The murine SP-C promoter directs type II cell-specific expression in transgenic mice

Stephan W. Glasser, Susan K. Eszterhas, Emily A. Detmer, Melissa D. Maxfield, and Thomas R. Korfhagen

Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Submitted 6 July 2004 ; accepted in final form 24 November 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

surfactant protein C; type II cells


PULMONARY SURFACTANT is a phospholipid and protein mixture that lines the gas-exchanging regions of the lung and prevents alveolar collapse during respiration (10). Pulmonary surfactant is synthesized and secreted in the alveolus by distinct cuboidal type II cells. The composition, alveolar concentration, and intracellular reservoir of pulmonary surfactant is precisely maintained in the normal lung. Alterations in surfactant composition or homeostasis can inhibit normal respiratory function. Neonatal respiratory distress is due to inadequate surfactant levels. Surfactant dysfunction can also result from microbial, particulate, or gaseous injury causing vascular leak that inhibits surfactant function. Surfactant inactivation contributes to the adult respiratory distress syndrome (16). Mutations that alter surfactant protein (SP) components result in interstitial lung disease or surfactant dysfunction (19).

Although phospholipids comprise the bulk of surfactant, essential functions are fulfilled by surfactant-associated proteins. Whereas SP-A and SP-D are components of innate immunity (13, 15), the hydrophobic proteins SP-B and SP-C augment the surface properties of surfactant phospholipids (25). SP-C is a highly hydrophobic 34-amino acid peptide that is proteolytically processed from a 197-amino acid precursor. The processed form of SP-C is associated with surfactant phospholipids through an {alpha}-helical structure spanning a lipid bilayer. Hydrophobic properties of SP-C are further enhanced by palmitoylation of two NH2-terminal cysteine residues at positions 5 and 6. Several clinical studies and laboratory investigations indicate that SP-C contributes to surfactant function. Surfactant replacement extracts enriched for SP-C are highly effective in the treatment of neonatal respiratory distress syndrome. A synthetic replacement surfactant consisting of surfactant phospholipids supplemented with a recombinant SP-C peptide as the only protein component restores pulmonary function in several animal models of surfactant-depleted acute lung injury (11). Addition of purified SP-C to synthetic phospholipid preparations lowers the surface activity to levels approximating whole native surfactant preparations (24).

The identification of naturally occurring SP gene mutations in humans and the development of gene knockout models in mice have provided insight into "nonsurfactant" functions. Correct temperospatial expression of the SP-C gene and subsequent processing of SP-C and its release from the type II cell is essential to normal pulmonary homeostasis. Mutations that alter normal intracellular pro-SP-C processing have been identified in cases of familial idiopathic interstitial pneumonitis (19, 23). Affected individuals within a family can present with highly variable onset and severity of clinical disease suggesting a complex etiology due to environmental influences or unknown modifier genes. SP-C-deficient mice generated by targeted gene inactivation develop a progressive interstitial lung disease consisting of cellular infiltrates and alveolar remodeling with fibrotic lesions in the most severely affected animals (8).

In the adult lung, SP-C mRNA expression is detected at high levels only in type II alveolar epithelial cells, whereas other SP genes are expressed in bronchiolar epithelial cells (SP-A and SP-B) and subsets of tracheal gland cells (SP-A and SP-D) as well as type II cells. Thus SP-C is the only gene expressed only in type II cells and has served as a marker of type II cell differentiation in the mammalian lung. In cell transfection experiments, the SP-C promoters are activated by an overlapping set of transcription factors that include thyroid transcription factor (TTF)-1, hepatocyte nuclear factors, GATA-6, and nuclear factor I (NFI). Analysis of SP gene promoters in transgenic mice has been used to correlate the arrangement of in vitro cis-active sites to actual in vivo regulation as well as to identify new cis-active regions, such as developmental control and chromatin-dependent enhancers that may go undetected in transient cell experiments.

