Laboratory of Metabolism (T.N., S.K.) and Laboratory of Experimental Carcinogenesis (C.L.K-W., N.C.P.), National Cancer Institute, NIH, Bethesda, Maryland 20892; and Genaera Corp. (Y.Z., R.C.L.), Plymouth Meeting, Pennsylvania 19462
Address all correspondence and requests for reprints to: Shioko Kimura, Ph.D., Building 37, Room 3E-24, NIH, Bethesda, Maryland 20892. E-mail: shioko{at}helix.nih.gov
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ABSTRACT |
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INTRODUCTION |
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T/EBP/NKX2.1 is expressed in the lung, thyroid, and ventral forebrain during embryogenesis (18, 19, 20), suggesting an involvement of T/EBP/NKX2.1 in developmental processes. In fact, suppression of T/EBP/NKX2.1 by antisense oligonucleotides in vitro using lung organ cultures abrogated normal branching morphogenesis (21). Targeted disruption of the T/ebp/Nkx2.1 locus resulted in immediate postnatal death from respiratory failure caused by profoundly hypoplastic lungs (22). In addition to the lung pathology, these mice lack thyroid and pituitary glands and exhibit severe defects, such as hypothalamus and basal ganglia, in the ventral forebrain (22, 23, 24, 25, 26). Thus, T/EBP/NKX2.1 appears to serve as one of the master regulatory genes responsible for organogenesis of the thyroid, lung, and ventral forebrain. However, the exact impact of the developmental block resulting from inactivation of the T/ebp/Nkx2.1 locus on structural morphogenesis and differentiation of the cells in these organs remains unclear.
In the lung, T/EBP/NKX2.1 is expressed in all epithelial cells early in
pulmonary morphogenesis, but the expression becomes progressively
restricted to alveolar type II and Clara cells (24).
Analyses of the T/ebp/Nkx2.1-null mouse suggested that
T/EBP/NKX2.1 may function in the establishment of pattern formation and
pulmonary morphogenesis during early embryonic development. The lack of
T/EBP/NKX2.1 expression leads to the condition called tracheoesophageal
fistula, in which the trachea and esophagus share a common tube
(23). The main stem bronchi bifurcate from this common
structure, connecting to severely hypoplastic lungs. These phenotypes
found in the T/ebp/Nkx2.1-null mouse must be related to the
ability of T/EBP/NKX2.1 to activate and/or suppress specific downstream
target genes. One such category of target genes consists of SP-A, -B,
and -C and uteroglobin/CCSP in the lung, which are not expressed in
T/ebp/Nkx2.1-null embryo lungs (23). These
genes, however, are not known to have morphoregulatory function. In
T/ebp/Nkx2.1-null embryo lungs, expression of some
extracellular matrix proteins and their cellular receptors, including
collagen type IV and -integrins, and some growth factors such as
Vegf3 and Bmp4, are reduced or absent (23, 24). Whether the abnormal phenotype in
T/ebp/Nkx2.1-null embryo lungs is entirely or
partially attributable to the reduction or absence of expression of
these genes remains to be examined.
In the current study, a potential T/ebp/Nkx2.1 target gene, a mouse Ugrp1 that encodes a uteroglobin/CCSP-related protein, was cloned using suppressive-subtractive hybridization between RNAs isolated from wild-type and T/ebp/Nkx2.1-null embryo lungs (27). UGRP1 showed an amino acid sequence similar to that seen in the uteroglobin/CCSP family of proteins that are characterized as dimeric secretory proteins of unknown function (28, 29, 30, 31), although in the case of uteroglobin/CCSP, an involvement in regulating inflammation has been suggested (31, 32, 33, 34, 35, 36, 37). UGRP1 is expressed in all epithelial cells during pulmonary morphogenesis and may be involved in inflammation. Analyses of the expression and promoter function of the Ugrp1 gene suggests that it is a downstream target gene regulated by the T/EBP/NKX2.1 homeodomain transcription factor.
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RESULTS |
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Sequence Characteristics of UGRP1- Encoded Polypeptides
To isolate a full-length UGRP1 cDNA, a mouse adult lung cDNA
library was screened and eight clones with a positive hybridization
signal were identified in 1 x 106
recombinant phage. After cloning and sequencing, three appeared to
contain full-length cDNAs. Two additional cDNAs that differ at their
C-terminal sequences were isolated by RT-PCR. These were used to
classify the transcripts into three types (A, B, and C), with type A
being the full-length cDNA obtained through the library screening. The
three polypeptides (A, B, and C) consist of 91, 113, and 139 amino
acids, respectively (Fig. 1A). cDNAs for
type B and C transcripts were not found by cDNA library
screening, suggesting that these two transcripts may be rare. Computer
analyses revealed that the first 21 residues of the UGRP1 polypeptide
may function as a signal sequence for targeting the protein to a
secretory pathway (Fig. 1B
).
