15-17).
Despite efforts by researchers, effects of retinoids on airway
epithelia are not completely understood. We propose that retinoids
induce differentiation through sequential changes in gene expression.
This is based on observations that retinoid administration coincides
with airway cell differentiation.
To test this hypothesis, we employed a two-color-based differential
hybridization on a high density cDNA microarray membrane, which
contained 30,000 cDNA spots (18, 19). These cDNA clones were
individually selected from a pool of cDNA libraries created from
human tracheobroncheal epithelial (TBE) cells. The membrane was
differentially hybridized with cDNA probes derived from
ATRA-treated and -depleted cultures of primary human TBE cells. Several
ATRA-responsive cDNA clones were identified, and one of them, the
DD4 clone, encoded the cDNA sequence of a novel gene. In
this report, we characterize the expression of this novel gene in
airway epithelia. Results from our studies suggest that the
DD4 clone encodes a novel gene product that corresponds to a
secretory protein specifically produced by epithelial cells of the
upper respiratory tract. Based on the characteristics of this gene, we
subsequently named this gene spurt (secretory
protein in upper respiratory
tracts).
Sources of Human Airway Tissues and Cells--
Human nasal,
tracheobronchial, and lung tissues were obtained from the University of
California at Davis Medical Center or from the Anatomic Gift Foundation
(Laurel, MD) with consent. The Human Subject Review Committee of the
University of California at Davis approved all procedures involved in
tissue procurement. Excised tissues were transported to the laboratory
in an ice-cold, minimal essential medium (Sigma). Primary human
TBE cells were isolated from these tissues by a protease dissociation
procedure and cultured in a serum-free hormone-supplemented medium as
previously described (1, 17). Normally, the cells were grown in 6-well tissue culture plates either without additional substratum (TC) or with
collagen gel substratum (CG) or in air-liquid biphasic culture insert
chambers (TranswellTM chamber, Corning/Costar 3450, Corning, NY) without (BI) or with collagen gel substratum (BI-CG). The
modified serum-free medium contained 1:1 of Ham's F-12/Dulbecco
modified Eagle's medium and supplemented with insulin (5 µg/ml),
transferrin (5 µg/ml), epidermal growth factor (10 ng/ml),
dexamethasone (0.1 µM), cholera toxin (20 ng/ml), bovine
hypothalamus extract (15 µg/ml), and ATRA (30 nM). This
growth-optimized culture medium allows airway epithelial cells to
express mucociliary differentiation in culture within 2-3 weeks of
incubation (15, 20, 21), especially under BI-CG conditions. For both BI
and BI-CG culture conditions, the cells were immersed in the culture
medium for 7 days, and then the TranswellTM chambers were
lifted up in between the air and liquid interface for the remaining
days in culture. For dose response and time course experiments, the
cultures were maintained for 7-10 days without ATRA supplementation
followed by the addition of ATRA at various concentrations (1-1000
nM) as described in the experiments. For ATRA-depleted
cultures, the cells were plated in the medium without ATRA.
The immortalized normal human TBE cell line HBE1 (22) was obtained from
Dr. J. Yankaskas (University of North Carolina, Chapel Hill, NC). HBE1
cells were maintained in serum-free Ham's F-12 medium supplemented
with six hormonal supplements as described previously (17). To induce
mucous cell differentiation in this cell line, ATRA (30 nM)
was added to the medium, the cells were plated on collagen gel-coated
TranswellTM chamber and maintained in an air-liquid interface as
described above.
RNA Isolation and Northern Blot Hybridization--
At varying
times and treatments, the cells were harvested for total RNA isolation
by a single-step acid guanidium thiocyanate extraction method (23). For
Northern blot hybridization, equal amounts of total RNA (20 µg/lane)
were subjected to electrophoresis on a 1.2% agarose gel in the
presence of 2.2 mM formaldehyde and transferred overnight
to a Nytran N+ nylon membrane. The transferred RNA was fixed and
cross-linked to the membrane by UV cross-linking using UV Stratalinker
2400 (Stratagene, La Jolla, CA). The cDNA probes were labeled with
[ Identification of ATRA-responsive Genes in Human TBE
Cells--
Thirty thousand cDNA clones were derived from primary
human TBE cells that had been cultured for more than 3 weeks under an air-liquid interface culture condition in the completed medium containing both the hormonal supplements and ATRA. Under this in
vitro culture system, the human TBE cells differentiated into a
mucociliary epithelium resembling that seen in vivo. The
cDNA clones were packaged using a pBK CMV phagemid packaging system (Stratagene, La Jolla, CA).
From the 30,000 cDNA clones, we developed a high density microarray
membrane (3.1 × 4.6 cm, Nytran N+ nylon membrane; Schleicher & Schuell). This 30,000 cDNA high density microarray membrane was
hybridized simultaneously with two-color cDNA probes derived from
ATRA-treated (magenta) and untreated (cyan) cultures of primary human
TBE cells as described before (18). Based on a quantitative ratio of
cyan/magenta either greater than 5 or less than 0.2, clones, including
our novel gene, were selected for further characterization.
In Situ Hybridization--
cDNA clones obtained from the
original phage library screening were converted to phagemids according
to the manufacturer's protocol (Stratagene). The recombinant plasmid
was linearized with EcoRI or XhoI to generate
antisense and sense templates, respectively. The linearized templates
were in vitro transcribed with T7 and T3 RNA polymerases
using MAXIscriptTM according to the manufacturer's
recommendations (Ambion Inc., Austin, TX) to produce
35S-UTP-labeled antisense and sense cRNA probes,
respectively. In situ hybridization was carried out as
described previously (24-26).
