Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice

Constantin C. Adams1,*, M. Nurul Alam1,*, Barry C. Starcher2, and Vijayakumar Boggaram1

Departments of 1 Molecular Biology and 2 Biochemistry, University of Texas Health Science Center at Tyler, Tyler, Texas 75708-3154


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Surfactant protein B (SP-B) is expressed tissue specifically in the lung and is developmentally regulated. To identify genomic regions that control SP-B expression, we analyzed SP-B promoter activity in transgenic mice containing rabbit SP-B 5'-flanking DNA fragments linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Results showed that whereas the -2,176/+39-bp fragment failed to express CAT, shorter fragments of -730/+39 and -236/+39 bp expressed CAT tissue specifically in the lung. Further deletion of 5'-flanking DNA to -136 bp resulted in no expression of CAT. Immunostaining demonstrated that both -730/+39- and -236/+39-bp regions expressed CAT specifically in alveolar type II and Clara cells. The -236/+39-bp region expressed CAT at a significantly lower level than the -730/+39-bp region. CAT expression in mice containing the -730/+39-bp region was detected in embryonic day 14 lung and attained maximum levels in day 18 lung, indicating that the developmental expression of CAT was similar to that of SP-B. These data show that the DNA elements necessary for cell type-specific expression are located within -236/+39 bp of the SP-B gene. Additionally, these data suggest that the -2,176/-730- and -730/-236-bp regions contain the DNA elements that repress and enhance SP-B gene transcription, respectively.

gene regulation; transcription; type II cell; Clara cell; respiratory distress syndrome


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SURFACTANT, A LIPOPROTEIN COMPLEX, is synthesized and secreted by the alveolar type II epithelial cells of the lung. Surfactant maintains the integrity of the alveoli during respiration by reducing surface tension at the alveolar air-tissue interface (11) and serves important roles in host defense in the lung (26). Deficiency of surfactant is associated with the occurrence of respiratory distress syndrome in preterm infants (2), the leading cause of neonatal morbidity and mortality in developed countries. Surfactant protein B (SP-B), a 9-kDa hydrophobic protein, is essential for the maintenance of the biophysical properties and physiological functioning of surfactant. SP-B promotes the adsorption and spreading of surfactant phospholipids (22) and stabilizes the phospholipid monolayer formed on the alveolar surface (8). Deficiency of SP-B due to a frame shift mutation in the coding region is associated with fatal respiratory failure in infants with congenital alveolar proteinosis (18). Targeted disruption of the SP-B gene causes respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (7).

SP-B mRNA is expressed in a cell type-specific manner by the alveolar type II and bronchiolar epithelial cells of the lung (20, 30) and is under developmental and multifactorial regulation (28). Glucocorticoids and cAMP increase, whereas tumor necrosis factor-alpha decreases SP-B mRNA expression (3).

Our laboratory (15) previously found that a minimal promoter containing -236/+39 bp of rabbit SP-B gene is necessary and sufficient for high-level expression of the chloramphenicol acetyltransferase (CAT) reporter gene in NCI-H441 cells, a cell line with the characteristics of bronchiolar epithelial cells (Clara cells). The minimal SP-B promoter supported high-level CAT expression in a lung cell type-specific manner in cell cultures, suggesting that it contained a cell- or tissue-specific enhancer (15). Our studies also identified binding sites for Sp1, Sp3, thyroid transcription factor-1 and hepatocyte nuclear factor-3 transcription factors in the minimal SP-B promoter that acted in a cooperative or combinatorial manner to maintain SP-B promoter activity (16).

Genomic regions and gene regulatory elements that direct cell-specific and developmental expression of the SP-B gene have not been identified. In this study, we analyzed SP-B promoter activity in mice containing SP-B 5'-flanking DNA fragments linked to the CAT gene. Analysis of CAT expression in transgenic mice showed that, whereas the -2,176/+39-bp region failed to support CAT expression, shorter fragments consisting of -730/+39 and -236/+39 bp expressed CAT in a cell- or tissue-specific manner in the lung. Further deletion of 5'-flanking DNA to -136 bp resulted in no expression of CAT in lung and other tissues. CAT expression was restricted to alveolar type II and bronchiolar (Clara) epithelial cells. The developmental expression of CAT in mice containing the -730/+39-bp region of the SP-B gene was similar or identical to endogenous SP-B gene expression.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid construction and generation of transgenic mice. Plasmid pBLCAT6 containing the -2,176/+39-bp fragment of the SP-B gene was digested with HindIII and KpnI or SacI to release SP-B-CAT fusion DNA constructs containing -2,176/+39- and -730/+39-bp fragments of the SP-B gene. Plasmid pSKCAT containing the -236/+39-bp fragment of the SP-B gene was digested with XbaI and KpnI to release a SP-B-CAT fusion DNA construct containing the -236/+39-bp fragment of the SP-B gene. Plasmid pBLCAT6 containing -136/+39 bp of SP-B 5'-flanking DNA was digested with HindIII and KpnI to obtain a SP-B-CAT construct containing the -136/+39-bp SP-B fragment. The linear SP-B-CAT fusion DNA fragments were purified by agarose gel electrophoresis and dissolved in 10 mM Tris · HCl, pH 7.4, at a concentration of ~100 ng/ml.