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Several mouse and human expressed sequence tag (EST) sequences that
demonstrate similarities to the mouse Ugrp1 were also
identified. Using RT-PCR with a part of the EST sequences as primers, a
mouse homologous gene (Ugrp2) and human orthologous genes to
each mouse gene (UGRP1 and -2) were obtained. The
human UGRP1 and -2 and mouse UGRP2 show 81%, 41%, and 33% amino acid
sequence identity, respectively, to mouse UGRP1 (Fig. 1C), which
suggests that they constitute a new gene family.
Ugrp1 Genomic Structure and Alternatively Spliced
Transcripts
To analyze Ugrp1 genomic structure and to define
the origin of the three types of transcripts, a mouse bacterial
artificial chromosome genomic library was screened using the
full-length type A cDNA as a probe. The mouse Ugrp1 gene is
composed of three exons and two introns. All of the exon/intron
boundaries match the consensus sequence for RNA splicing.
Interestingly, the whole or N-terminal half of intron 2 can be
alternatively retained in the transcripts, which appears to be
responsible for the production of types B and C transcripts (Fig. 1A).
Alternatively, they could represent incompletely spliced RNA
transcripts. In the type B transcript, the N-terminal half of intron 2
encodes an additional 22 amino acids that are inserted at residue 85 of
the type A polypeptide. In the type C transcript, the intron 2 sequence
is completely retained, which results in 33 unique amino acids at its C
terminus after residue 85 because of a stop codon present in the intron
sequence.
Chromosomal Localization of the Ugrp1 Gene
Mouse chromosome spreads that were hybridized with biotin or
digoxigenin-labeled genomic probes had specific fluorescent signals at
identical sites on chromosome 18 in 40 of 50 metaphases randomly
selected for recordings (Fig. 2). This
was the only site with a double symmetrical fluorescent signal.
Occasionally, single randomly distributed fluorescent spots were
observed. Twenty-five metaphases without overlapping chromosomes
were analyzed by imaging of 4',6-diamidino-2-phenylindole
(DAPI)-enhanced G-like banding. Symmetrical fluorescence signal
was localized at region 18C-D, where we assigned the location of the
Ugrp1 gene. This region is homologous with human chromosome
5q31-q34 (40, 41): multiple disorders such as cortisol
resistance, refractory macrocytic anemia, 5q syndrome, and Treacher
Collins mandibulofacial dystosis are located in this region
(42). Translocations specific for acute lymphoblastic
leukemia are also localized to this region (43). This
region is further known to contain at least one asthma susceptibility
locus (44, 45, 46, 47).
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The presence of alternatively spliced transcripts was confirmed by
RT-PCR using various combinations of primers and E18.5 wild-type embryo
lung mRNAs as a template or the individual A, B, and C cDNA clones as
control templates. In embryo lungs, a fragment corresponding to type A
(167 bp) and both type B (130 bp) and type C (394 bp) transcripts was
demonstrated using exon 2 (P1)- and exon 3 (P2)-specific, and intron 2
5' region (P3)- and exon 3 (P2)-specific primer pairs, respectively
(Fig. 3C). A faint but clear band corresponding to type C transcript
(410 bp) was exhibited by using exon 1 (P5)- and intron 2 3' region
(P4)-specific primer pairs. Although the signal from RT-PCR is not
necessarily proportional to the expression level, these data again
support our finding that type A transcript is most abundant.
Ugrp1 Promoter Is
Trans-Activated by T/EBP/NKX2.1
To determine whether Ugrp1 promoter sequences
are responsive to activation by T/EBP/NKX2.1, a DNA fragment containing
the 5' flanking region of the mouse Ugrp1 gene was isolated
and sequenced (Fig. 4A). A major
transcription initiation site was determined by the 5' rapid
amplification of cDNA ends method using adult mouse lung mRNA as a
template. A TATA box is located at position -26 bp. Four minimum
consensus sequences for a possible T/EBP/NKX2.1 binding site (CTNNAG)
(9) were identified at positions -255, -182, -120, and
-37 bp within 307 bp of the upstream sequences.