Cloning and Sequencing of spurt cDNA--
A human airway
epithelial cell-specific cDNA library was screened to isolate
spurt cDNA using a
The sequence data were analyzed with both Geneworks
(IntelliGenetics, Inc., Mountain View, CA) and Lasergene (DNASTAR
Inc., Madison, WI) software programs as well as with the online GCG software package SeqWeb (GCG, Madison, WI). Sequence homology to
published sequences in public data bases was analyzed and determined by
the BLASTn or BLASTp programs at the National Center for Biotechnology Information through internet services.
5'-Primer Extension and 5'-Rapid Amplification of cDNA
Ends--
The sequence of the full-length cDNA was determined by
5' extension of RNA from human TBE cells cultured under the BI-CG
condition with 30 nM ATRA, using an end-labeled antisense
primer (5'-GGCTAACAGCCCGTAGAAGAC-3') and the dideoxynucleotide chain
termination method according to the manufacturer's recommendations
(Promega, Madison, WI). A base pair ladder was sequenced in parallel
with the 5' extension product TaqtrackTM sequencing kit
(Promega).
For 5'-rapid amplification of cDNA ends, the marathon kit
(Clontech) was used to synthesize the first strand
cDNA from mRNA (1 µg) isolated from primary cultures of human
TBE cells, which were cultured under air-liquid interface culture
conditions and 30 nM ATRA supplement for 21 days (17).
Oligo(dT) primers were used to initiate first strand cDNA
synthesis. This was followed up by second strand cDNA synthesis and
adaptor ligation to terminal ends. PCR with nested primers was
performed for product amplification. The PCR products were subcloned
into the TA vector (Invitrogen, San Diego, CA) for cloning and DNA sequencing.
In Vitro Transcription-Translation of spurt
Protein--
spurt protein was transcribed and translated
in vitro by combining the MAXIscriptTM T7/T3 kit and Retic
Lysate IVT kit (Ambion). Template DNA was obtained by subcloning the
ORF fragment of spurt cDNA into a pcDNA3 expression
vector (Invitrogen). 1 µg of DD4 (spurt) DNA template was
then mixed with the corresponding RNA polymerase, rNTPs, and
transcription buffer, and the 20-µl reaction mixture was incubated
for 45 min at 37 °C.
In vitro transcribed RNA (0.5 µg/reaction) was then
labeled with 4 µl (1200 Ci/mmol) of [35S]methionine
(ICN), reticulocyte lysate, and master mix containing 200 mM creatine phosphate, 3 M potassium acetate,
10 mM MgCl2, and an amino acid mixture (2 mM) (Ambion) for 60 min at 30 °C. Adding 1/10
volume of 1 mg/ml RNase A and continuing the incubation at 30 °C for
10 min more terminated the translation reaction. Four volumes of
Laemmli SDS sample buffer (2.5% SDS, 0.1 M Tris, pH 6.8, 10% glycerol, 350 mM 0.025% Construction and Transfection of a spurt FLAG-tagged Fusion
Protein--
A fusion protein with FLAG attached to the N terminus of
spurt protein was made to identify the size of
spurt protein. For FLAG tagging, the entire coding region of
the novel cDNA was fused in frame 3' to a FLAG tag in the vector
pFLAG CMV2 (Sigma). To prepare the FLAG-spurt expression
vector, the ORF fragment of spurt was amplified by PCR using
appropriate primers (forward Primer DD4FLAG-1F,
5'-ACGAATTCGTTTCAAACTGGGGGCCTC-3'; reverse primer DD4FLAG-2R,
5'-TGCTCTAGAGGCACATGGGATGTTACAC-3') containing EcoRI
and BglII sites at the 5'- and 3'-ends, respectively. The PCR products were inserted into EcoRI/BglII sites
of the pFLAG vector (Sigma). Modification was made to place a perfect
Kozak sequence (5'-CCACC-3') (28) immediately upstream of the
presumed translational start codon of the ORF of spurt. The
vector-insert junction was verified by nucleotide sequencing. For
transient transfection studies, 0.5 µg of FLAG-spurt
plasmid was transfected into 5 × 104 HBE cells in a
35 mM Petri dish with LipofectAMINE (Invitrogen). Two days
post-transfection, one dish from each transfection group was harvested
for protein isolation and Western blot analysis.
Polyclonal Antibody Production and Western Blot Analysis--
A
16-mer oligopeptide antigen was synthesized (Research Genetics, Inc.,
Huntsville, AL) using the deduced amino acid sequence from
Arg165 to Cys180 of spurt. The
peptide was conjugated to multiple antigen peptide to increase its
antigenicity, and rabbit-based polyclonal antibodies were generated as
described before (29). The specificity of the polyclonal antiserum was
determined by enzyme-linked immunosorbent assay and Western blot analysis.
For Western blot analysis, cultured cells were harvested as described
(30, 31). Supernatant protein concentrations were determined by the
method of Lowry using the Bio-Rad Dc assay. Equal protein
amounts were subjected to discontinuous SDS-polyacrylamide gel
electrophoresis according to Laemmli (27). The proteins were blotted
onto polyvinylidene difluoride or Nytran membranes according to the
manufacturer's recommendations with a semi-dry blotting apparatus
(Schleicher & Schuell) at 120 mA/45 min/10-cm2 gel surface
area. Western hybridization was done using a Vectastain ABC kit (Vector
Laboratories, Inc., Burlingame, CA) and the appropriate primary and
secondary antibodies.