In transgenic mice generated with human SP-C genomic DNA, 3.7 kb of the human SP-C promoter directed lung-specific expression of a diphtheria toxin A or chloramphenicol acetyl transferase (CAT) reporter gene in transgenic mice (9, 14). Subsequent experiments demonstrated that this region of DNA directed developmental transgene expression similar to the endogenous gene and that expression was sustained in bronchiolar and alveolar type II cells of the adult lung (26). Further dissection of the elements critical for type II cell-specific transcription have proven complicated by the bronchoalveolar pattern of expression. To discern what elements are essential for accurate temporal spatial expression in the lung, we have analyzed segments of the mouse SP-C gene promoter in transgenic mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SP-C-CAT constructs and transgenic mice. A 4.8-kb fragment of murine SP-C genomic DNA was isolated from a 129/J library and subcloned into the promoterless pBLCAT 6 expression plasmid. The –318- and –230-bp deletion constructs were generated by restriction digestion of the 4.8-kb fragment. The 13-kb SP-C-CAT construct was generated by ligation of an 8.2-kb SalI-BamHI genomic fragment to a unique 5' BamHI site in the proximal 4.8-kb SP-C promoter fragment (12). Restriction digestion at unique sites in the flanking multicloning sites was used to liberate the transgene constructs. Transgene constructs were purified from plasmid vector DNA by electrophoresis through low-melt agarose gels. DNA was recovered and purified for microinjection using Magic PCR columns (Promega, Madison, WI). DNA was resuspended in 5 mM Tris·HCl, pH 7.9, and provided to the Cincinnati Children's Hospital transgenic core facility. Founder (F0) SP-C-CAT mice were identified by genomic Southern blot using a 550-bp fragment of the CAT gene as probe or by PCR of genomic DNA isolated from tail clips. F0 mice were bred to FVB/N mice (Taconic, Germantown, NY) to establish heterozygous F1 animals that were bred to produce mice homozygous for the transgene. All mice were maintained in microisolator housing within a barrier facility. Sentinel mice were negative for both bacterial and viral pathogens. Studies were reviewed and approved by the Cincinnati Children's Hospital Institutional Animal Use and Care Committee.

CAT enzyme assay of tissue extracts. Organs were removed and homogenized in 500 µl of 1 M Tris·HCl, pH 7.4 (adult mice), 200 µl embryonic day 16 (ed 16) or 100 µl (ed 13 animals). Organs from adult or ed 16 mice were homogenized with a conventional tissue homogenizer or with a microgrinder fitted to 1.5-ml Eppendorf vials for the organs of ed 13 mice. Homogenates were heated at 65°C for 5 min and centrifuged to remove cell debris. CAT assays were normalized to uniform protein concentration from organ extracts and run as previously described in detail (9).

Cell culture and selection for stable transfection. MLE-15 cells were maintained in HITES media supplemented with 4% FBS. MLE-15 cells are mouse lung epithelial tumor cells with features of type II cells. Cells were plated in six-well dishes at a density of 105 cells/well. Cotransfections included 0.01 pmol of the plasmid pGKneo, 0.125 pmol of pGL-2, or 0.18 pmol of SP-C-luciferase (luc) constructs complexed with Fugene-6 transfection reagent (Boehringer Mannheim). Cells were held at 37°C and 5% CO2 overnight, and the media were removed and replaced with selection media (HITES, 4% FBS, and 350 µg/ml G418, a neomycin analog). Media were changed every 2 days. Cells were harvested at 6 days for luciferase assays or trypsinized and replated in the G418 selection media to expand the stable, transfected cell populations. Passage 2 of the mixed populations of these G418-resistant colonies were frozen as stock cultures.

In situ hybridization. Lungs of adult mice were inflation fixed at 20-cm pressure with freshly prepared 4% paraformaldehyde. Animals taken for developmental time points were fixed by immersion after the thoracic cavity was opened. Tissue was embedded in paraffin, and 4-µm sections were cut. 35S-labeled riboprobes for bacterial CAT and murine SP-C were synthesized from pGEM plasmids. Probe synthesis, hybridization, and wash conditions have been previously described (26). Specificity of CAT hybridization was established by comparison of antisense CAT probe hybridization to nontransgenic lung and hybridization signal from CAT and SP-C sense strand probes. Equivalent total counts of CAT or SP-C riboprobes were used in individual hybridizations.