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Six Ugrp1 promoter-luciferase constructs (PGL3-18,
-67, -147, -190, -242, and -907) (Fig. 4A) were used to map
regions responsible for Ugrp1 transcriptional activity by
cotransfecting into NCI-H441 or HeLa cells with either a
pCMV4-T/EBP/NKX2.1 expression plasmid or a control pCMV4 vector (Fig. 5
, A and B). NCI-H441 cells endogenously
express T/EBP/NKX2.1, whereas HeLa cells do not. In NCI-H441 cells, a
construct containing from +72 to -147 bp of the 5' flanking sequence
(-147) showed similar luciferase activity with and without
cotransfection of expression plasmid (Fig. 5A
). Construct -190
demonstrated approximately twice the activity as construct -147 when
control pCMV4 vector was present and approximately 4-fold higher
activity when cotransfected with T/EBP/NKX2.1 expression plasmid
vs. control vector. This increase of activity by
cotransfection of the expression plasmid was probably attributable to
an insufficient amount of endogenous T/EBP/NKX2.1 present in NCI-H441
cells for full activity. A similar phenomenon was reported previously
(48). In the case of HeLa cells, constructs -147 and
-190 exhibited approximately 5-fold and 10-fold increases in
luciferase activity, respectively, by cotransfection of T/EBP/NKX2.1
expression plasmid compared with the control vector alone (Fig. 5B
).
Such an increase in activity was not observed with the -67 construct.
The increase in the activity of construct -147 was obtained by
cotransfection of T/EBP/NKX2.1 in HeLa cells but not in NCI-H441 cells.
The mechanism for this discrepancy is unclear, although it could be
partly related to the presence of endogenously expressed T/EBP/NKX2.1
in NCI-H441 cells, a possibility that remains to be examined.
Nevertheless, these results indicate that T/EBP/NKX2.1-binding elements
necessary to activate Ugrp1 gene transcription may be
located between -190 and -67 bp. The nucleotide sequence of this
region contains two consensus T/EBP/NKX2.1-binding sites (Fig. 4A
). We
focused our attention on these sites, because protected region I is
located upstream of the -242 construct and protected region IV was
unable to form specific DNA-protein complexes with NCI-H441 cell
nuclear extracts using gel shift analysis (see below).
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Characterization of UGRP1 Proteins
The polypeptides of uteroglobin/CCSP family members form a
homodimer or heterodimer (28). To examine whether the
three types of UGRP1 polypeptides are capable of forming a homodimer,
the cDNAs for each polypeptide were fused to a c-myc epitope
tag (39 amino acids including the linker sequence) at the 3' end and
were expressed individually in COS-1 cells. Immunoblot analyses with
anti-c-myc antibody clearly demonstrated that in cell
lysate, both polypeptides A and B are mainly present in the dimeric
form, as seen in nonreducing conditions, which is reduced to the
monomeric form in reducing conditions (Fig. 7). A small amount of the monomeric forms
of polypeptides A and B were also found in nonreducing conditions. In
conditioned medium, only homodimers were detected for polypeptides A
and B, indicating that only dimeric forms may be directed to the
secretory pathway. In contrast, no band corresponding to polypeptide C
was ever detected in either the cell lysate or the medium under any of
the conditions examined, despite the effort to keep the procedures of
transient expression and immunoblot analyses consistent for all three
polypeptides. This may indicate that type C cDNA is transcribed in the
COS-1 cells at lower efficiency compared with types A and B cDNAs, or
that the transcript encoding polypeptide C has different translation
efficiency, and/or the protein is more susceptible to degradation.
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DISCUSSION |
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UGRP1 mRNA is detected in the lungs of mouse embryos as early as E12.5, a few days after the onset of T/EBP/NKX2.1 expression (E9E9.5) (19). T/EBP/NKX2.1 is responsible for morphogenesis and cellular differentiation of the distal lung compartments, and it appears to be one of the key regulators of early lung development (23, 24). Northern blot and immunohistochemical analyses indicate that UGRP1 is predominantly expressed in the lung, although expression is also found at very low but detectable levels in the thyroid. Expression in the lung is localized in the epithelial cells of the airways. Analyses of the Ugrp1 gene and its transcripts showed that at least three transcripts are produced, possibly through an alternative splicing event in which the second intron is either spliced or totally or partially retained in mature mRNAs (50). All three Ugrp1 transcripts are expressed in embryonic lungs, with the type A transcript being most abundant. Whether these three transcripts are expressed in distinct temporal and spatial patterns and exert different functions during lung morphogenesis remains to be determined.