Sputum Sample Preparation--
Sputum samples were collected
from subjects using a protocol approved by the University of California
at Davis Institutional Human Subjects Review Board. Subjects were
randomly asked to generate sputum during a routine visit to a general
pulmonary clinic. Sputum generation was directly observed to ensure
collection of an adequate specimen. The samples were discarded if the
fluid viscosity was more consistent with saliva compared with sputum.
All expectorated sputum samples were immediately stored on ice. During
preparation, sputum samples were placed in a shaking water bath at
37 °C for 20 min to ensure complete homogenization and in the
presence of a protease inhibitor mixture that included 1 µM iodoacetamine, 25 µg/ml aprotinin, 10 µM leupeptin, 5 mM phenylmethylsulfonyl fluoride, and 10 mM dithiothreitol to reduce protein
degradation and disulfide cross-links (Sigma). The samples were spun to
separate gel/mucin/cells from the soluble phase (32, 33). The
protein concentration of the soluble portion of the sputum sample was determined by Lowry's method (Bio-Rad). From the soluble portion of
the sputum samples, 30 µg of total protein was used for SDS-PAGE and
Western blotting.
Isolation of an ATRA-responsive Gene--
A high density DNA
microarray membrane containing 30,000 cDNA clones derived from
cDNA libraries of human TBE cells was used to differentially screen
for ATRA-responsive genes (18, 19). One of the ATRA-inducible novel
genes, DD4, was selected for further studies. Northern blot
hybridization with RNA isolated from primary human airway epithelial
cultures treated with or without ATRA (30 nM) confirmed
that the expression of DD4 is strongly induced by ATRA (Fig.
1A). The RNA staining gel
(Fig. 1B) confirmed equal loading of the RNA samples and
lack of any RNA degradation. To further confirm the ATRA-responsive
nature of DD4, we examined DD4 induction at a
variety of ATRA concentrations and times. Northern blot hybridization
revealed that the DD4 message was strongly induced by ATRA
in a dose-dependent manner (Fig.
2A). The induction could be
seen at concentrations higher than 30 nM. DD4 gene
expression was detected 7 days after starting retinoid treatment, which
indicates that induction by retinoids is a late event (Fig.
2B). Other ATRA derivatives, such as
all-trans-retinol and 9-cis-retinoic acid, also
stimulate the expression of this novel gene (data not shown).
In addition, cell culture conditions had an effect on DD4 mRNA
expression levels. The highest induction of DD4 message was observed when cultured cells were maintained on collagen gel under air-liquid interface culture conditions (BI-CG). Other culture conditions, such as plating on CG substratum alone or with an air-liquid interface alone (BI), resulted in lower levels of DD4 gene
expression. Expression was the lowest in cells plated on a TC plastic
surface. Despite these changes in DD4 gene expression, quantitative results demonstrated that ATRA enhanced DD4
mRNA expression at least 10-fold in all culture conditions (Fig.
1C).
Expression Pattern of spurt mRNA in Normal Tissues--
To
identify the expression pattern of the DD4 gene in normal
tissues, we employed a multiple-tissue Northern blot hybridization. Using a human tissue distribution membrane consisting of total RNA
derived from various normal human tissues, Northern blot analysis revealed significant DD4 message levels in the trachea,
bronchi, and lung. No hybridization signal was observed in RNA samples harvested from other tissues (Fig. 3).
Similar results were obtained using the human DD4 probe
against a murine multiple tissue distribution membrane, which included
14 different mouse tissues. DD4 expression in the mouse was
observed only in the nasal septum, trachea, and lung (data not shown).
In addition, nasal epithelial cells freshly obtained from human nasal
turbinates and nasal polyps exhibited relative high DD4
expression levels by Northern blot analysis. These observations suggest
that DD4 expression is specific to the conducting
airways.
In situ hybridization demonstrated that DD4
mRNA is specifically expressed in the secretory ducts and
submucosal glands of normal human tracheobronchial tissue sections
(Fig. 4). For comparison, in
situ hybridization with a sense cRNA probe did not display an
observable signal when compared with background (data not shown). In situ hybridization of tissue sections from "normal"
patients showed low abundance DD4 message in the surface
epithelium. Furthermore, the expression level of DD4 is
highest in the trachea and decreases progressively from the proximal
(bronchial region) to distal (bronchiolar region) airways in the human
lung (Fig. 5). This pattern closely correlates with previous reports on serous cell distribution patterns in the respiratory tract (12, 34).
Expression Pattern of spurt mRNA in COPD
Tissues--
Importantly, although the gene expression of
DD4 was generally confined to the submucosal gland region of
normal human tracheobronchial tissues, we were able to detect elevated
DD4 message in the airway surface epithelia in all tissue
sections obtained from four patients with chronic obstructive pulmonary
disease (COPD). For in situ hybridization, we labeled
DD4 riboprobes by an enzymatic method, obtaining concurrent
views of both staining pattern and histological structure. The observed
positive staining using antisense probe (Fig.
6A) indicated specific DD4
gene expression when compared with the negative staining of the sense
probe (Fig. 6B). DD4 message was elevated in both
the surface epithelium and the submucosal glands as shown in Fig. 6
(C and D). These results suggest that an increase
in DD4 mRNA expression is associated with COPD.
