UGRP1, a Uteroglobin/Clara Cell Secretory Protein-Related Protein, Is a Novel Lung-Enriched Downstream Target Gene for the T/EBP/NKX2.1 Homeodomain Transcription Factor

Tomoaki Niimi1, Catherine L. Keck-Waggoner2, Nicholas C. Popescu, Yuhong Zhou, Roy C. Levitt and Shioko Kimura

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A novel gene that is down-regulated in lungs of T/ebp/Nkx2.1-null mouse embryos has been identified using a suppressive-subtractive hybridization method. The gene product is a secreted protein, forms a homodimer, and exhibits an amino acid sequence similar to that seen in the uteroglobin/Clara cell secretory protein family of proteins. This gene, designated Ugrp1 (uteroglobin-related protein 1), consists of three exons and two introns and produces three transcripts by alternative splicing. The Ugrp1 gene was localized by fluorescence in situ hybridization to mouse chromosome 18 at region 18C-D; this region is homologous with human 5q31-34, where one of the asthma susceptibility genes has been assigned. UGRP1 mRNA is predominantly expressed in the lung, with low levels of expression in the thyroid. Expression in the lung is detectable as early as embryonic day 12.5 and increases markedly by embryonic day 16.5. In T/ebp/Nkx2.1-null embryo lungs, UGRP1 expression was significantly reduced as assessed by RT-PCR analysis. Cotransfection assays using a T/EBP/NKX2.1 expression construct with Ugrp1 promoter-luciferase reporter constructs confirmed that T/EBP/NKX2.1 regulates Ugrp1 gene activity at the transcriptional level. Thus, Ugrp1 is a downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor. Changes in UGRP1 mRNA levels in lungs from antigen-sensitized mice suggest the possible involvement of UGRP1 in inflammation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
T/EBP (THYROID-SPECIFIC ENHANCER-BINDING protein)/NKX2.1, also known as TTF1 (thyroid transcription factor 1), is a homeodomain-containing DNA-binding protein that was originally characterized as a transcription factor regulating thyroid-specific expression of genes, including thyroglobulin (1), thyroid peroxidase (2, 3, 4), TSH receptor (5, 6), and Na/I symporter (7) genes. T/EBP/NKX2.1 also controls the transcription of genes specifically expressed in the lung, such as those encoding surfactant proteins (SP)-A (8), B (9), and C (10) and uteroglobin/Clara cell secretory protein (CCSP) (11, 12). The tissue-specific pattern of expression of these genes is believed to be conferred by a unique combination of T/EBP/NKX2.1 along with other transcription factors, including PAX8 and TTF2 in the thyroid (3, 13) and hepatocyte nuclear factor 3 (HNF3) and the HNF3/forkhead homolog (HFH) in the lung (9, 14). Pax8 is a member of the murine Pax family of paired domain-containing genes, which is expressed in the developing kidney, neural tube, and developing and adult thyroid (15). TTF2 is a forkhead domain-containing protein expressed in pituitary and thyroid glands (16). HNF3 and HFH are members of the winged helix/forkhead transcription factor gene family (17). HNF3{alpha} and -ß, and HFH4 and –8, are expressed in developing and adult mouse lungs (17).

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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of the T/EBP/NKX2.1 Downstream Target Gene
To isolate putative T/EBP/NKX2.1 downstream target genes, a suppressive subtractive hybridization method was used to generate a cDNA library of clones that were enriched in the lungs of embryonic day (E) 16.5 wild-type embryos vs. T/ebp/Nkx2.1-null embryos. The lungs of gene-targeted embryos are severely hypoplastic and do not exhibit any characteristics that are present once proximal lung morphogenesis has occurred (24). Thus, clones identified in the suppressive-subtractive cDNA library may include genes that are regulated by a differentiation process in addition to the effects of T/EBP/NKX2.1 deficiency. One hundred ninety-two clones were initially chosen that were then probed with forward-subtracted cDNAs (wild-type) or reverse-subtracted cDNAs (mutant). Twenty-seven clones that gave stronger signals with the forward-subtracted probe than with the reverse-subtracted probe were subjected to virtual Northern blotting analyses. Five clones were found to be differentially expressed. Sequence analyses revealed that one of the clones encodes a polypeptide exhibiting sequence similarity to the uteroglobin/CCSP family of proteins (28, 29, 38, 39). Therefore, we refer to this gene as UGRP1 (uteroglobin-related protein 1).