Despite the sequence similarity, UGRP1 is clearly different from uteroglobin/CCSP, as revealed by the following evidence: 1) in the lung, UGRP1 expression is found in the trachea, bronchus, and bronchioles, whereas uteroglobin/CCSP is expressed only in bronchus and bronchioles, but not in the trachea; 2) the mouse Ugrp1 gene is localized on chromosome 18C-D, which is homologous with human chromosome 5q31-q34, whereas all known human members of the uteroglobin/CCSP gene family are localized on chromosome 11q12 (31).
Uteroglobin/CCSP is believed to function as a regulator of inflammation in the lung. This belief is based on several findings, such as the inhibition of PLA2 activity, the binding of PLA2 substrate (Phosphatidylcholine/phosphatidylinositol), and the location of the gene in the proximity of other genes involved in the regulation of inflammation (31, 32, 33, 34). The antiflammin domain exhibits potent antiinflammatory and immunomodulatory activities and appears to be responsible for the PLA2-inhibitory activity of uteroglobin/CCSP (28, 31).
Although the functions of UGRP1 and UGRP2 are unknown, we examined the relationship between the expression of these genes and allergic lung inflammation for a number of reasons: 1) UGRP1 expression is highly enriched in naive lung; 2) a potential antiinflammatory role has been described for uteroglobin/CCSP (28, 35); and 3) the human UGRP1 gene is likely to be localized to chromosome 5q31-q34, a genomic region known to contain at least one asthma susceptibility locus (44, 45, 46, 47). Allergic models were chosen because they are well characterized and reproducibly associated with TH2 cytokine-mediated inflammatory responses in the lung (51, 52, 53). Our results demonstrated that antigen-induced lung inflammation appears to be associated with decreased expression of UGRP1 and UGRP2. Steroid treatment in vivo increased their expression toward baseline, or to levels found in naive animals. Similar results were observed for uteroglobin/CCSP in vivo after lipopolysaccharide (LPS)-induced acute lung inflammation (35). LPS caused a marked reduction in uteroglobin/CCSP expression in bronchoalveolar lavage fluid and lung homogenates. In this case, at high LPS concentration, the decrease of uteroglobin/CCSP level was thought to be associated with a reduction in the number of Clara cells caused by damage to the cells secondary to pulmonary inflammation, and possibly with the intravascular leakage of the protein across the disrupted bronchoalveolar blood barrier (35). Furthermore, dexamethasone pretreatment failed to prevent the LPS-induced changes in uteroglobin/CCSP levels (35). Reduced lung expression of uteroglobin/CCSP was also reported in mouse after bacterial infection (36) and in human asthmatic patients (37). In the current study, the expression pattern of UGRP1 and UGRP2 after antigen treatment appears to be consistent with that observed for uteroglobin/CCSP. The decreased expression of UGRP1 and UGRP2 could be attributable to a different mechanism(s) from that reported for uteroglobin/CCSP. Nevertheless, UGRP1 and UGRP2 are down-regulated by TH2 inflammatory cytokines that are in turn down-regulated by steroid treatment. Further studies are required to determine the mechanism of decreased expression of UGRP1 and UGRP2 related to inflammation.
The T/EBP/NKX2.1 was found to trans-activate the mouse Ugrp1 gene promoter. DNase I footprinting analyses of the promoter region and cotransfection experiments of Ugrp1-luciferase reporter constructs with the T/EBP/NKX2.1 expression plasmid delineated a minimal region of the Ugrp1 gene promoter that is sufficient to mediate T/EBP/NKX2.1-activated transcription. This region contains two consensus T/EBP/NKX2.1-binding elements, 5'-CTNNAG-3' (9). Mutation of this motif in the binding sites interfered with T/EBP/NKX2.1 binding to the site and reduced its ability to activate transcription. Because construct -190 mut 2 showed a larger decrease in activity compared with -190 mut 1, it appears that the T/EBP/NKX2.1-binding site at -120 bp is more important for the promoter activity than that located at -182 bp, yet both are required for full activity. EMSAs confirmed that T/EBP/NKX2.1 interacts with the two binding sites. The requirement of two T/EBP/NKX2.1 binding sites for full promoter activity has been reported in the SP-B and -C promoters (9, 10).