Relative copy number estimation. Genomic DNA was isolated from tail clips of control and candidate SP-C-CAT transgenic mice. Five micrograms of DNA were digested with the restriction enzyme EcoRI and separated on 0.8% agarose gel for Southern blot analysis. A 550-bp CAT fragment was labeled with {alpha}-dCTP by nick translation and used as the hybridization probe. Blots were hybridized overnight and washed at a final stringency of 0.5x SSC at 65°C and subjected to autoradiography. Films were scanned, and relative densitometer values of the SP-C-CAT diagnostic bands were compared.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mouse lines were generated with two large fragments of genomic DNA from the mouse SP-C gene directing CAT reporter gene expression. One construct incorporated 4.8 kb of DNA upstream of the basal promoter. By sequence comparison, the mouse 4.8-kb DNA fragment was equivalent to a 3.7-kb human SP-C promoter fragment used in previous transgenic studies. Analysis of the 4.8-kb SP-C promoter in vivo could potentially distinguish expression differences due to species-specific variation between mouse and human sequences. A second construct was made with 13 kb upstream of the promoter to functionally identify any distal enhancer sequence that would contribute to type II cell specificity or that would silence bronchiolar expression seen with the human 3.7-kb SP-C promoter.

Tissue-specific expression of 4.8-kb SP-C-CAT transgenes. Three 4.8-kb SP-C-CAT transgene founder mice were identified by genomic Southern blot. Two of the 4.8-kb SP-C-CAT founders were bred to establish lines 4.4 and 6.1. Tissue extracts were prepared from organs of adult mice homozygous for the transgene from both lines and assayed for CAT enzyme activity. High levels of CAT activity were detected only in lung extracts of both 4.4 SP-C-CAT and 6.1 SP-C-CAT mice and not in other tissues. Lung and other organ extracts were uniformly negative from nontransgenic control mice (Fig. 1). The murine 4.8-kb promoter segment targeted lung-specific gene expression.



View larger version (56K):
[in this window]
[in a new window]
 
Fig. 1. Organ-specific expression of the mouse 13- and 4.8-kb surfactant protein C-chloramphenicol acetyl transferase (SP-C-CAT) transgenes. CAT enzyme assays were performed as described in MATERIALS AND METHODS with tissue extracts prepared from organs of the 4.8 SP-C-CAT transgene-positive mice line 4.4 (A), age-matched nontransgenic FVB/N mice (B), and 13-kb SP-C-CAT line (C). Lane 1, lung; lane 2, heart; lane 3, liver; lane 4, kidney; lane 5, spleen; lane 6, muscle; lane 7, salivary gland; lane 8, no extract, negative control (C only). Significant CAT enzyme activity is detected only in lung extract of transgene-positive lung from 4.8 SP-C-CAT and 13 SP-C-CAT mice (arrows). Nearly identical lung-specific expression was obtained with tissue extracts of a second 4.8 SP-C-CAT transgenic line 6.1 (not shown). Presented data are representative of four 4.8 SP-C-CAT mice and eight 13 SP-C-CAT mice.

 
Cell-specific expression of 4.8-kb SP-C-CAT transgenes. The cellular sites of 4.8-kb SP-C-CAT expression from lines 4.4 and 6.1 in the lung were determined by in situ hybridization with CAT riboprobes. Sections of adult lung and ed 13 and ed 16 lung from both 4.4 and 6.1 mice were compared with the pattern of expression for the endogenous SP-C gene. Strong focal alveolar CAT expression was detected in sections of adult lung from both lines (Fig. 2). Bronchiolar expression was absent, whereas CAT expression was restricted to alveolar cells and overlapped expression of SP-C. These findings from two independent founder lines indicate that the 4.8-kb region is sufficient to restrict expression to the adult type II cells.



View larger version (113K):
[in this window]
[in a new window]
 
Fig. 2. 13- and 4.8-kb SP-C promoter regions direct SP-C-CAT transgene expression in alveolar epithelial cells of adult transgenic mice. CAT transgene mRNA and endogenous SP-C mRNA expression was detected using in situ hybridization. A and B: corresponding dark- and bright-field images of SP-C antisense hybridization. C–F: images of CAT antisense hybridization. Arrowheads indicate bronchiolar structures that are negative for SP-C or CAT hybridization. C and D: dark- and bright-field images of 4.8 SP-C-CAT antisense hybridization results. E and F: images of 13 SP-C-CAT results. Both CAT and SP-C probes hybridize in a focal pattern to alveolar sites consistent with the location of type II cells. Presented micrographs are representative of sections from 4 mice.