Molecular Characterization of Full-length spurt cDNA--
Our
original search in the GenBankTM data base found three
expressed sequence tag (EST) clones, (GenBankTM accession
numbers AA315985, N27741, and N23239) with nucleotide sequences
homologous to the preliminary DD4 sequence (954 bp) (Fig.
7A). To further characterize
the full-length cDNA sequence of DD4, several different
approaches were used. These approaches included further cDNA
library screening, rapid amplification of cDNA ends, and 5'-end
primer extension.
Through cDNA library screening, we isolated four
DD4-like clones (clone DD4-C1 to DD4-C4). Sequence
information of these cDNA clones revealed the consensus cDNA
sequence of the DD4 gene. Various products of rapid
amplification of cDNA ends were carried out to further confirm the
sequence information of this gene.
To identify the transcription start site, 5'-end primer extension was
performed (Fig. 7B). These results indicate that the transcription start site is at least 12 bp upstream of the longest cDNA clone, DD4-C4. We obtained similar results using Eukaryotic Neural Network Promoter Prediction (NNPP/Eukaryotic) software to
theoretically predict the promoter region and transcription start site
(data not shown). However, the smear pattern seen with our primer
extension results suggested incomplete primer extension. We found that
there were several consecutive guanidine residues in the region of the
transcription start site. This potentially will interfere with the
capability of reverse transcriptase to fully extend to the upstream
5'-end of the actual starting site. In support of this, additional data
from recently released EST clones (BG546713, BG570614, BG538707, and
BG537983) suggest that the actual transcriptional starting site is five base pairs upstream of our original results.
Based on PCR data, we confirmed two alternatively spliced products of
the DD4 gene (Fig. 7C). Using the same set of
primers, we were able to amplify two products, 1090 and 1035 bp long,
that differ by 55 bp at the 3'-untranslated end. Both amplified clones contain an open reading frame of 256 amino acid residues. For both
products, the region adjacent to the translational initiation codon
contains a favorable Kozak sequence (28). Both cDNA products contain the same polyadenylation signal, AAUAAA, located 19 bp upstream
of the polyadenylation site. Although the 55-bp size difference of the
two transcripts is distinguishable using reverse transcription-PCR
amplification methods, we are unable to resolve the two transcripts
with Northern blot analysis.
Characterization of Protein Expression--
The peptide sequence
analyses suggested that both DD4 cDNAs encode a protein
with 256 amino acid residues. The predicted isolectric point of
DD4 is 5.497, and the calculated molecular mass is
26,711 daltons. To confirm the accuracy of the deduced amino acid
sequence, we used in vitro transcription-coupling
translation. This approach demonstrated a major translation product at
25 kDa in SDS-PAGE (Fig. 8A,
lane 3). The slight discrepancy between the in
vitro translated product and the predicted molecular mass of 26.7 kDa is probably due to the migration behavior of DD4 protein
in gel. In addition, we performed transient transfection studies using a chimeric construct of pFLAG fused in frame to the coding sequence of
the DD4 gene under the direction of a CMV promoter. Western blot analysis using anti-FLAG M5 antibody demonstrated a single protein
product at the 27-kDa position. Mock (no transfection) and control
transfections (control pFLAG vector with no cDNA insert) yielded no
detectable signal (Fig. 8B).
The DD4 Gene Product Is a spurt Protein--
To analyze the
protein structure of DD4, several web-based protein
structure analysis software programs were used. SignalP identifies
secretory signal peptides and predicts the possible cleavage site of
putative signal peptide sequences (35). Based on the analysis by this
program of the deduced amino acid sequence, the human DD4
gene contains a distinct signal peptide sequence in its first 19 amino
acids (data not shown). In addition, SignalP analysis predicted
Ala19 to Gln20 as the most likely cleavage
site. Another software program, PSORT, detects the sorting signals of
proteins and predicts their subcellular locations (36). The results
obtained from PSORT suggested a 33.3% probability that
spurt is extracellularly secreted (data not shown). Both of
these analyses strongly suggest that DD4 is a secretory protein.
Hydrophobicity analysis, based on the method of Kyte and Doolittle
(59), predicted the hydrophilic/hydrophobic regions of DD4 (Fig. 9A).
Based on this analysis, an oligopeptide that corresponds to a highly
hydrophilic, highly antigenic region of 16 amino acids with high
surface probability (Arg165-Cys180) was
synthesized and used as an immunogen to generate polyclonal antibodies
in rabbit. Using anti-DD4 antibody, DD4 protein
was detected from cell culture supernatants collected from
the apical region of the culture chamber in both human bronchial
epithelial HBE1 cell lines and primary cultured airway epithelial cells
maintained in 30 nM ATRA. Sputum samples also demonstrated
the presence of DD4 protein (Fig. 10).
Similar Western blot analyses were performed using preimmune serum or
antibody neutralization with DD4-specific antigen. The
observed staining patterns confirmed not only anti-DD4 antibody specificity but also the secretory characteristics of DD4 because its gene product could be detected in naturally
secreted human airway sputum. A nonspecific upper band exists in both
preimmune and anti-DD4 antisera. Based on these results, we
named DD4 gene the secretory protein
in upper respiratory tracts:
spurt.
spurt Protein Expression Is Elevated in COPD Patients--
Our
initial analysis of sputum samples revealed that spurt
expression was higher in the sputum of patients with COPD. To further characterize this observation, we examined sputum samples from additional normal subjects and COPD patients for the presence of
spurt. Using the anti-spurt specific
(anti-DD4 specific) antibody, we consistently observed
higher amounts of spurt in sputum samples obtained from COPD
patients compared with normal subjects (Fig. 10B). These
results were consistent with our in situ hybridization results (Fig. 6), which demonstrated higher levels of spurt
mRNA in COPD patients.