The intensity of the supershifted band resulting from the use of anti-TTF1(T/EBP/NKX2.1) monoclonal antibody was much less for probes II and III than that for oligonucleotide C, an effect that was observed using either NCI-H441 nuclear extracts or recombinant T/EBP/NKX2.1. This finding may be related to differences in T/EBP/NKX2.1 protein recognition by the anti-TTF1(T/EBP/NKX2.1) antibody among protein complexes formed with DNA probes II and III and oligonucleotide C. DNase I footprinting-protected region IV that was obtained using bacterially expressed recombinant T/EBP/NKX2.1 did not produce any specific protein-DNA-shifted band when examined by gel shift analyses using NCI-H441 nuclear extracts. This region does not have a typical T/EBP/NKX2.1-binding consensus sequence. It is not clear why this discrepancy exists. Possibly, T/EBP/NKX2.1 binds to the region when no other protein is around, as seen in our footprinting analyses. It is also not clear why the -147 mut construct exhibited an incomplete reduction of luciferase activity when T/EBP/NKX2.1 expression plasmid was cotransfected into HeLa cells, a response not seen with the -190 mut 3 construct in NCI-H441 cells. It is possible that the massive amount of expressed T/EBP/NKX2.1 may successfully compete with other proteins to unmask and bind to region IV, leading to a slight activation. This could be the case in both HeLa and NCI-H441 cells. However, the effect in NCI-H441 cells may not be as pronounced as in HeLa cells as a result of the levels of endogenously expressed T/EBP/NKX2.1. Other transcription factors, such as HNF-3 family members and the HFHs, are known to be involved in the expression of lung- specific genes, including SP-B and uteroglobin/CCSP (9, 14, 54). It remains to be determined if HNF-3 and HFH transcription factors are also involved in mouse Ugrp1 gene promoter activity.
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MATERIALS AND METHODS |
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Suppressive-subtractive hybridization and differential screening were performed using the PCR-Select cDNA Subtraction Kit (CLONTECH Laboratories, Inc.) and the PCR-Select Differential Screening Kit (CLONTECH Laboratories, Inc.), respectively, according to the manufacturers instructions. Clones that hybridized with only the forward-subtracted probe were selected for virtual Northern blot analyses, which uses cDNAs instead of RNAs as a source of expressed genes. The membrane containing cDNAs synthesized by the SMART PCR cDNA Synthesis Kit was prehybridized at 60 C in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.) for 30 min and hybridized in fresh buffer with denatured random primer-labeled probe at 60 C for 3 h. After hybridization, the blot was washed twice in 2 x 0.15 M NaCl and 15 mM Na citrate, pH 7.0 (SSC), containing 0.1% SDS at room temperature for 10 min followed by once with 0.1x SSC containing 0.1% SDS at 50 C for 20 min. The filter was then exposed to a PhosphorImager screen overnight. Signal intensities were analyzed using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). Differentially expressed clones were subjected to DNA sequencing analysis.
Cloning and DNA Sequencing
An adult mouse lung cDNA library in the ZAPII vector
(Stratagene, La Jolla, CA) was screened by plaque
hybridization using cDNA isolated from suppressive-subtractive
hybridization as a probe. Hybridization was carried out at 65 C in 6x
SSC, 0.5% SDS, 5x Denhardts solution, and 0.1 mg/ml denatured
salmon sperm DNA for 16 h. The membrane was washed twice with 2x
SSC containing 0.1% SDS at room temperature for 10 min and once with
0.1x SSC containing 0.1% SDS at 55 C for 30 min. Positive plaques
were picked from plates and subjected to secondary and tertiary
screenings. The Ugrp1 genomic DNA was isolated from a mouse
bacterial artificial chromosome genomic library (Incyte Genomics, St.
Louis, MO) using labeled UGRP1 cDNA as probe.
The cDNAs encoding mouse UGRP2 and human UGRP1 and -2 were isolated by RT-PCR using total RNAs prepared from adult mouse and human lungs (Ambion, Inc., Austin, TX), respectively, and primers based on EST sequences that exhibited similarities to the mouse UGRP1 cDNA sequence. In the case of mouse UGRP2 cDNA, a mouse lung cDNA library was also screened using a fragment obtained by RT-PCR as probe. The identities of both cDNA clones obtained by RT-PCR and library screening were confirmed by sequencing. Sequencing was performed using an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit and a model 377 DNA sequencer (PE Applied Biosystems, Foster City, CA).
The nucleotide sequences reported in this paper appear in the GenBank databases under the following accession numbers: UGRP1 type A mRNA, AF274959; type B mRNA, AF274960; type C mRNA, AF274961; mUGRP2, AF313456, EST AI391046; hUGRP1, AF313455, EST AI355612, EST AI355302; hUGRP2, AF313458, EST AW974727.