Transgenic mice were generated at the Transgenic Technology Core Facility (University of Texas Southwestern Medical Center, Dallas, TX). Transgenic offspring were identified by dot blot analysis of tail DNA with the use of full-length CAT cDNA as a probe. The number of copies (c) of transgene integrated per genome of transgenic mice was determined by quantitative DNA dot blot assay, with CAT cDNA as the standard.

Dot and Southern blot analysis of tail DNA. Genomic DNA from mouse tails (0.6-1 cm) was isolated with a DNA isolation kit from 5 Prime right-arrow 3 Prime (Boulder, CO) or QIAGEN (Valencia, CA), and the DNA concentration was determined by measuring absorbance at 260 nm. Dot and Southern blot analyses of DNA were carried out according to standard methods with 5-10 µg of genomic DNA and Hybond N+ (Amersham) as the transfer membrane. The membranes were hybridized with 32P-labeled full-length CAT cDNA as the probe, washed under high-stringency conditions, and exposed to X-ray film to obtain an autoradiograph.

CAT enzymatic assay and CAT ELISA. Mouse tissues were suspended in 5 volumes (vol/wt) of cold 0.25 M Tris · HCl, pH 7.8, containing 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin and leupeptin, and 0.5 µg/ml of benzamidine and homogenized with a high-speed homogenizer (Tekmar Tissuemizer). Homogenates were freeze-thawed and centrifuged at 12,000 g for 5 min, and the supernatants were removed and stored at -70°C. The CAT activity of the tissue extracts was determined by the liquid scintillation counting method (24) after the extracts were heated to 60°C for 10 min to inactivate the endogenous acetylase. CAT levels were also measured by ELISA with a CAT ELISA kit (Boehringer Mannheim).

Determination of protein. The protein concentration of the tissue extracts was determined by the Bradford (5) method with the Bio-Rad protein assay reagent and bovine serum albumin as the protein standard.

RNA isolation and Northern blot analysis. Total RNA from tissues was isolated by the acid-phenol method with TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. The RNA was separated by electrophoresis on agarose gels (1%) containing 20 mM MOPS and 1.1% formaldehyde and transferred to Hybond N+ membranes by capillary action with saline-sodium citrate (20×) as the transfer solution. The membranes were hybridized with 32P-labeled full-length mouse SP-B cDNA and CAT cDNA probes, washed, and exposed to X-ray film to obtain an autoradiograph.

Immunohistochemical localization of CAT and SP-B. Adult mice were killed, and their lungs were perfused with saline to remove blood. The perfused lungs were instilled with formalin or ExCell fixative for 20 min under 20 cmH2O pressure and then removed and stored in the fixative for 24 h. The fixed lungs were dehydrated and embedded in paraffin, and serial 6-µm sections were cut for histology. Tissue sections were serially incubated with polyclonal CAT (5 Prime right-arrow 3 Prime), human SP-B (Chemicon International, Temecula, CA), or nonimmune IgG antibodies at 1:200 dilution; biotinylated secondary antibody; and horseradish peroxidase-labeled streptavidin (Innovex Biosciences, Richmond, CA). Sections were then stained with 3-amino-9-ethylcarbazole (AEC) according to the kit instructions and counterstained with hematoxylin. For double immunostaining, the sections were stained first with AEC for CAT and then with TrueBlue substrate (Kirkegaard and Perry, Gaithersburg, MD) for SP-B.

Developmental regulation of CAT. F1 heterozygous male transgenic mice were mated with wild-type female mice, and the female mice were examined daily for the copulatory plug. The gestational age of the embryos was estimated based on the day that the copulatory plug was first detected in the female (day 0). The female mice were killed at different stages of gestation, and the embryos were collected. Lungs were removed for the preparation of extracts with the aid of a dissecting microscope.