Functional Analysis of spurt Protein--
To elucidate the
function of spurt protein, various bioinformatics search
methods were performed, including InterPro Scan, ScanProsite,
ProfileScan, pfam HMM search, and PATTINPROT. Using a Smith-Waterman
algorithm, the putative spurt amino acid sequence has 27.3%
homology to the C-terminal domain of the bactericidal/permeability increasing protein (BPI) that is located at chromosome 20q11.23 (37,
38) (Fig. 11A). Furthermore,
when reverse position-specific BLAST (39) was performed to search
against the Conserved Domain Data Base, spurt aligns to a
conserved domain named BPI 1 that is derived from the N-terminal domain
of BPI (SMART accession number SM0328) (40-42) (Fig. 11B).
In addition, preliminary findings demonstrated a conserved
phosphorylation site for casein kinase II from Ser190 to
Asp193.
In this study, we utilized a microarray assay to identify an
ATRA-inducible, novel secretory protein, spurt, which is
secreted by conducting airway epithelia. Our results indicate retinoids regulate spurt mRNA and protein levels in cultured
airway epithelial (TBE) cells. We observed that ATRA significantly
increases spurt gene expression. Increased expression occurs
after several days of ATRA treatment and with higher doses of retinoids.
Furthermore, we demonstrated that specific cell culture manipulations
enhance spurt expression. Expression of spurt
increased in a progressive fashion as the culture conditions were
changed from tissue culture plates alone (TC) to biphasic conditions
without and with collagen gel substrata. Optimized expression of
spurt message occurred when airway epithelial cells were
plated on a collagen gel substratum with air-liquid interface culture
conditions (BI-CG). These data suggest that expression of
spurt occurs as the airway epithelial cell layer develops
toward the more mature stages of differentiation.
In support of this idea, it has been observed that the most suitable
culture conditions to promote airway mucociliary differentiation in vitro include the combination of an air-liquid interface,
a collagen gel substratum, and retinoid supplementation (1). Under
these culture conditions, human airway epithelial cells develop into a
fully differentiated cell layer with multiple mucociliary functions. In
the absence of one or more of these conditions, the differentiated
features are not as extensive. In addition, spurt gene
expression in vivo appears to be limited to specialized cells in the human airway. In situ hybridization of lung
tissue and Northern blot analyses of RNA samples from various human
tissues demonstrated an abundance of spurt message in the
upper respiratory tract. Gene expression was particularly high in
serous cells within the submucosal glands.
Based on the sequence information obtained from our genomic cloning
effort and information in the public data base, full-length spurt sequence consists of nine exons and eight introns that
spans a genomic DNA region of 7,313 bp (spurt genomic clone
GenBankTM accession number AF421369). Because the matched
BAC clone, bA49G10, is designated to contig 158 of chromosome 20, the
corresponding cytogenetic chromosome position of the spurt
gene is assigned to 20q11.21. In addition, we have found that two
spurt transcripts, a short and a long form, exist in EST
data bases and our airway epithelial cell specific cDNA library.
These two transcripts differ at the 3'-untranslated region. This
observation supports the proposal that there is alternative splicing of
spurt in the 9th exon that generates two
transcripts with the same ORF. How this alternative splicing affects
the function and expression of the spurt gene is currently unknown.
It is necessary to point out that several human EST clones identified
in GenBankTM and other sequence data bases show high
sequence alignments with our spurt clone, but all of them
have shorter inserts than ours and contain only partial sequence
information. Importantly, although several different researchers
independently identified spurt-like EST clones, all of the
EST clones were identified in cDNA libraries from human lung or
olfactory epithelium. These observations further support our finding
that spurt is a human airway-specific gene.
To elucidate the putative function of spurt, protein
structural analyses were performed using several software packages
including Geneworks, Lasergene, GCG, and web-based search sites. These
analyses predicted spurt to be a globular protein with
slightly hydrophobic properties. Furthermore, protein analyses by
SignalP predicted a 19-amino acid secretory signal peptide at the
N-terminal region of spurt. This signal peptide sequence is
homologous to other secretory proteins such as the von Ebner minor
salivary gland protein (46, 47), and the parotid secretory protein
precursor (48, 49). Based on this information, we generated a specific polyclonal antibody, spurtAb2, to detect the protein
expression of spurt. Examination of sputum samples confirmed
the secretory nature of the protein. We easily detected
spurt protein in sputum samples by Western blot analyses
using spurtAb2 antibody.
Although we have identified spurt to be an upper
airway-specific secretory protein, the function of spurt is
still unknown. Despite an extensive blast search, no human protein with
known function shows more than 40% homology to the deduced amino acid sequence of spurt. A separate group identified a "mouse
unknown protein," plunc (palate,
lung, and nasal epithelium clone)
(43) with 74% homology to the human spurt deduced amino
acid sequence. Mouse plunc was identified through mRNA
differential display methods as a gene up-regulated during palate
closure of the developing mouse embryo. Similarly, Iwao et
al. (44) identified a novel human lung-specific gene,
LUNX (lung-specific X protein), to
be a potential molecular marker for detection of micro-metastasis in
non-small cell lung cancer. However, the protein properties and
function of plunc were not described in these papers.