Determination of the Transcription Start Site
The transcription start site of the mouse Ugrp1 gene
was determined with the SMART RACE (rapid amplification of cDNA ends)
cDNA Amplification Kit (CLONTECH Laboratories, Inc.) using
adult mouse lung total RNA. DNA sequence analyses indicated the
presence of multiple transcription start sites. Because the most clones
(8 of 16) had the exact sequence (91 bp upstream from ATG), we refer to
this site as the major transcription start site.
Chromosomal Mapping
A Ugrp1 probe of 11 kb of genomic DNA labeled with
biotin or digoxigenin was used for in situ hybridization of
chromosomes derived from mouse spleen cultures. Conditions of
hybridization, detection of hybridization signals, digital image
acquisition, processing, and analysis, and direct fluorescent signal
localization on banded chromosomes were performed as previously
described (55). To confirm the identity of chromosomes,
preparations were rehybridized with mouse chromosome 18 painting probe,
and previously observed labeled metaphases were recorded.
RNA Analyses
Reverse transcription of mRNAs was carried out in a final volume
of 20 µl containing 2 µg of total RNA, 4 µl of 5 x
first-strand synthesis buffer (Invitrogen Life Technologies, Carlsbad, CA), 1 µl of a mixture of four
deoxynucleoside triphosphates (2.5 mM each), 2 µl of 0.1
M dithiothreitol, and 100 ng of random primers. After
incubation at 37 C for 2 min, 200 units of SuperScript II reverse
transcriptase (Invitrogen Life Technologies) was added,
and the incubation was continued for 60 min at 37 C. Single-stranded
cDNAs in 0.1 µl of the reaction mixture were amplified by PCR using
AmpliTaq DNA polymerase (PE Applied Biosystems) under the
following conditions: denaturation at 94 C for 30 sec, annealing at
60 C for 30 sec, and extension at 72 C for 1 min, for 30 or 25 cycles
when total RNAs or plasmids were used as template, respectively. The
oligonucleotide primers used to detect UGRP1 and uteroglobin/CCSP
transcripts were as follows (see Fig. 1A for UGRP1): P1,
5'-GTAGAACATCTGGTGACAGG-3'; P2, 5'-CAGCCAGAGTGAGCAAATCC-3'; P3,
5'-TCCCTGGGAGAAGCCTTTGC-3'; P4, 5'-GGAGTCCCTGGGATATGCAC-3'; P5,
5'-GACTGCATTCCAAAGTCCCG-3'; uteroglobin/CCSP forward,
5'-CTACAGACACCAAAGCCTCC-3'; uteroglobin/CCSP reverse,
5'-AAGGAGGGGTTCGAGGAGAC-3' (38).
Northern blotting was carried out using a multiple mouse tissue Northern blot (CLONTECH Laboratories, Inc.) or total RNAs isolated from adult mouse lung and thyroid. The blots were hybridized with a full-length UGRP1 cDNA as a probe. Hybridization was performed in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.) at 68 C for 2 h. The membrane was washed twice with 2 x SSC containing 0.1% SDS at room temperature for 10 min and twice with 0.1 x SSC containing 0.1% SDS at 55° C for 20 min, followed by exposure to x-ray film at -80 C.
For the analyses of UGRP1, UGRP2, and uteroglobin/CCSP expression levels in uterus, mice were daily injected ip with progesterone (3 mg/kg) in PBS or PBS alone for 4 d, and RNA was prepared on d 5.
Luciferase Plasmid Construction and Site- Directed
Mutagenesis
A 9-kb BglII fragment containing 0.9 kb of the 5'
flanking sequence of mouse Ugrp1 genomic DNA was subcloned
into the BamHI site of pBluescript II, and PCR was performed
with T7 primer (5'-GTAATACGACTCACTATAGGGC-3') and a Ugrp1
gene-specific primer (5'-TGCCTGTGATGTTTTCCGGG-3'; +85 to +66). The PCR
product was subcloned into pCR2.1 (Invitrogen Life Technologies), and an XbaI-BamHI fragment
from this plasmid was inserted into the
NheI-BglII site of the pGL3-Basic luciferase
reporter vector (Promega Corp., Madison, WI) to generate
the pGL3-907 plasmid. This construct was further digested with
KpnI and MluI for preparation of deletion
plasmids using Exonuclease III (New England Biolabs, Inc.,
Beverly, MA) and S1 nuclease (Invitrogen Life Technologies). Six deletion constructs (PGL3-18, -67, -147,
-190, -242, and -907) were sequenced to determine the exact
sequences.