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. 1AGo). 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. 1BGo).



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Figure 1. Mouse UGRP1

A, Scheme of the mouse Ugrp1 gene structure showing the organization of exons and introns and three types of transcripts. Solid boxes represent exons. The translation initiation and termination codons, including one within intron 2, are indicated. Sequences in intron 2 that are retained in types B and C transcripts are shown by a thick line. A thin jagged line shows sequences that are spliced out in mature mRNAs. The size of each cDNA and polypeptide is indicated on the right. Arrows indicate the positions of primers used for RT-PCR analysis (P1–P5). B, The UGRP1 type A amino acid sequence is aligned with those of mouse uteroglobin/CCSP (mUG/CCSP) (38 ), human mammaglobin A (hMAM-A) (29 ), and rat prostatein C3 (rPSC3) (39 ). Identical residues are shaded. Asterisks indicate the conserved cysteine and lysine residues present in the uteroglobin/CCSP gene family (28 ). The antiflammin region in the uteroglobin/CCSP (28 ) and the predicted UGRP1 signal sequence are shown by a bracket and a line above the alignment, respectively. C, The UGRP1 type A amino acid sequence is aligned with those of mouse UGRP2 and human UGRP1 and -2. Identical and conserved residues are shown in black and shaded boxes, respectively.

 
A BLAST search of the type A amino acid sequence for similar proteins exhibited similarities to the uteroglobin/CCSP family of proteins. Mouse UGRP1 has overall amino acid sequence identity of 25%, 18%, and 27% to mouse uteroglobin/CCSP (38), human mammaglobin A (29), and rat prostatein C3 (39), respectively (Fig. 1BGo). Significant similarity was found in the signal sequence at the N terminus and amino acid residues 63–72, the area called antiflammin, which is believed to be responsible for PLA2-inhibitory activity of the uteroglobin/CCSP (28). The UGRP1 signal sequence exhibits a particularly high similarity to that of rat prostatein C3.

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. 1CGo), 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. 1AGo). 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. 2Go). 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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Figure 2. Fluorescence in Situ Hybridization for Ugrp-1 Gene Chromosome Localization

A, Digital image of a normal mouse metaphase showing G-like banding after enhancement of DAPI counterstain with chromosome 18 labeled at region 18C-D. B, Subsequent painting with chromosome 18 probe.

 
Expression of UGRP1
Using type A cDNA as a probe, UGRP1 expression was examined in adult mouse tissues by Northern blot analyses (Fig. 3AGo). A single 0.5-kb transcript corresponding to type A was clearly detected in the lung. Because T/EBP/NKX2.1 is expressed in the thyroid, this tissue was also examined for UGRP1 expression. Longer exposure did reveal expression in the thyroid. No clear signal was found for types B and C transcripts, further suggesting that types B and C transcripts are expressed at low levels (data not shown). Because uteroglobin/CCSP was originally identified in pregnant rabbit uterus and is induced by progesterone (28, 31), mouse uterus with and without progesterone treatment was examined for the expression of UGRP1, UGRP2, and uteroglobin/CCSP. RT-PCR analysis did not detect any of these transcripts (data not shown). Thus, UGRP1, UGRP2, and uteroglobin/CCSP mRNAs are neither expressed nor induced by progesterone in mouse uterus, at least under the conditions used.