Cell culture and transfections. MLE-12 cells, a mouse lung epithelial cell line, were maintained in HITES (hydrocortisone, insulin, transferrin, estrogen, and selenium) medium containing penicillin (100 U/ml) and streptomycin (100 µg/ml) and supplemented with 2% fetal bovine serum. These cells express detectable levels of SP-B and SP-C and therefore may have characteristics of alveolar type II cells (29). MLE-12 cells were transfected by liposome-mediated DNA transfer with LipofectAMINE. The conditions for transfection were similar to those previously described for NCI-H441 cells by our laboratory (15).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of SP-B-CAT transgenic mice. To identify the genomic regions that control cell type-specific and developmental expression of the SP-B gene, we constructed SP-B-CAT fusion genes containing -2,176/+39-, -730/+39-, -236/+39-, and -136/+39-bp fragments of rabbit SP-B 5'-flanking DNA (Fig. 1). The SP-B-CAT fusion DNA constructs were injected into mouse oocytes to generate transgenic mice, and the transgenic founder mice were bred with wild-type mice to establish independent lines of transgenic mice.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic representation of surfactant protein B (SP-B):chloramphenicol acetyltransferase (CAT) constructs used for the generation of transgenic mice. Solid lines, SP-B genomic DNA containing different lengths of 5'-flanking DNA and 39 bp of first exon; solid boxes, CAT gene; arrows, position of the transcription start site and direction of transcription.

Expression of CAT in transgenic mice. We analyzed SP-B promoter activity in transgenic mice by measuring tissue expression of CAT activity (Table 1). Results showed that CAT activity was not detected in the lung extracts of mice from seven independent lines derived from the -2,176/+39-CAT construct. Analysis of CAT activity in other tissues, such as liver, kidney, intestine, stomach, trachea, spleen, heart, and skeletal muscle, in mice from two independent lines indicated the absence of CAT expression. Tissue-specific expression of CAT was also analyzed by Northern blotting. Consistent with the results of tissue expression of CAT activity, Northern blot analysis showed no expression of CAT mRNA in lung and other tissues (data not shown). These data indicated that the -2,176/+39-bp region of the SP-B gene is not capable of directing CAT expression in lung or other tissues.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   CAT expression in various tissues of adult F1 heterozygous SP-B TG mice

To determine if the lack of transgene expression was due to an alteration of the introduced transgene by degradation or rearrangement, we determined the size of the transgene integrated into the genome by Southern blot analysis. Tail DNA of seven independent lines of mice was digested with EcoRI (EcoRI cuts the transgene at nucleotide 303 of CAT cDNA) and analyzed by Southern blotting with 32P-labeled CAT cDNA. Results indicated that a DNA fragment of ~4 kb was hybridized in the DNA digests of mice containing more than one copy of the transgene (data not shown). These data are consistent with the integration of the intact transgene linked in a tandem head-to-tail array at the site of integration and rule out degradation or rearrangement of the transgene. It is a common finding in transgenic mice that when multiple copies integrate, they are always found linked in a tandem head-to-tail array at the site of integration (19).

Analysis of CAT expression in F1 heterozygous mice from three independent lines derived from the -730/+39-CAT construct revealed that CAT activity was expressed in a tissue-specific manner in the lung (Table 1). CAT activity in other tissues examined was not significantly higher than background, indicating lack of expression. Transgenic line 1, with three to six copies of the transgene, expressed CAT at a significantly higher level than the other lines (Table 1). Tissue-specific expression of CAT activity in an adult F1 heterozygous mouse from line 1 (3-6 c) is shown in Fig. 2. Southern blot analysis of tail DNA from transgenic line 1, with 32P-labeled CAT cDNA indicated that the integrated transgene had not undergone any degradation or rearrangement (data not shown). Of the four independent lines of mice that were positive for the -236/+39-bp construct, only one line (line 3) expressed CAT in a tissue-specific manner in the lung. CAT activity in the other tissues was similar to that in corresponding tissues of a wild-type littermate and was considered background. The -236/+39-bp region expressed CAT at a significantly lower level compared with the -730/+39-bp region (Table 1). CAT activity was not detected in lung and other tissues from four independent lines of mice derived from the -136/+39-bp construct, indicating the inability of the -136/+39-bp SP-B region to direct expression.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2.   Tissue-specific expression of SP-B-730:CAT transgene. CAT activity in various tissues from an adult F1 heterozygous mouse from transgenic line 1 and lung tissue [Lu (wt)] from a wild-type littermate was determined and normalized to protein content of tissue extracts. Lu, lung; He, heart; In, intestine; Ki, kidney; Li, liver; Mu, muscle; Sp, spleen; St, stomach; Tr, trachea. Data are means ± SD of duplicate measurements. Identical expression patterns were observed in 2 other independent lines of transgenic mice.