 
Development expression of 4.8-kb SP-C-CAT transgenes. Tissue CAT assays and in situ analysis were used to assess whether developmental expression was encoded by the 4.8-kb SP-C-CAT transgene. Extremely low levels of lung-specific CAT activity was detected in extracts from ed 13 embryos. Significantly higher levels of transgene CAT activity was detected in lung extracted from ed 16 embryos (Fig. 3). Low-level 4.8-kb SP-C-CAT expression was localized by in situ hybridization over the epithelial cells at the distal boundary of the developing tubules in ed 13 lungs (Fig. 4). CAT or SP-C expression was not detected over large proximal airways or mesenchymal tissue. At ed 16, the relative level of CAT signal was increased relative to ed 13 and was confined to the numerous small distal airway epithelia, consistent with apparent increased branching (Fig. 5). Whereas the developmental pattern of 4.8-kb SP-C-CAT transgene expression was similar to the pattern of SP-C expression, in situ signals of 4.8-kb SP-C-CAT expression were modest in comparison with the intensity of endogenous SP-C signal.



View larger version (57K):
[in this window]
[in a new window]
 
Fig. 3. CAT activity in tissue extracts during mouse embryonic development. CAT assays were performed using extracts prepared from 4.8 SP-C-CAT transgenic mice at embryonic day 13 (A) and embryonic day 16 (B) and nontransgenic mice (C). Arrows indicate specific acetylated chloramphenicol reaction products. CAT activity was barely detected in day 13 lung and was abundant in day 16 lung extracts. Organ extracts in A and C: lane 1, liver; lane 2, heart; lane 3, intestine; lane 4, lung. Organ extracts in B: lane 1, liver; lane 2, heart; lane 3, intestine; lane 4, kidney; lane 5, stomach; lane 6, lung. Presented data are representative of 4 mice from 4.8-kb SP-C-CAT transgenic line.

 


View larger version (124K):
[in this window]
[in a new window]
 
Fig. 4. Expression of 4.8 SP-C-CAT transgene at embryonic day 13. Dark-field images of sense (A) and antisense (B) SP-C hybridization. Sense (C) and antisense (D) images of CAT hybridization. Magnification, x4. Weak CAT and strong SP-C signals are detected by antisense probes in peripheral tubules. E and F are high magnification. Arrowheads indicate central tubule that is negative for CAT expression, and small arrows indicate peripheral airways positive for CAT expression. Magnification, x20.

 


View larger version (155K):
[in this window]
[in a new window]
 
Fig. 5. Expression of 4.8 SP-C-CAT transgene at embryonic day 16. CAT antisense hybridization (A, C, and E) and SP-C antisense hybridization (B, D, and F) were performed as described in MATERIALS AND METHODS. CAT expression was detected in developing distal epithelium similar to the pattern of SP-C transcripts. At high magnification, CAT and SP-C signals are concentrated over peripheral airways and extinguished in central airway epithelia (arrowheads in C–F, x20). E and F are the corresponding bright-field images for the dark-field images in C and D to illustrate proximal to distal CAT transgene and endogenous SP-C expression. CAT transgene expression is significantly increased over levels seen on embryonic day 13. Photomicrographs are representative of sections of line 4.4 mice.

 
Lung- and cell-specific expression of 13-kb SP-C-CAT transgenes. Four transgenic lines were established with the 13-kb SP-C-CAT construct. CAT reporter gene activity was lung specific (Fig. 1C). Transgene expression was detected in a focal pattern throughout the parenchyma. No aberrant bronchiolar expression was detected (Fig. 2, E and F). The number of alveolar sites of transgene expression varied between 13 SP-C-CAT lines similar to the findings obtained from the 4.8-kb SP-C-CAT lines. The extended 13 kb of SP-C DNA, therefore, do not appear to harbor enhancers that confer uniform expression among all type II cells. Whereas the cis-active elements in the SP-C sequences confer lung- and type II cell-specific expression, other chromatin-dependent elements associated with the SP-C locus may be required to elicit uniform transcription in all type II cells.

Specificity of truncated 0.32 SP-C-CAT transgene expression. To map essential cis-active regulatory regions, eight founder transgenic lines were established from constructs consisting of 318 or 230 bp of 5' DNA. Tissue CAT activity was not detected from the 230-bp SP-C-CAT lines. Very weak CAT activity was detected in lung extracts from the 318-bp SP-C-CAT line 1.3 (Fig. 6). This finding localizes an essential cis-active determinant of SP-C transcription to the proximal –318 bp of the SP-C promoter. The relative transgene copy number for 4.8 SP-C-CAT and 318 SP-C-CAT lines was compared. Both 4.8 SP-C-CAT lines 4.4 and 6.1, expressed at high levels, were extremely low copy number (line 4.4, 1 copy) or low copy number (line 6.4, 3 copies). The only 318 SP-C-CAT line to express had 30 copies relative to 4.8 SP-C-CAT line 4.4. Therefore, the high-level 4.8 SP-C-CAT expression did not reflect an increased transgene copy number.