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Figure 3. UGRP1 Expression

A, Northern blot analyses of adult mouse tissues. Left, CLONTECH Laboratories, Inc. multiple Northern blot. Right, Tissue RNAs isolated from adult mouse. Note that the result in the right panel was exposed to x-ray film for 3 d compared with overnight in the left panel. B, Differential expression of mRNA by RT-PCR in E12.5 and E16.5 embryonic lungs prepared from wild-type (+/+) and T/ebp/Nkx2.1-null mutant (-/-). ß-Actin was used as internal control. C, Expression of three types of transcript. The primer positions are indicated in Fig. 1AGo. PCR was carried out using RNAs prepared from E18.5 wild-type mouse embryo lungs (RT) with type A (A), type B (B), and type C (C) cDNAs as controls. A 1-kb DNA Ladder (Invitrogen Life Technologies) is indicated on the left.

 
UGRP1 expression was also examined using RT-PCR with exon 1 (P5)- and exon 3 (P2)-specific primer pairs on E12.5 and E16.5 embryonic lung mRNAs obtained from wild-type and T/ebp/Nkx2.1-null mice (Figs. 1AGo and 3BGo). In wild-type embryo lungs, a band corresponding to type A transcript (380 bp) was barely detected at E12.5 but became intense by E16.5. In contrast, markedly reduced expression was noted in E16.5 T/ebp/Nkx2.1-null embryo lungs. This may reflect the absence of an inductive effect of T/EBP/NKX2.1 and/or the absence of differentiated cells that normally express the gene.

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. 3CGo). 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. 4AGo). 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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Figure 4. Mouse Ugrp1 Gene Promoter Analyses

A, Sequence of the mouse Ugrp1 gene promoter. Arrowheads indicate the positions of the -242, -190, -147, -67, and -18 deletion constructs used in transfection analyses. The minimal T/EBP/NKX2.1-binding consensus sequences (CTNNAG) (9 ) are shown in boldface. The TATA sequence is boxed. The bent arrow (+1) indicates the major transcription start site. DNase I footprint-protected regions I through IV are underlined. B, DNase I footprint. Results of both coding (left) and noncoding (right) strands are shown in the presence of a constant amount of bacterially expressed T/EBP/NKX2.1 (2 µg) or BSA (30 µg) as a control protein with 0.33 and 1 or 0.11 and 0.33 U of DNase I, respectively. The protected areas marked I through IV are indicated by lines on the right. The dideoxy sequencing reaction product (ATGC) was used as a size marker. The nucleotide positions are indicated along the ladder.

 
To precisely localize the T/EBP/NKX2.1 binding sites, DNase I footprinting analyses were carried out using bacterially expressed recombinant T/EBP/NKX2.1 and the 307-bp mouse Ugrp1 gene promoter sequence (Fig. 4AGo). Four protected regions were obtained in which regions II and III contained consensus T/EBP/NKX2.1 binding sites at -182 and -120 bp, respectively (Fig. 4BGo).

Six Ugrp1 promoter-luciferase constructs (PGL3-18, -67, -147, -190, -242, and -907) (Fig. 4AGo) 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. 5Go, 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. 5AGo). 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. 5BGo). 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. 4AGo). 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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Figure 5. Transfection Analyses of the Mouse Ugrp1 Gene

A, Deletion analyses of the mouse Ugrp1 gene promoter. The relative luciferase activity of NCI-H441 cells transiently transfected with the indicated deletion or mutant constructs is shown based on the activity obtained with the basic vector measured as 1 in the presence of coexpressed pCMV4-T/EBP/NKX2.1 (black bars) or pCMV4 (white bars). Data are means of at least three experiments (duplicate samples) ± SD. B, Deletion analyses of the mouse Ugrp1 gene promoter in HeLa cells. The relative luciferase activity was expressed as described in B. C, Scheme of mutant constructs. Asterisks indicate base changes.