Tissue-specific expression of CAT mRNA. Because CAT expression in mice (3-6 c) derived from the -730/+39-CAT transgene was highest compared with the other lines, we analyzed tissue-specific expression of CAT mRNA by Northern blotting. Results showed that similar to the endogenous SP-B mRNA, CAT mRNA expression was detected only in the lung (Fig. 3).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3.   Tissue-specific expression of SP-B-730:CAT transgene and the endogenous SP-B mRNA. Total RNA (30 µg) isolated from different tissues of an adult F1 heterozygous mouse from transgenic line 1 was analyzed by Northern blotting with 32P-labeled full-length mouse SP-B cDNA (A) or CAT cDNA (B) as a probe. After hybridization with CAT cDNA, the membrane was stripped and hybridized with SP-B cDNA. C: ethidium bromide staining of 28S and 18S RNAs in total RNA samples.

Western blot analysis of the tissue extracts of transgenic mice with polyclonal antibodies to CAT identified a protein of 24 kDa in the lung extracts, which was consistent with the results of the Northern blot analysis (data not shown). No protein of similar size was detected in the lung extracts of a wild-type littermate.

Cellular expression of the CAT transgene. SP-B mRNA is expressed in the alveolar type II and bronchiolar epithelial cells of adult lungs (20, 30). In the adult rabbit lung, approximately equal concentrations of SP-B mRNA were detected in alveolar type II and bronchiolar epithelial cells (30). We examined the cellular expression of CAT in transgenic mice by immunohistochemical analysis. Serial sections of the lung tissues of adult transgenic mice for the -730/+39-bp (line 1, 3-6 c), -236/+39-bp (line 3, 3-6 c), and wild-type littermates were processed for immunohistochemical analysis with polyclonal antibodies to CAT and SP-B.

Results for the -730/+39-bp transgenic mice showed that the majority of alveolar epithelial cells that expressed SP-B were located at the corners of the alveoli (Fig. 4A). Immunostaining for CAT was also found predominantly in alveolar epithelial cells located at the corners of the alveoli (Fig. 4B). Nearly all of the cells lining the bronchiolar epithelium stained positive for SP-B (Fig. 4C) and CAT (Fig. 4D). Staining for CAT was not detected in lung tissues of wild-type littermates (Fig. 4, E and F). Tissue sections stained with nonimmune IgG showed little or no reaction, indicating low background (data not shown). Double immunostaining showed that many but not all of the cells that stained positive for CAT also stained for SP-B (data not shown). The extensive staining for SP-B and CAT in the bronchiolar epithelium was consistent with the finding that Clara cells comprise 90% of the total cell population in the bronchiolar epithelium (21). Staining for CAT was detected in the alveolar type II and bronchiolar (Clara) epithelial cells of -236/+39-bp SP-B transgenic mice, but the intensity was less compared with -730/+39-bp transgenic mice (data not shown).


View larger version (156K):
[in this window]
[in a new window]
 
Fig. 4.   Immunohistochemical localization of CAT and SP-B expression in lung tissues of SP-B-730 transgenic mice. Lung tissue from an adult F1 transgenic mouse from line 1 (3-6 copies) was fixed in formalin and then embedded in paraffin. Serial sections (6 µm) were mounted on slides and immunostained with polyclonal antibodies to human SP-B (A and C) or CAT (B and D). Immune complexes were visualized with 3-amino-9-ethylcarbazole substrate, resulting in the deposition of reddish precipitate. After immunostaining, tissue sections were counterstained with hematoxylin. Arrows indicate staining for SP-B in alveolar type II cells (A; original magnification, ×325) and Clara cells (C; original magnification, ×240) and for CAT in alveolar type II cells (B; original magnification, ×325) and Clara cells (D; original magnification, ×240). Staining for CAT in lung tissue from a wild-type littermate was negative (E; original magnification, ×80 and F; original magnification, ×160).

Developmental expression of CAT mRNA. We investigated the developmental expression of CAT to determine if the -730/+39-bp region of the SP-B gene was capable of controlling the developmental expression of the CAT gene in a manner similar to that in the endogenous SP-B gene. CAT expression in the lungs of embryonic day 14, 15, 16, and 18 and newborn transgenic mice was analyzed by CAT assay to investigate the developmental regulation of CAT expression (Fig. 5). CAT expression was detected in embryonic day 14 lungs and increased markedly as a function of development to reach maximal levels by embryonic day 18. The expression levels of CAT in newborn mice were not significantly different from those in embryonic day 18 mice. Investigation of the developmental expression of CAT by ELISA produced similar results (data not shown). The levels of CAT expression in embryonic lungs in a given family varied considerably even though they were expected to have identical sites of integration and copy numbers of the transgene.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   Developmental expression of SP-B-730:CAT transgene. Male mice from transgenic line 1 (3-6 copies) were mated with wild-type female mice. Female mice were killed at the specified days of gestation, and fetal lungs were collected for measurement of CAT activity. Each data point represents CAT activity in lung extracts of individual fetal or newborn mice.