View larger version (92K):
[in this window]
[in a new window]
 
Fig. 6. Lung-specific CAT expression in mice carrying the 318 SP-C-CAT transgene. CAT assays were performed using tissue extracts from adult 318 SP-C-CAT transgenic mice (A) and nontransgenic extracts (B). Tissue extract: lane 1, liver; lane 2, muscle; lane 3, kidney; lane 4, heart; lane 5, lung; lane 6, no extract. Arrows indicate acetylated chloramphenicol due to CAT enzyme activity.

 
Increased 4.8-kb promoter activity is due to chromatin. Whereas the 4.8-kb promoter was far more active in transgenic mice than the 318-bp promoter, the 4.8-kb promoter construct was only slightly more active than the 318-bp promoter in transient cell transfection experiments (12). To determine whether the increased activity of 4.8-kb SP-C promoter activity in vivo could be explained by a chromatin-dependent mechanism, 4.8-kb SP-C-luc and 318-bp SP-C-luc gene expression was compared when stably integrated in cells. Stable transfected MLE-15 cells were generated by cotransfection with a pGKneo plasmid and selection of neomycin (G418)-resistant colonies. Equal molar amounts of 318-luc or 4.8-luc DNA were used in transfections. A similar number of G418-resistant colonies were obtained in each of seven independent transfections, indicating that efficiency of integration was nearly identical with both 318-SP-C-luc and 4.8-SP-C-luc constructs. In each experiment, between 200 and 500 G418-resistant colonies were harvested per plate so that measured reporter activity reflected numerous independent integrations. Luciferase assays were normalized to total cell protein. Luciferase reporter activity was 90-fold higher in extracts from 4.8-kb SP-C-luc-transfected colonies than in the 318-bp SP-C-luc cell extracts. In contrast, the 4.8-kb SP-C-luc luciferase activity was only seven- to eightfold higher than 318-bp SP-C-luc activity in transient transfection experiments (Fig. 7). This finding indicates that incorporation of the 4.8-kb SP-C DNA into chromatin structure stimulates enhancer activity by more than an order of magnitude. Virtually no luciferase activity was detected in cells transfected with the promoterless luciferase plasmid, indicating that potential stimulation of luciferase activity due to integration of test DNA adjacent to active but unrelated cellular promoters was minimal.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7. Comparison of 4.8-kb and 318-bp promoters in permanent MLE cell lines and transiently transfected MLE cells. Luciferase (luc) activity from 318 SP-C-luc transfections was set at 1. 4.8 SP-C-luc activity is reported as fold increase over 318 SP-C-luc activity in transient expression experiments (n = 5) or stable transfected cells (n = 7). P < 0.01 comparing 4.8-kb to 318-bp values.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SP-C is distinguished from other SP by being expressed only in alveolar type II cells and serves as a specific type II cell marker during lung maturation or injury response. SP-C gene expression has also been analyzed in vitro in cell transcription assays and in vivo transgenic models to ascertain mechanisms regulating SP-C gene transcription. The mouse SP-C gene promoter was analyzed in transgenic mice with the goal of identifying cis-active regions that support alveolar type II cell expression. Previous transgenic studies using the human SP-C promoter demonstrated lung-specific expression but were complicated by the finding that human 3.7-kb SP-C transgenes did not recapitulate the expression pattern of the endogenous SP-C gene. The human SP-C transgene was expressed in alveolar type II cells and robustly in bronchiolar cells (7, 26). Analysis of the mouse SP-C promoter constructs in transgenic mice identified promoter regions that direct cell-appropriate expression, a proximal region essential for in vivo promoter activity, and a distal chromatin-dependent regulatory domain that functions as an enhancer.