 
EMSAs were performed with nuclear extracts from NCI-H441 cells to define the function of possible T/EBP/NKX2.1-binding elements at -182 and -120 bp (Fig. 6Go). Oligonucleotides [Fig. 6AGo, probe II (-200 to -173 bp) and probe III (-136 to -113 bp)], each containing a T/EBP/NKX2.1 consensus binding site (CTNNAG) (9), formed a specific protein-DNA complex that was competed away by the addition of 100- and 500-fold excesses, respectively, of unlabeled specific oligonucleotide but not by a nonspecific oligonucleotide (Fig. 6Go, B and C, left). Oligonucleotides (Fig. 6AGo, probe II mut and probe III mut), each containing a mutated T/EBP/NKX2.1-binding site, did not compete for complex formation, whereas oligonucleotide C, containing the T/EBP/NKX2.1-binding site identified in the rat thyroglobulin gene (1), did compete. Anti-TTF1(T/EBP/NKX2.1) monoclonal antibody produced a faint but distinct supershifted band (Fig. 6Go, B and C, right). When recombinant T/EBP/NKX2.1 was used, incubation with anti-TTF1(T/EBP/NKX2.1) monoclonal antibody resulted in a supershift (Fig. 6DGo). The supershifted bands using probes II and III were faint but had the same mobility as the bands using oligonucleotide C as a probe. The third T/EBP/NKX2.1 consensus binding site found at -37 bp did not produce any specific protein-DNA complex, indicating that T/EBP/NKX2.1 does not bind to this site (data not shown). Interestingly, when the DNase I footprinting-protected area IV was used in gel shift analyses using NCI-H441 nuclear extracts, no specific protein-DNA complex was obtained (data not shown). Thus, T/EBP/NKX2.1 appears to bind to the T/EBP/NKX2.1 consensus binding sites at -182 and -120 bp of the mouse Ugrp1 gene promoter.



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Figure 6. EMSAs

A, Oligonucleotide sequences used as probes: probe II, -200 to -173 bp in the mouse Ugrp1 gene promoter; probe III, -136 to -113 bp in the mouse Ugrp1 gene promoter; probe II mut, T/EBP/NKX2.1-binding site mutated in probe II; probe III mut, T/EBP/NKX2.1-binding site mutated in probe III. Oligonucleotide C was taken from the rat thyroglobulin (rTG) promoter, which had been identified as a T/EBP/NKX2.1-binding site (1 ). Putative T/EBP/NKX2.1 consensus and mutated sequences are shown in boldface and underlined. B, Left, NCI-H441 nuclear extracts were incubated with 32P-labeled probe II. The specifically retarded band is shown by an arrow. Competition assays were performed with 100- and 500-fold excesses of unlabeled specific (s), nonspecific (ns), probe II mut (mut), or oligonucleotide C (c) oligonucleotides. Right, Antibody supershift analysis. One microliter of anti-TTF1(T/EBP/NKX2.1) monoclonal antibody was added (Ab +). A supershifted band is marked with an asterisk. C, Left, NCI-H441 nuclear extracts were incubated with labeled probe III. Competition assays were performed with a 100-fold excess of unlabeled specific (s), nonspecific (ns), probe III mut (mut), or oligonucleotide C (c). Right, Antibody supershift analysis. One microliter of anti-TTF1(T/EBP/NKX2.1) monoclonal antibody was added (Ab+). A supershifted band is marked with an asterisk. Note that in the antibody supershift analyses in B and C, faint but clearly supershifted bands were observed. D, Labeled probe II, probe III, and oligonucleotide C were incubated with (+) or without (-) recombinant T/EBP/NKX2.1 in the presence or absence of anti-TTF1(T/EBP/NKX2.1) monoclonal antibody. A supershifted band is indicated by an arrow.

 
To confirm the functional relationship between two T/EBP/NKX2.1 consensus binding sites and Ugrp1 gene transcriptional activity, mutations were introduced into T/EBP/NKX2.1-binding sites at -182 and -120 bp of the reporter constructs (Figs. 5CGo and 6AGo). In the presence of the T/EBP/NKX2.1 expression vector, mutation at -182 bp (-190 mut 1) and -120 bp (-190 mut 2) resulted in approximately one half and one fourth of the luciferase activity compared with no mutation (-190) (Fig. 5AGo). When both binding sites were mutated (-190 mut 3), trans-activation by T/EBP/NKX2.1 was completely abolished. Construct -147 mut, which contains only one T/EBP/NKX2.1-binding site at -120 bp that is mutated, exhibited a 50% reduction in luciferase activity compared with construct -147 when T/EBP/NKX2.1 was coexpressed in HeLa cells (Fig. 5BGo). These data demonstrate that elements that mediate the transcriptional activation of T/EBP/NKX2.1 are located at positions -182 and -120 bp in the 5' region of the mouse Ugrp1 gene.