Analysis of SP-B promoter function in MLE-12 cells. Immunostaining of lung tissues from mice containing the -236/+39-bp region of the SP-B promoter demonstrated staining for CAT in both alveolar type II and Clara cells, although the intensity of staining was less than that in the mice containing the -730/+39-bp region. These data suggested that cis-DNA elements within -236 bp are capable of directing transgene expression in both alveolar type II and Clara cells. We analyzed SP-B promoter function in MLE-12 cells by deletion mapping to determine if SP-B promoter fragments confer a pattern of regulation similar to that in NCI-H441 cells and transgenic mice. The results showed that deletion of the 5'-flanking SP-B DNA from -2,176 to -730 and -236 bp increased promoter activity by 30%; however, further deletion to -136 bp reduced activity by >80% (Fig. 6). The pattern of SP-B promoter regulation in MLE-12 cells is similar to that observed in H441 cells (15), suggesting that the cis-DNA elements that control promoter activity in H441 cells also control its activity in MLE-12 cells.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Deletion analysis of SP-B 5'-flanking DNA in MLE-12 cells. Plasmids containing SP-B promoter fragments 3'-end at +39 bp, linked to the CAT gene were transiently transfected into MLE-12 cells, and after 48-60 h of incubation, CAT activity of cell extracts was determined. CAT activities were normalized to cotransfected beta -galactosidase activity to correct for variations in transfection efficiency. Data are means ± SE of >4 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SP-B mRNA is expressed in a highly cell type-specific manner by the alveolar type II and bronchiolar epithelial cells of the lung (20, 29). Although analysis of SP-B promoter expression by transfection studies has indicated that promoter fragments of human (4, 27), rabbit (15), and mouse (6) SP-B genes contain the cis-DNA elements necessary for cell-specific expression, their role in conferring lung cell-specific expression has not been investigated. Furthermore, the regulatory DNA regions that control developmental expression of the SP-B gene have not been identified.

Our laboratory previously determined (15) that rabbit SP-B 5'-flanking DNA sequences within -2,176 bp are capable of expressing the CAT reporter gene in NCI-H441 cells and that a minimal promoter sequence comprising -236/+39 bp was necessary and sufficient for high-level expression of the CAT gene. In the present study, we analyzed SP-B promoter activity in transgenic mice to identify the DNA regions necessary for cell-specific and developmental regulation of expression. Transgene expression was not detected in lung and other tissues of several independent lines of mice harboring the -2,176/+39-bp region of the SP-B gene. The lack of expression of the transgene may not be related to the site of integration or the copy number of the transgene because similar results were found in mice derived from seven independent lines. Although we did not determine the site of integration of the transgene in each line, it is highly likely that each line had a unique integration site because the microinjected DNA integrates randomly in individual embryos (19). Southern blot analysis did not indicate modification of the injected DNA by degradation or rearrangement. Taken together, these data indicated that the -2,176/+39-bp sequence of the SP-B gene is not capable of expressing the transgene.

The inability of the -2,176/+39-bp region of the SP-B gene to express CAT suggests the presence of silencer elements within the -2,176-bp sequence. These DNA elements can bind transcriptional repressors to promote closed chromatin structure, leading to the inhibition of gene transcription. Our previous transfection study (15) showed that deletion of the -2,176-bp fragment of SP-B 5'-flanking DNA to -730 and -236 bp increased CAT expression by nearly twofold (15), suggesting the existence of putative silencer elements in the upstream region. The results from transgenic mice are consistent with the data from transfection studies that indicate the presence of putative silencer elements within the -2,176/-730-bp region of SP-B 5'-flanking DNA.