Site-directed mutagenesis of a potential T/EBP/NKX2.1-binding site was introduced into the pGL3-190 and -147 plasmids using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The following primers were used to make pGL3-190 mut 1 and mut 2 and -147 mut plasmids: mut 1, 5'-GGTGCCAGAACATTTCTCTACGGGAGACTACTTCTGTG-3' and 5'-CACAGAAGTAGTCTCCCGTAGAGAAATGTTCTGGCACC-3' (complementary strand); mut 2, 5'-GTGGAAAACCCTTCCTAATGTTTAGTTAGGAAGATTGCCCTG-3' and 5'-CAGGGCAATCTTCCTAACTAAACATTAGGAAGGGTTTTCCAC-3' (complementary strand).
Transfection and Reporter Gene Assays
The human lung adenocarcinoma cell line NCI-H441 was maintained
in RPMI 1640 medium containing 10% FCS. HeLa cells were cultured in
MEM containing 10% FCS. Cells in 12-well plates at 5070% confluence
were transfected using Effectene transfection reagent
(QIAGEN, Valencia, CA) with 250 ng of reporter plasmid, 25
ng of expression vector, and 25 ng of pCH110 (Amersham Pharmacia Biotech, Uppsala, Sweden) as an internal control. After 48
h, the cells were harvested in Reporter Lysis Buffer (Promega Corp.), and the lysates were assayed for ß-galactosidase and
luciferase activities using the High-Sensitivity ß-Galactosidase
Assay Kit (Stratagene) and the Luciferase Assay System
(Promega Corp.), respectively. To correct for transfection
efficiency, luciferase activity was normalized to ß-galactosidase
activity. Relative luciferase activity of various mouse
Ugrp1 promoter constructs was expressed based on the
activity of pGL3-Basic in the presence of the same
trans-activating plasmid as 1. Data are mean values of at
least three experiments (duplicate samples) ±
SD.
DNase I Footprinting
A 5' end-labeled probe of the 307-bp mouse Ugrp1
promoter region was generated by PCR using pGL3-907 as a template and
a sense primer (5'-AAAGGATCCTATAGGAAAGCATTCCTCTC-3') and an antisense
primer (5'-AAACTCGAGTGATGGCTGCTTTTCCTCAG-3'). Recombinant T/EBP/NKX2.1
protein was produced according to the manufacturers instructions
(Novagen, Madison, WI) using the pET-30a (+)TTF1(T/EBP/NKX2.1)
expression vector (kindly provided by Dr. Leonard Kohn, Ohio
University, Athens, OH). The DNase I footprinting reaction was
performed using a SureTrack Footprinting Kit (Amersham Pharmacia Biotech). Briefly, recombinant T/EBP/NKX2.1 protein (2 µg), or
BSA (30 µg) as a control, was incubated with 20,000 cpm of probe for
30 min and subjected to DNase I digestion for 1 min at room
temperature. The DNA fragments were separated on 6% polyacrylamide, 7
M urea sequencing gels using the dideoxy
sequencing reaction product (fmol DNA Sequencing System; Promega Corp.) as a size marker.
EMSAs
Nuclear extracts of NCI-H441 cells were prepared as described
(56). Nuclear extracts (15 µg) or the expressed
T/EBP/NKX2.1 protein (500 ng), and when indicated, unlabeled
oligonucleotide competitor DNAs, were preincubated in 23 µl of gel
mobility shift assay buffer (10 mM HEPES-KOH, pH 7.9, 50
mM KCl, 0.6 mM EDTA, 5 mM
MgCl2, 10% glycerol, 5 mM
dithiothreitol, 0.7 mM phenylmethylsulfonyl fluoride, 2
µg/µl pepstatin A, 2 µg/µl leupeptin, and 87 µg/µl
poly[dI-dC] [Amersham Pharmacia Biotech]) for 10 min
on ice. An oligonucleotide probe (1 x 105
cpm) was added to the mixture, and the mixture was incubated for an
additional 30 min at room temperature. For antibody supershift
analyses, 1 µl of anti-TTF-1 (T/EBP/NKX2.1) monoclonal antibody (Lab
Vision Corp., Fremont, CA) was added, and the incubation was continued
for an additional 1 h. Protein-DNA complexes were separated from
free probe by 5% nondenaturing PAGE. After electrophoresis, the gel
was blotted onto Whatman 3MM paper, dried, and exposed to
x-ray film.