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. 7Go). 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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Figure 7. Expression of UGRP1 Products in COS-1 Cells

Cell lysates and conditioned media from the culture of COS-1 cells individually expressing c-myc-tagged types A, B, and C polypeptides were separated by SDS-PAGE in reducing (R) and nonreducing (NR) conditions and then immunoblotted with anti-c-myc antibody. The sizes of protein markers are shown on the left. The bands assumed to be monomers (m) and dimers (d) are indicated.

 
UGRP1 Expression in Lung Airways and Its Possible Involvement in Inflammation
Polyclonal antibody against mouse UGRP1 was raised using bacterially expressed UGRP1 type A polypeptide. The antibody specificity for UGRP1 was examined by Western blotting using c-myc epitope-tagged UGRP1 and uteroglobin/CCSP polypeptides expressed in COS-1 cells, in which anti-UGRP1 antibody reacted with only UGRP1 (Fig. 8AGo). Wild-type newborn embryo lungs were then subjected to immunohistochemistry using this antibody. Positive immunostaining for UGRP1 was clearly found in the epithelial cells of the trachea, bronchus, and bronchioles, whereas immunostaining for uteroglobin/CCSP was detected only in the bronchus and bronchioles, but not the trachea (Fig. 8BGo). These results further indicate that the anti-UGRP1 antibody does not cross-react with uteroglobin/CCSP. Most UGRP1 immunopositive cells in the bronchioles are Clara cells. The T/ebp/Nkx2.1-null embryo lungs did not have any positive staining, as expected (data not shown).



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Figure 8. Analyses Using UGRP1 Antibody

A, Specificity of anti-UGRP1 antibody. Western blotting was performed using c-myc epitope-tagged UGRP1 and uteroglobin/CCSP proteins expressed in COS-1 cells, which were treated with anti-UGRP1 (left) and c-myc (right) antibodies. Note that UGRP1-expressed protein reacted clearly, albeit faintly, with both anti-UGRP1 and c-myc (open arrowhead) antibodies, whereas uteroglobin/CCSP-expressed protein reacted only with c-myc antibody. B, Immunostaining of UGRP1 (a–c) and uteroglobin/CCSP (d–f). a and d, trachea; b and e, bronchus (arrows show bronchioles); c and f, bronchioles. Magnification: a, b, d, and e, x150; c and f, x300.

 
To obtain information regarding UGRP1 function, Northern analyses were performed using RNAs isolated from whole lungs of naive animals, antigen-exposed animals, antigen-exposed and dexamethasone-treated animals, and animals treated with dexamethasone in the absence of antigen exposure (Fig. 9Go). Antigen-induced inflammation in BALB/c mice was associated with a reduction in UGRP1 expression compared with naive animals. Although the expression of UGRP1 returned toward normal with dexamethasone treatment in antigen-exposed animals, dexamethasone treatment alone did not change the level of UGRP1 expression compared with naive animals. UGRP2 was also examined; it showed a similar pattern of changes to UGRP1, although there was less of a reduction in UGRP2 expression apparent after antigen treatment. In contrast to UGRP1, dexamethasone treatment of healthy animals (no antigen exposure) increased UGRP2 expression by approximately 20% when normalized for ß-actin levels. To further ensure that our results were significant, we probed the same RNAs for mCLCA3 (gob-5), a member of a gene family of calcium-activated chloride channel proteins (49). In contrast to UGRP1, and consistent with the literature (49), this gene was not expressed in lungs of BALB/c naive animals, was up-regulated in lungs of antigen-exposed animals, and expression was slightly reduced by dexamethasone treatment of these antigen-exposed animals (data not shown). Thus, the down-regulation of lung UGRP1 and UGRP2 steady-state expression by antigen is an unexpected and novel finding in this T helper cell (TH)2 cytokine-based antigen model.