Tissue CAT assays and CAT in situ hybridization experiments demonstrated that 4.8 kb of mouse SP-C promoter and 5'-flanking DNA targeted lung and alveolar cell-specific expression in adult mice. In an independent study, the mouse 4.8-kb SP-C promoter was used to restore SP-B expression in lungs of SP-B–/– mice. SP-B mRNA and protein expression was type II cell specific for two of the three lines and was detected only in a subset of the alveolar type II cells (17). The murine 4.8-kb promoter segment is the sequence equivalent to the 3.7-kb human promoter wherein three regions of homology are conserved between the two species (7). The bronchiolar expression obtained from the human promoter likely results from altered assembly of murine transcription complexes on human binding sites or from displacement of complexes that silence SP-C expression in bronchiolar cells. The number of alveolar cells expressing 4.8 SP-C-CAT in the transgenic lines was reduced relative to sites of endogenous SP-C expression, recapitulating results obtained with the human SP-C promoter. This consistent finding suggested either that additional enhancers with type II cell activity were missing or that uniform type II cell expression is context dependent, requiring sequences and higher-ordered chromatin unique to the SP-C locus. Such modifiers have been documented and include matrix attachment regions that tether chromatin loops at highly transcribed gene loci, locus control regions that modify gene expression over distance and/or insulator elements that limit active gene transcription and selective alteration of histone acetylation (6). Transgenic mice were generated with extended 13 kb of SP-C promoter and upstream DNA to test whether extended sequences harbored such type II cell normalizing elements. Each of four separate 13-kb SP-C-CAT founder lines supported alveolar (type II) cell-specific expression. Surprisingly, the number of CAT-positive alveolar cells varied between each founder line, with one line having 79% of cells expressing the CAT gene compared with the number of cells expressing SP-C. Expression from the other three 13-kb SP-C-CAT lines had diminished number of sites of expression similar to the 4.8 SP-C-CAT lines. The current analysis with the extended promoter does not rule out intron or 3' enhancer(s) that would confer uniform type II cell expression. The structural features of the SP-C locus limit the possibility of 3' regulatory elements wherein the SP-C gene terminates adjacent to the promoter of a new gene. The sequence immediately 3' of SP-C exon 6 is G-C rich (82%) and serves as the promoter of the bone morphogenetic protein (BMP)-1 gene, with the BMP-1 transcription start site only 700 bp downstream of the SP-C gene (22). The BMP-1 gene encodes an essential developmental procollagenase C gene. The BMP-1 exon 1 is followed by extended CA dinucleotide repeats that are highly polymorphic. The structural features of the unusual sequence immediately 3' of the SP-C gene suggest that 3' cis-active regulation of the SP-C gene is unlikely.

Extremely low level but lung-specific CAT activity was detected when the mouse 4.8-kb SP-C promoter was deleted to 318 bp. This finding indicates that significant enhancer activity is located in the deleted distal region. The nature of the enhancer is unclear, but initial cell transfection experiments suggest that it is responsive to higher-ordered DNA structure. When previously tested in transient cell transfection, only modestly elevated 4.8 SP-C-CAT expression levels were seen compared with 318 SP-C-CAT activity (12). By selecting for stable chromosome integration, the relative expression of the 4.8 SP-C to the –318-bp SP-C promoter was 90-fold greater than differences detected in transient assays. This finding is consistent with strong 4.8 SP-C vs. 318 SP-C expression in transgenic mice and indicates chromatin association is necessary for activity of an enhancer upstream of 318 SP-C sequence. The 318 SP-C-CAT transgenes were present at >10-fold higher copy number than in 4.8 SP-C-CAT lines. The high-level expression of 4.8 SP-C-CAT lines was, therefore, not simply due to increased number of transgenes. It is unknown whether integration increases the efficiency of the weak enhancer activity detected in transient expression or if integration activates a separate enhancer. Analysis of the human 3.7 SP-C promoter produced similar results wherein the 3.7-kb SP-C promoter was extremely weak in transient assays but directs vigorous expression as an integrated transgene. Two regions of homology to the human 3.7-kb sequence are found in the upstream region at ~–4,700 to –4,100 bp and –3,500 to –2,800 bp (7). These conserved sequences between species are potential candidates for the chromatin enhancer element(s).