Western Blot Analysis
Three forms of cDNAs encoding type A, B, and C proteins were
amplified by PCR and inserted into EcoRI and XhoI
sites of pcDNA3.1/Myc-His(+) A vector (Invitrogen Life Technologies). Transient transfection into COS-1 cells was
performed using Effectene transfection reagent (QIAGEN).
After 2 d, cells and conditioned media were collected, separated
on 13% SDS-polyacrylamide gels under reducing and nonreducing
conditions, and electrophoretically transferred to nitrocellulose
membrane (Schleicher & Schuell, Inc., Keene, NH). The
filter was incubated in PBS containing 5% skim milk and then for
1 h with 250-fold-diluted c-myc 9E10 polyclonal
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
The filter was washed in PBS containing 0.1% Tween 20, incubated with
horseradish peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech), and then washed with the same buffer.
Protein bands were detected using an ECL Western Blotting Detection
Reagent (Amersham Pharmacia Biotech).
Immunohistochemistry
A cDNA segment encoding the mature 70 amino acids of UGRP1 type
A polypeptides was prepared by PCR with the use of full-length UGRP1 as
a template and subcloned into bacterial expression vector pET-32a (+)
(Calbiochem-Novabiochem Corp., La Jolla, CA), placing the
UGRP1 sequence in frame downstream of a hexahistidine tag. The tagged
UGRP1 peptide was expressed in Escherichia coli BL21(DE3) by
induction with 1 mM isopropylthio-ß-galactoside
for 5 h. Cells were collected and lysed in native conditions, and
tagged peptide was purified on a nickel-nitrilotriacetic acid agarose
column followed by SDS-PAGE. The purified peptide was used to prepare
UGRP1 antibody in rabbits (Macromolecular Resources, Fort Collins, CO).
The antimouse uteroglobin/CCSP antibody was a kind gift of Dr. Anil
Mukherjee (National Institute of Child Health and Human Development,
Bethesda, MD). Immunohistochemistry was carried out using 2,000- and
1,000-fold dilutions of UGRP1 and uteroglobin/CCSP antibodies,
respectively, and the Vectastain ABC Rabbit Elite Kit
(Vector Laboratories, Inc., Burlingame, CA).
Mouse Sensitization
Female BALB/cJ mice (Jackson Laboratories, Bar
Harbor, ME), 67 wk old, were used in these studies. They were housed
in a controlled environment with a 12-h light/12-h dark cycle and had
access to food and water ad libitum. The animals were
treated in accordance with Public Health Service guidelines under a
protocol approved by the Genaera Corporation Institutional Animal Care
and Use Committee.
Mice were sensitized with ip administrations of a mixture of
Aspergillus fumigatus extract (Bayer Corp.,
Elkhart, IN; 200 µg/mouse) and alum (Imject; Pierce Chemical Co., Rockford, IL; 2.25 mg/mouse) on study d 0 and 14 and
subsequent intranasal administrations of 25 µl of A.
fumigatus extract [final concentration, 1:50 (wt/vol) in 10%
glycerol] while under light inhaled anesthesia on study d 24, 25, and
26. Mice that were not sensitized or treated were designated
"naive" (see Fig. 9). Sensitized mice were treated ip with either
1) dexamethasone-21-phosphate (Sigma, St. Louis, MO) at a
dose of 2.5 mg/kg twice per wk for a total of nine administrations
(Fig. 9
, Af + Dex), or 2) saline (0.9% sodium chloride injectable,
USP; Baxter, Co., Deerfield, IL) as vehicle control (Fig. 9
, Af). Mice
were also treated with dexamethasone-21-phosphate alone on the same
schedule. On study d 28, mice were killed and lung tissues were
harvested (n = 12 per group) and immediately frozen in liquid
nitrogen for later analyses of mRNA. The RNAs collected were probed for
multiple end points, including UGRP1, UGRP2, and mCLCA3 (gob-5) (Zhou,
Y., and R. C. Levitt, unpublished observation).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: The University of Texas, M.D. Anderson Cancer
Center, Smithville, Texas 78957.
Abbreviations: CCSP, Clara cell secretory protein; DAPI, 4',6-diamidino-2-phenylindole; E, embryonic days; EST, expressed sequence tag; HFH, HNF3/forkhead homolog; HNF, hepatocyte nuclear factor; LPS, lipopolysaccharide; SP, surfactant protein; SSC, 0.15 M NaCl and 15 mM Na citrate, pH 7.0.
Received for publication October 17, 2000. Accepted for publication July 27, 2001.
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REFERENCES |
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