Recently, a human sequence homologous to mouse plunc and
human spurt was reported in a brief communication by Bingle
and Bingle (45). Similar to LUNX, the human plunc
sequence is highly homologous to the short form of spurt
with some sequence discrepancies in both the coding and noncoding
regions. One important difference regarding the expression of human
spurt and the expression of human plunc was the
induction by retinoids. spurt was originally identified in
our microarray study as an ATRA-responsive gene. This induction was
further confirmed by Northern blot analyses as shown in our data.
However, Bingle's research group described high levels of
plunc RNA expression in cultured airway epithelial cells
without any retinoid treatment and showed that this expression was not
altered by retinoid treatment as indicated in the manuscript. Although
we observed higher expression levels of spurt mRNA in human nasal turbinates and trachea, we did not identify a significant amount of spurt RNA expression in primary cultures of normal
nasal epithelial or tracheobronchial epithelial cells under
retinoid-depleted culture conditions. In addition, we did not
demonstrate the expression of spurt or plunc-like
message in NCI-H647 cell lines obtained from the ATCC. This cell line
was shown by Bingle's group to be the only cell line constitutively
expressing plunc message. The reason for these discrepancies
is currently unclear.
Based on our protein structural analyses of spurt, two
functional motifs have been identified. One of them is a conserved phosphorylation site from Ser190 to Asp193 for
casein kinase II, which is known to regulate cellular responses through
phosphorylation and is associated with a number of substrates, including topoisomerase I, p53, and protein phosphatase 2A (50-52). Currently, we have not determined whether spurt is
phosphorylated. However, based on the similarity of the molecular
weight obtained from amino acid sequence estimations and the mobility
of spurt in SDS-PAGE, we concluded that phosphorylation is
less likely.
The second "putative" domain identified in spurt shows
amino acid sequence homology to both the N-terminal and C-terminal domains of BPI. BPI has been implicated in bactericidal processes (53-57). The presence of a BPI-like domain in spurt protein
suggests an antibacterial role for this novel protein. Consistent with this idea, our in situ hybridization studies demonstrated
that spurt is frequently expressed in the serous cells of
secretory ducts and submucosal glands. Some serous cell secretions,
such as lysozyme, are known to have antibacterial activity. It is
interested to note that plunc, a spurt-like mouse
gene, has been shown to be expressed in mouse thymus, a tissue that
harbors antimicrobial gene products (58). These observations suggest
that spurt and plunc represent members of a new
bactericidal gene family.
To better understand the role of spurt in human disease, we
compared the level of spurt expression between normal
subjects and subjects with chronic lung diseases. Based on in
situ hybridization, we observed a higher level of spurt
mRNA expression in submucosal gland serous cells of patients with
COPD. In addition, the submucosal gland ducts and airway epithelial
surface of lung tissue from COPD patients also displayed expression of
spurt mRNA. We were unable to demonstrate
spurt mRNA expression in the airway epithelial surface
of lung tissue obtained from normal patients. Similarly, Western blot
analysis demonstrated that spurt protein expression is
significantly elevated in all sputum samples obtained from COPD
patients compared with normal subjects. We postulate that the recurrent
infections seen in patients with chronic lung diseases such as
emphysema and chronic bronchitis results in the up-regulation of airway
bactericidal proteins such as spurt. This higher expression of spurt involves both increased induction of the gene in
submucosal gland serous cells that normally express the gene as well as
the induction of cells in the submucosal gland ducts and airway surface epithelia that normally do not express spurt.
In conclusion, we have shown that spurt is a novel upper
airway-specific secretory protein that is induced by retinoids. Two alternatively spliced forms of spurt exist. spurt
has putative antibacterial functions, and the expression of this
protein is increased in lung diseases such as chronic bronchitis and
emphysema. The mechanisms and purpose of this up-regulation are
actively being investigated in our laboratory.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (ICN, Costa Mesa, CA) to a specific
activity of ~2 × 109 dpm/µg with a Ready-To-Go
random primer labeling kit (Amersham Biosciences). The membranes were
prehybridized in hybridization solution (6× SSC, 0.5% SDS, 0.01 M EDTA, 0.5% disodium pyrophosphate, 5× Denhardt's
solution) at 68 °C for a minimum of 4 h followed by
hybridization in the same solution plus specific probe at 68 °C for
16-20 h (overnight). Hybridized membranes were washed once with 2×
SSC, 0.1% SDS for 10 min at room temperature and twice with 1× SSC,
0.1% SDS for 30 min at 68 °C. Following the second wash, the
membranes were checked for excessive radioactivity, and, if necessary,
washed in 0.1× SSC and 0.1% SDS for various times at 68 °C.
PhosphorImager screens (Molecular Dynamics and Amersham Biosciences)
and/or X-Omat film (Eastman Kodak Co.) were exposed to hybridized
membranes for various times. ImageQuant analysis software was used for
quantitation of Northern hybridization signals (Molecular Dynamics). To
study the novel gene expression patterns in normal tissues, Northern
blots containing 20 µg/lane of total RNA from various human tissues
were prepared, and Northern analysis was carried out as described above.