In related studies, a transgenic approach has been used to localize cis-active regions that direct lung-specific expression of the SP-A and SP-B promoters. These studies indicate that the arrangement and nature of enhancer activity are different for each SP gene. A 4-kb segment of the rabbit SP-A promoter directed lung-specific transgene expression. Deletion to –991 bp resulted in transgene expression in several additional organs. This result suggests that unlike SP-C, a distal silencing element actively contributes to correct SP-A gene expression. A second region that mediates bronchoalveolar SP-A expression is mapped to the –378- to –47-bp region (2). The authors were unable to separate alveolar and bronchiolar regulation of SP-A expression. Three recent reports have assessed SP-B promoter activity in transgenic mice. A –1,039- to +431-bp segment of the human SP-B promoter generated bronchoalveolar expression and low-level thyroid, trachea, and intestine expression (21). A separate study of the human SP-B promoter demonstrated that 1.5 kb directs lung and intestine expression in mice. An enhancer was mapped to the –500- to –331-bp region (27). The –236- to +39-bp region of the rabbit SP-B promoter directed bronchoalveolar selective expression and transcriptional repression mapped in the distal –2,176- to –730-bp region (1). In all of these studies, ontogenic expression was shown to parallel the endogenous gene. It will be intriguing to learn whether in vivo mutational analysis can map regulatory regions that determine ontogenic expression distinct from cell-selective expression. Although the SP-A and SP-B promoter studies provide initial insights into elements required for bronchoalveolar expression, identification of cis-elements may be affected by species sequence variation as seen with the SP-C promoters.

The essential 318-bp SP-C sequence conferring lung-specific activity identified in this study spans a region where multiple factors interact to influence SP-C transcription in vitro. The homeobox transcription factor TTF-1 (also termed Nkx2.1) trans-activates the 318-bp SP-C promoter (12). With the use of mutagenesis assays, NFI binding sites were identified and shown to be required for SP-C promoter activity (3). Cotransfection experiments demonstrated that NFI and TTF-1 synergistically activate 318 SP-C promoter activity (4). TTF-1 also directly interacts with the transcription factor TAZ to enhance 318 SP-C promoter activity (20) and interacts cooperatively with GATA-6 to increase SP-C expression (18). In addition, TTF-1/GATA-6 interactions enhance SP-A activity (5). Deletion mapping of the human SP-C promoter in transgenic mice showed that the equivalent TTF-1-responsive region was required for lung-specific expression in vivo, similar to the current findings (7). Thus the combined in vitro promoter activation studies and functional in vivo mapping implicate a critical role for the TTF-1-responsive region in controlling the level and cell-specific expression of SP-C.

The current study demonstrates that 4.8 kb of promoter sequence activates and sustains alveolar cell-specific expression in the lungs of transgenic mice. A proximal 318-bp region containing known cis-active NFI, TTF-1, and GATA-6 binding sites is critical for lung-specific expression. High-level expression from the 4.8-kb promoter is mediated by an element that is dependent on a chromatin environment. The pattern of CAT expression directed by 4.8 SP-C promoter was alveolar type II cell specific. The aberrant bronchiolar component of expression obtained with the human 3.7 SP-C promoter in transgenic mice is due to species/sequence differences. The 3.7 SP-C promoter has been used successfully to express a variety of bioactive reporter molecules in lungs of transgenic mice. The 4.8-kb murine SP-C promoter may have further utility in directing expression restricted to just type II cells in vivo and thus provides a useful tool for developing transgenic models.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-50046 and HL-58795.


    ACKNOWLEDGMENTS
 
The authors acknowledge the technical assistance of Danielle Eiseman and Michael Burhans, Dr. Jeffrey Whitsett for critical reading and editing of the manuscript, and Ann Maher for manuscript preparation.

Present address of S. K. Eszterhas: Dept. of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. W. Glasser, Cincinnati Children's Hospital Medical Center, Div. of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: steve.glasser{at}cchmc.org