Because the -2,176/+39-bp region of SP-B 5'-flanking DNA failed to express the CAT gene in the transgenic mice, we sought to determine if shorter fragments of SP-B 5'-flanking DNA, -730/+39 and -236/+39 bp, were capable of expressing CAT gene in the transgenic mice. Our laboratory showed in a previous study (15) that a minimal promoter region comprising -236/+39 bp of the SP-B gene supported high-level expression of the CAT reporter gene in H441 cells. CAT expression was detected in a tissue-specific manner in the lungs of all three independent lines of transgenic mice harboring the -730/+39-bp region. These data indicated that cis-acting DNA elements within the -730/+39-bp region of SP-B gene 5'-flanking DNA are capable of directing tissue-specific expression of the CAT gene in the lung. Because all three independent lines of transgenic mice expressed CAT specifically in the lung, the findings cannot be related to the integration site or the copy number of the transgene.

The -236/+39-bp SP-B region expressed CAT at a significantly lower level than the -730/+39-bp region. Consistent with these data, immunohistochemical staining showed diminished CAT expression in alveolar type II and Clara cells of -236/+39-bp transgenic mice. As in the case of the -730/+39-bp construct, CAT activity was not detected in any tissue examined except lung, indicating that strict tissue-specific control of expression was maintained. Further deletion of the SP-B promoter to -136 bp resulted in no expression of CAT. These data are consistent with the results of our previous cell transfection study (15) that demonstrated the presence of the cis-DNA elements necessary for lung cell-specific expression within the -236/+39-bp region of the SP-B gene.

SP-B promoter function was regulated similarly in both H441 cells and MLE-12 cells, suggesting that the same cis-DNA elements control cell-specific expression of the SP-B promoter in alveolar type II and Clara cells. This conclusion is further supported by the deletion mapping studies of SP-B promoter function in transgenic mice. The markedly lower expression of CAT in mice harboring the -236/+39-bp construct compared with the -730/+39-bp construct suggested that sequences within the -730/-236-bp region contain enhancer elements necessary for the optimal expression of SP-B promoter activity. The lack of CAT expression from the -136/+39-bp region indicates that upstream sequences contain important cis-DNA elements and is consistent with the results of in vitro transfection studies that showed that the -236/-136-bp region contains functional cis-DNA elements for thyroid transcription factor-1 and Sp1/Sp3 binding (14). cis-DNA elements within -236/-136- and -136/-27-bp regions are necessary for SP-B promoter activity in H441 cells in vitro (14, 15).

The promoter activity of the -236/+39-bp region in transgenic mice was significantly lower compared with the activity we (15) previously observed in NCI-H441 cells in vitro. The relatively low level of expression of the -236/+39-bp promoter in transgenic mice could be due to the loss of upstream enhancer elements that were not detected in the in vitro cell culture system. Alternatively, the differences in the promoter activities between the in vitro (transient expression) and in vivo (transgenic animal) systems may be related to the state of the introduced DNA. Whereas the DNA was not integrated into the chromosome under conditions of the transient expression assay, it was stably integrated into the chromosome in transgenic mice. Because CAT expression was detected in both alveolar type II and Clara cells of mice containing the -236/+39-bp region, albeit at lower levels compared with the mice containing the -730/+39-bp region, the decrease in promoter activity could not have been due to the loss of type II cell-specific enhancer elements but rather due to the loss of enhancer elements that increase expression in both alveolar type II and Clara cells.

Genomic DNA regions that control cell- or tissue-specific and developmental regulation of other genes that are expressed in the pulmonary epithelium, such as Clara cell secretory protein (CCSP), SP-C, and SP-A, have recently been identified. A 2.25-kb fragment of the 5'-flanking region of the rat CCSP gene directed lung- and Clara cell-specific expression of the human growth hormone (hGH) reporter gene (12). In a separate study (13), a -2,338/+49-bp fragment of 5'-flanking DNA of the rat CCSP gene directed expression of the CAT reporter gene in the lungs and tracheae of transgenic mice (25). Although the CCSP genomic DNA fragment maintained Clara cell-specific expression of the hGH reporter gene, the developmental expression of the transgene was not maintained (13). In other studies, it was found that even though the -166-bp region of the mouse CCSP gene was sufficient to direct Clara cell-specific expression of the transgene, enhancer sequences between the -803- and -166-bp regions were required for maximal expression of the transgene (23). As little as 215 bp of human SP-C 5'-flanking DNA were found to be capable of directing alveolar type II cell-specific expression of CAT gene (10).

A DNA fragment consisting of 378 bp of 5'-flanking DNA of the rabbit SP-A gene directed hGH expression in alveolar type II and bronchiolar epithelial cells of transgenic mouse lungs (1). The DNA fragment also specified appropriate developmental regulation of the hGH gene (1). However, ectopic expression of the hGH gene was detected in heart, thymus, and spleen. Sequences upstream of the 378-bp region, -4,000/-991 bp, appeared to be required to prevent ectopic expression while maintaining lung-specific expression of the hGH gene (1).