ZAP cDNA library that was custom-made in our laboratory. The cDNA library was derived from human primary cultures of airway epithelial cells grown under BI-CG
conditions in the presence of 30 nM ATRA for 3 weeks. DNA sequencing was carried out at the Institutional DNA Core Facility (Department of Plant Genetics, University of California at Davis) using
the fluorescence-labeled automatic sequencing approach and separated by
the ABI Prism model 377 automated DNA sequencer (Applied Biosystems,
Foster City, CA). Four sequence primers (DD4 SEP 1-4) at different
positions of DD4 were used as follows: DD4-SEP1, 5'-GACGTCAGTGATTCCTGGCC-3'; DD4-SEP2, 5'-TCCAGAAGACCTTGAATGGG-3'; DD4-SEP3, 5'CGTGTGCCCTCTGGTCAATG-3'; and DD4-SEP4,
5'-TAGGTGAGGCACATGGATG-3'.
-mercaptoethanol,
0.1% bromphenol blue) were added (27). The translated products were
separated by electrophoresis in a 12% SDS-polyacrylamide gel. For
imaging, dried gels were exposed to x-ray film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 1.
DD4 gene expression is increased
by ATRA in primary human TBE cell cultures. Northern blots of RNA
from cultured human TBE cells were hybridized to probes specific to
DD4. A, primary human TBE cells were cultured
under four different culture conditions (TC, CG, BI, and BI-CG) for 14 days with (lanes 2, 4, 6, and
8) and without (lanes 1, 3,
5, and 7) supplementary 3 × 10 8 M ATRA as described above. B,
the staining gel of the transferred membrane that was used in
A showing the 28 and 18 S bands, which confirms equal RNA
loading. C, quantitative results that represent
DD4 gene expression levels after normalization with18
S.
View larger version (75K):
[in a new window]
Fig. 2.
The ATRA-induced increase in DD4
gene expression is dose- and time-dependent.
Northern blots of RNA from cultured human TBE cells were hybridized to
probes specific either to human DD4 or, as an internal
control (Con), 18 S rRNA. A, primary human TBE
cells were cultured under BI-CG conditions for 14 days with increasing
concentrations of ATRA as shown. B, primary human TBE cells
were cultured under BI-CG conditions with 3 × 10 8
M ATRA for the indicated periods. The lower
panels of A and B are the same membranes
rehybridized with 18 S ribosomal cDNA probe to normalize RNA
loading.
View larger version (57K):
[in a new window]
Fig. 3.
DD4 gene expression in humans is specific to
the upper airways. Northern blot analysis was performed for
DD4 gene expression with total RNA isolated from various
human tissues. Total RNA (20 µg/lane) from human tissues was
separated by formaldehyde, 1.2% agarose gel electrophoresis.
Hybridization was performed as described above, followed by
autoradiography. DD4 gene expression was observed in RNA isolated from
the lung (lane 2), bronchi (lane 4), trachea
(lane 5), nasal turbinates (lane 11), and nasal
polyps (lane 12). There was no DD4 gene expression seen in
the heart (lane 1), kidney (lane 3), liver
(lane 6), brain (lane 7), stomach (lane
8), small intestine (lane 9), or colon (lane
10).
View larger version (73K):
[in a new window]
Fig. 4.
DD4 message is localized to the
tracheal submucosal glands and secretory ducts. Radiolabeled
antisense DD4 RNA probes were prepared as described above
and hybridized to human tracheal tissue sections. DD4
message is localized to the secretory ducts (A and
B) or submucosal glands (C and D) of
the trachea. Original optical magnification, 400×.
View larger version (57K):
[in a new window]
Fig. 5.
DD4 message in vivo
is specific to the human upper airways. In situ
hybridization of human airway surface epithelia was performed using
radiolabeled antisense DD4 RNA probes as described above.
The intensity of DD4 signal in airway surface epithelia
progressively decreased from the upper airways (A and
D) to the lower airways (C and F) as
shown. Original optical magnification, 400×.
View larger version (135K):
[in a new window]
Fig. 6.
Increased expression of DD4 mRNA occurs
in the epithelium of a diseased airway. In situ
hybridization of human trachea tissue sections from a patient with
COPD. A, positive hybridization with DD4
antisense probe was seen in both the surface epithelium and the
submucosal gland region in the trachea of a COPD patient (100×).
B, the same serial tissue sections as in A were
hybridized with DD4 sense probe and did not show observable
signal in either the surface epithelium or the submucosal glands
(100×). Higher magnification clearly demonstrates the positive
staining of DD4 message in the surface epithelium
(C) and submucosal glands (D) (200×).
DD4 message was elevated in both the surface epithelium and
the submucosal glands compared with normal tissue sections (Fig.
5).
View larger version (64K):
[in a new window]
Fig. 7.
Two DD4 cDNA sequences
exist with alternative splicing at the 3'-untranslated end.
A, cartoon representation of the full-length human
DD4 cDNA. The proposed open reading frame (bold
arrow) and the position of the primer used for 5'-extension are
shown. The positions of our four independently isolated cDNA clones
and three GenBankTM EST clones are shown relative to the
human DD4 (spurt) cDNA. B, results
of 5'-end primer extension experiments using a 22-mer primer sequence
antisense to the 95-116-bp region of the DD4 cDNA.