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Figure 9. UGRP1 and UGRP2 Expression in Antigen-Exposed Mouse Lungs

Left, RNAs were collected from lungs of naive mice (not antigen exposed or treated), mice exposed to antigen and treated with saline only (Af), or mice exposed to antigen and treated with dexamethasone (Af + Dex). Right, RNAs were collected from lungs of naive mice or mice treated with dexamethasone alone (Dex) (not antigen exposed). Approximately 3.5 µg of total RNA was used per lane. Full-length UGRP1 and UGRP2 cDNAs were used as probes.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A potential T/EBP/NKX2.1 target gene, Ugrp1, mainly expressed in the lung, was cloned. The presence of orthologous and homologous genes in mouse and human suggests that they constitute a new gene family. The UGRP1 amino acid sequences show similarities to those of the uteroglobin/CCSP gene family of proteins (28, 29, 31, 38, 39). Uteroglobin/CCSP, the main member of the gene family, is a progesterone-inducible, homodimeric secretory protein expressed in many organs, such as uterus, lung, mammary gland, and prostate (28, 31). Western blotting analyses indicate that UGRP1 is a secreted protein and may function as a homodimer, as seen with uteroglobin/CCSP. The uteroglobin/CCSP family proteins are characterized by two conserved cysteine residues at the N- and C-terminal regions that are required to form a dimer and a lysine residue located between them (28, 31). The area containing the conserved lysine residue is called antiflammin in the uteroglobin/CCSP family (28, 31). The amino acid similarities between UGRP1 and uteroglobin/CCSP family proteins are significant in the areas of signal peptide and antiflammin, although overall similarities are low and two conserved cysteine residues are absent in UGRP1.

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 (E9–E9.5) (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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of the Lung-Specific Gene Ugrp1 by Suppressive-Subtractive Hybridization
T/ebp/Nkx2.1 (+/-) mice were bred to generate T/ebp/Nkx2.1-null embryos (22). Embryos were obtained by dissection of pregnant mice at E16.5, and genotyping was performed by PCR using yolk sacs. Noon on the day when the vaginal plug was detected was considered stage E0.5. Total RNA was isolated from lungs of null mutant embryos (driver) and wild-type embryos (tester) using the ULTRASPEC RNA Isolation System (Biotecx Laboratories, Houston, TX) and used as a template to synthesize double-stranded cDNAs using a SMART PCR cDNA Synthesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA).

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 manufacturer’s 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 {lambda}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 Denhardt’s 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. 1AGo 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 50–70% 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 manufacturer’s 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), 6–7 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. 9Go). 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. 9Go, Af + Dex), or 2) saline (0.9% sodium chloride injectable, USP; Baxter, Co., Deerfield, IL) as vehicle control (Fig. 9Go, 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 = 1–2 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).


    ACKNOWLEDGMENTS
 
We thank Drs. Frank Gonzalez and Taro Akiyama for helpful discussions and critical review of the manuscript, Dr. Anil Mukherjee for providing anti-uteroglobin/CCSP polyclonal antibody, Dr. Leonard Kohn for providing the pET-30a (+)-TTF1(T/EBP/NKX2.1) bacterial expression vector, Dr. Jerrold Ward for performing immunohistochemical analyses, and Christine Weiss for her expert technical assistance with the animals models.


    FOOTNOTES
 
1 Current address: Sekiguchi Biomatrix Signaling Project, ERATO Japan Science and Technology Corporation, c/o Aichi Medical University, Aichi 480-1195, Japan. Back

2 Current address: The University of Texas, M.D. Anderson Cancer Center, Smithville, Texas 78957. Back

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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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