In the present study, we found that the developmental expression of CAT activity was similar or identical to the expression of the endogenous SP-B mRNA (9). SP-B mRNA was detected in embryonic day 15 lung, and its levels increased markedly thereafter to reach maximal levels in embryonic day 18 lung (6). These data suggested that the SP-B genomic fragment -730/+39 bp contains necessary information to specify appropriate developmental regulation of CAT expression. CAT expression levels during development varied within members of a given family. The exact reasons for the variation in CAT expression levels within a family are not clear but may be caused by integration of the transgene at more than one site in the genome.

Immunostaining experiments demonstrated CAT expression in cells localized at the corners of the alveoli and in cells lining the bronchiolar epithelium in a manner similar to the expression of endogenous SP-B. Because alveolar type II cells are predominantly located at the corners of the alveoli (17) and Clara cells (21) make up the majority of cells lining the bronchiolar epithelium in the adult lung, the staining patterns identified cells expressing CAT as most likely alveolar type II and Clara cells.

In summary, our studies have shown that the -236/+39-bp region of the rabbit SP-B gene contains the cis-DNA elements necessary for alveolar type II- and bronchiolar (Clara) cell-specific expression and that the -730/-236-bp region contains elements that enhance the expression of the gene. Our data have also indicated that the -2,176/-730-bp SP-B region may contain elements that repress promoter activity. It remains to be determined whether cis-DNA elements and interacting trans-acting factors that have been identified as important for SP-B promoter function in vitro are also necessary for specifying spatial and temporal expression of the SP-B gene in transgenic mice.


    ACKNOWLEDGEMENTS

We thank Dr. Kathy Graves and John Ritter for the generation of the transgenic mice and Dr. Carole Mendelson and Meg Smith for helpful discussions.


    FOOTNOTES

* C. C. Adams and M. N. Alam contributed equally to this work.

This work was supported by National Heart, Lung, and Blood Institute Grant HL-48048.

Address for reprint requests and other correspondence: V. Boggaram, Dept. of Molecular Biology, Univ. of Texas Health Science Center, 11937 US Highway 271, Tyler, TX 75710 (E-mail: vijay.boggaram{at}uthct.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 March 2000; accepted in final form 17 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Alcorn, JL, Hammer RE, Graves KR, Smith ME, Maika SD, Michael LF, Gao E, Wang Y, and Mendelson CR. Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice. Am J Physiol Lung Cell Mol Physiol 277: L349-L361, 1999[Abstract/Free Full Text].

2.   Avery, ME, and Mead J. Surface properties in relation to atelectasis and hyaline membrane disease. Am J Dis Child 97: 517-523, 1959[ISI].

3.   Boggaram, V. Hormonal regulation of lung surfactant protein B and surfactant protein C gene expression in fetal lung. In: Endocrinology of the Lung: Development and Surfactant Synthesis, edited by Mendelson CR.. Totowa, NJ: Humana, 2000, p. 81-90.

4.   Bohinski, RJ, Huffman JA, Whitsett JA, and Lattier DL. Cis-active elements controlling lung cell-specific expression of human surfactant protein B gene. J Biol Chem 268: 11160-11166, 1993[Abstract/Free Full Text].

5.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

6.   Bruno, MA, Bohinski RJ, Carter JE, Foss KA, and Whitsett JA. Structure and function of the mouse surfactant protein B gene. Am J Physiol Lung Cell Mol Physiol 268: L381-L389, 1995[Abstract/Free Full Text].

7.   Clark, JC, Wert SE, Bachurski CJ, Stahlman MT, Stripp BR, Weaver TE, and Whitsett JA. Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc Natl Acad Sci USA 92: 7794-7798, 1995[Abstract].

8.   Cochrane, CG, and Revak SD. Pulmonary surfactant protein B (SP-B): structure-function relationships. Science 254: 566-568, 1991[ISI][Medline].

9.   D'Amore-Bruno, M, Wikenheiser KA, Carter JE, Clark JC, and Whitsett JA. Sequence, ontogeny, and cellular location of murine surfactant protein B mRNA. Am J Physiol Lung Cell Mol Physiol 262: L40-L47, 1992[Abstract/Free Full Text].

10.   Glasser, SW, Burhans MS, Esterhas SK, Bruno MD, and Korfhagen TR. Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice. Am J Physiol Lung Cell Mol Physiol 278: L933-L945, 2000[Abstract/Free Full Text].