Single base ladder markers (lanes 1-4) were compared with
the DD4 extended products, spurt and
spurta (lane 5). C, reverse
transcription-PCR confirmed two alternatively spliced forms of
DD4 (spurt) at the 3'-untranslated region. PCR
primers were designed to correspond to the DD4
3'-untranslated region upstream (5'-CATGCTGATCCACGGACTAC-3') and
downstream (5'-ACGCCTGGTGGGAAAGGAG-3') of the proposed alternative
splicing site. Amplified PCR products were separated by electrophoresis
on a 2% agarose gel and visualized by an UV/light gel documentation
system from Amersham Biosciences. Molecular markers at 250-bp
(lane 1) and 100-bp (lane 2) intervals served as
size references for our PCR products. PCR amplification was performed
with 0.2 µg of total RNA template from primary TBE cells under BI-CG
and 30 nM ATRA culture conditions (lanes 3 and
4). For positive control, we used full-length (lane
5, clone DD4-C1) and alternatively spliced (lane 6,
clone DD4-C3) DD4 cDNA clones. These results indicate that both the
long (spurt) and short (spurta) forms of
DD4 cDNA exist simultaneously in airway tissues.
View larger version (71K):
[in a new window]
Fig. 8.
Successful DD4 in vitro
transcription/translation and FLAG-tagged human DD4
protein expression. A, the complete
DD4 cDNA was cloned into a pcDNA3 expression vector
and translated in a reticulocyte cell lysate in the presence of
[35S]methionine. The reaction products were analyzed by
12% (w/v) SDS-polyacrylamide gel electrophoresis, followed by
autoradiography. The reaction was performed in the presence of capped
XeF-1 RNA (lane 1, positive control) and in the absence of
RNA (lane 2, negative control). The DD4
translated gene product was observed with an approximate molecular mass
of 25 kDa as indicated (lane 3). B, human HBE1
cells were transiently transfected with either no DNA (lane
1), pFLAG CMV2 empty expression vector (lane 2), or
pFLAG CMV2 expression vector inserted with the DD4 ORF
(lane 3), and protein extracts were analyzed by Western
hybridization as described above. The proteins were transferred to a
polyvinylidene difluoride membrane and were incubated with the
anti-FLAG antibody M5 at a 1:800 dilution. The arrow
corresponds to a FLAG-tagged DD4 fusion protein.
View larger version (44K):
[in a new window]
Fig. 9.
Secretory nature of human DD4
is recognized by a polyclonal anti-DD4 antibody
to DD4 protein expression in culture supernatants.
A, schematic representation of the human DD4
(spurt) protein that includes a hydrophilicity plot
(Kyte-Doolittle), an antigenic index (Jameson-Wolf), and a surface
probability plot (Emini). Lasergene software was used to generate the
analyses. The horizontal axis represents the corresponding amino acid
position of DD4 (spurt), and the vertical axis of
each plot represents arbitrary units. The amino acid residues that we
used to generate anti-DD4 (spurt) polyclonal
antibodies are marked in a shaded rectangular box, which
corresponds to a region of high hydrophilicity and high surface
probability (Arg165-Cys180). B,
HBE1 cells and human primary TBE cells were treated with and without
3 × 10 8 M ATRA in air-liquid interface
culture conditions. Supernatants from the apical region were collected
and analyzed by Western hybridization using anti-DD4
(spurt) antibody. As indicated by a hybridization band at
~25 kDa (arrow), increased protein expression was observed
in cell culture conditions that included ATRA.
View larger version (27K):
[in a new window]
Fig. 10.
DD4 (spurt)
protein secretion is increased in patients with COPD. A, the
secretion of spurt protein could be detected in clinical
sputum samples from a normal patient (lanes 1, 3,
and 5) and a patient with COPD (lanes 2,
4, and 6). Preimmune serum (lanes 3 and 4) was obtained from the same rabbit that generated the
anti-DD4 antibody (lanes 1 and 2). Antigen block
(lanes 5 and 6) was achieved using
anti-DD4 (spurt) antibody that was prehybridized
with DD4-specific oligopeptide antigen. B, the secretion of
spurt protein is elevated in clinical sputum samples from
COPD patients. Sputum samples were obtained from two normal subjects
(lanes 1 and 2) and six COPD patients
(lanes 3-8). Anti-DD4 (spurt)
antibody was used at a 1:1,000 dilution for Western blot
analyses.
View larger version (46K):
[in a new window]
Fig. 11.
Significant alignment of the
spurt and BPI amino acid sequences. A,
using a Smith-Waterman algorithm, our analysis revealed the
spurt amino acid sequence is homologous to the C-terminal
domain of rabbit BPI. B, the peptide sequence alignment of
spurt to the N-terminal domain of human BPI (BPI 1, CD:smart00328) using a reverse position-specific BLAST search. The
exact alignment of amino acids between spurt and BPI is
indicated as a solid line, and the dots represent
the alignment of two substitutable amino acids.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL35635, ES09701, ES06230 5F32HL09573, and HL04404), American Lung Association Grant RG-025L, and California Tobacco-related Disease Research Program Grants 7RT-0145, 10KT-0262, and 8KT-0092.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF417257, AF417256, and AF421369.
§ To whom correspondence should be addressed: Dept. of Environmental and Occupational Health, University of Pittsburgh, 3343 Forbes Ave., Pittsburgh, PA 15260. Tel.: 412-383-2157; Fax: 412-383-2123; E-mail: peterdi@pitt.edu.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M210523200
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ABBREVIATIONS |
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The abbreviations used are: ATRA, all-trans-retinoic acid; TBE, tracheobroncheal epithelial; TC, tissue culture; CG, collagen gel; ORF, open reading frame; COPD, chronic obstructive pulmonary disease; EST, expressed sequence tag; CMV, cytomegalovirus; BPI, bactericidal/permeability increasing protein; contig, group of overlapping clones.
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