11.   Goerke, J, and Clements JA. Alveolar surface tension and lung surfactant. In: Handbook of Physiology. The Respiratory System. Mechanics of Breathing. Bethesda, MD: Am. Physiol. Soc, 1986, vol. III, pt. 2, chapt. 16, p. 247-261.

12.   Hackett, BP, and Gitlin JD. Cell-specific expression of a Clara cell secretory protein-hGH gene in the bronchiolar epithelium of transgenic mice. Proc Natl Acad Sci USA 89: 9079-9083, 1992[Abstract].

13.   Hackett, BP, and Gitlin JD. 5' Flanking region of the Clara cell secretory protein gene specifies a unique temporal and spatial pattern of gene expression in the developing pulmonary epithelium. Am J Respir Cell Mol Biol 11: 123-129, 1994[Abstract].

14.   Margana, R, Berhane K, Alam MN, and Boggaram V. Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene. Am J Physiol Lung Cell Mol Physiol 278: L477-L484, 2000[Abstract/Free Full Text].

15.   Margana, RK, and Boggaram V. Rabbit surfactant protein B gene: structure and functional characterization of the promoter. Am J Physiol Lung Cell Mol Physiol 270: L601-L612, 1996[Abstract/Free Full Text].

16.   Margana, RK, and Boggaram V. Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3, TTF-1 and HNF-3 transcription facotors are necessary for lung cell-specific activation of SP-B gene transcription. J Biol Chem 272: 3083-3090, 1997[Abstract/Free Full Text].

17.   Mensah, EA, Kumar NM, Nielsen L, and Lwebuga-Mukasa JS. Distribution of alveolar type II cells in neonatal and adult rat lung revealed by RT-PCR in situ. Am J Physiol Lung Cell Mol Physiol 271: L178-L185, 1996[Abstract/Free Full Text].

18.   Nogee, LM, Demello DE, Dehner LP, and Colten HR. Brief report: deficiency of pulmonary surfactant protein B in congenital alveolar proteinosis. N Engl J Med 328: 406-410, 1993[Free Full Text].

19.   Overbeek, PA. Factors affecting transgenic animal production. In: Transgenic Animal Technology: A Laboratory Handbook, edited by Pinkert CA.. New York: Academic, 1994, p. 69-114.

20.   Phelps, DS, and Floros J. Localization of surfactant protein synthesis in human lung by in situ hybridization. Am Rev Respir Dis 137: 939-942, 1988[ISI][Medline].

21.   Plopper, CG. Clara cells. In: Lung Growth and Development, edited by McDonald JA.. New York: Dekker, 1997, p. 181-209.

22.   Possmayer, F. The role of surfactant-associated proteins. Am Rev Respir Dis 142: 749-752, 1990[ISI][Medline].

23.   Ray, MK, Magdaleno SW, Finegold MJ, and DeMayo FJ. Cis-acting elements involved in the regulation of mouse Clara cell-specific 10-kDa protein gene. In vitro and in vivo analysis. J Biol Chem 270: 2689-2694, 1995[Abstract/Free Full Text].

24.   Seed, B, and Sheen JY. A simple phase-extraction assay for chloramphenicol acetyltransferase activity. Gene 67: 271-277, 1988[ISI][Medline].

25.   Stripp, BR, Sawaya PL, Luse DS, Wikenheiser K, Wert SE, Huffman JA, Lattier DL, Singh G, Katyal SL, and Whitsett JA. Cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem 267: 14703-14712, 1992[Abstract/Free Full Text].

26.   Van Iwaarden, JF, and Van Golde LMG Pulmonary surfactant and lung disease. In: Surfactant Therapy for Lung Disease, edited by Robertson B, and Taeusch HW.. New York: Dekker, 1995, p. 75-92.

27.   Venkatesh, VC, Planer BC, Schwartz M, Vanderbilt JN, Tyler R, White RT, and Ballard PL. Chracterization of the promoter of human pulmonary surfactant protein B gene. Am J Physiol Lung Cell Mol Physiol 268: L674-L682, 1995[Abstract/Free Full Text].

28.   Weaver, TE, and Whitsett JA. Function and regulation of expression of pulmonary surfactant-associated proteins. Biochem J 273: 249-264, 1991[ISI][Medline].

29.  Wikenheiser KA, Vorbroker DK, Rice WR, Clark JC, Bachurski CJ, Oie HK, and Whitsett JA. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus large tumor antigen transgenic mice. Proc Natl Acad Sci USA 23: 11029-11033.

30.   Wohlford-Lenane, CL, and Snyder JM. Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal lung tissue by in situ hybridization. Am J Respir Cell Mol Biol 7: 335-343, 1992[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 280(4):L724-L731
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society