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adams CC, Alam MN, Starcher BC, and Boggaram V. Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice. Am J Physiol Lung Cell Mol Physiol 280: L724–L731, 2001.[Abstract/Free Full Text]
  2. Alcorn JL, Hammer RE, Graves KR, Smith ME, Maika SD, Michael LF, Gao E, Wang Y, and Mendelson CE. 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]
  3. Bachurski CJ, Kelly SE, Glasser SW, and Currier TA. Nuclear factor I family members regulate the transcription of surfactant protein-C. J Biol Chem 272: 32759–32766, 1997.[Abstract/Free Full Text]
  4. Bachurski CJ, Yang GH, Currier TA, Gronostajski RM, and Hong D. Nuclear factor I/thyroid transcription factor 1 interactions modulate surfactant protein C transcription. Mol Cell Biol 23: 9014–9024, 2003.[Abstract/Free Full Text]
  5. Bruno MD, Korfhagen TR, Liu C, Morrisey EE, and Whitsett JA. GATA-6 activates transcription of surfactant protein A. J Biol Chem 275: 1043–1049, 2000.[Abstract/Free Full Text]
  6. Felsenfeld G and Groudine M. Controlling the double helix. Nature 421: 448–453, 2003.[CrossRef][ISI][Medline]
  7. Glasser SW, Burhans MS, Eszterhas 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]
  8. Glasser SW, Detmer EA, Ikegami M, Na CL, Stahlman MT, and Whitsett JA. Pneumonitis and emphysema in SP-C gene targeted mice. J Biol Chem 278: 14291–14298, 2003.[Abstract/Free Full Text]
  9. Glasser SW, Korfhagen TR, Bruno MD, Dey C, and Whitsett JA. Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse. J Biol Chem 265: 21986–21991, 1990.[Abstract/Free Full Text]
  10. Goerke J. Pulmonary surfactant: functions and molecular composition. Biochim Biophys Acta 1408: 79–89, 1998.[ISI][Medline]
  11. Hafner D, Germann PG, and Hauschke D. Effects of rSP-C surfactant on oxygenation and histology in a rat lung-lavage model of acute lung injury. Am J Respir Crit Care Med 158: 270–278, 1998.[ISI][Medline]
  12. Kelly SE, Bachurski CJ, Burhans MS, and Glasser SW. Transcription of the lung-specific surfactant protein C gene is mediated by thyroid transcription factor-1. J Biol Chem 271: 6881–6888, 1996.[Abstract/Free Full Text]
  13. Korfhagen TR. Surfactant protein A-mediated bacterial clearance: SP-A and cystic fibrosis. Am J Respir Cell Mol Biol 25: 668–672, 2001.[Free Full Text]
  14. Korfhagen TR, Glasser SW, Wert SE, Bruno MD, Daugherty CC, McNeish JD, Stock JL, Potter SS, and Whitsett JA. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice. Proc Natl Acad Sci USA 87: 6122–6126, 1990.[Abstract/Free Full Text]
  15. Korfhagen TR, Sheftelyevich V, Burhans MS, Bruno MD, Ross GF, Wert SE, Stahlman MT, Jobe AH, Ikegami M, Whitsett JA, and Fisher JH. Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem 273: 28438–28443, 1998.[Abstract/Free Full Text]
  16. Lewis JF and Veldhuizen R. The role of exogenous surfactant in the treatment of acute lung injury. Annu Rev Physiol 65: 613–642, 2003.[CrossRef][ISI][Medline]
  17. Lin S, Na CL, Akinbi HT, Apsley KS, Whitsett JA, and Weaver TE. Surfactant protein B–/– mice are rescued by restoration of SP-B expression in alveolar type II cells but not Clara cells. J Biol Chem 274: 19168–19174, 1999.[Abstract/Free Full Text]
  18. Liu C, Glasser SW, Wan H, and Whitsett JA. GATA-6 and thyroid transcription factor-1 directly interact and regulate surfactant protein-C gene expression. J Biol Chem 277: 4519–4525, 2002.[Abstract/Free Full Text]
  19. Nogee LM. Alterations in SP-B and SP-C expression in neonatal lung disease. Annu Rev Physiol 66: 601–623, 2004.[CrossRef][ISI][Medline]
  20. Park KS, Whitsett JA, Di Palma T, Hong JH, Yaffe MB, and Zannini M. TAZ interacts with TTF-1 and regulates expression of surfactant protein-C. J Biol Chem 279: 17384–17390, 2004.[Abstract/Free Full Text]
  21. Strayer M, Savani RC, Gonzales LW, Zaman A, Cui Z, Veszelovszky E, Wood E, Ho YS, and Ballard PL. Human surfactant protein B promoter in transgenic mice: temporal, spatial, and stimulus-responsive regulation. Am J Physiol Lung Cell Mol Physiol 282: L394–L404, 2002.[Abstract/Free Full Text]
  22. Takahara K, Lee S, Wood S, and Greenspan DS. Structural organization and genetic localization of the human bone morphogenetic protein 1/mammalian tolloid gene. Genomics 29: 9–15, 1995.[CrossRef][ISI][Medline]
  23. Thomas AQ, Lane K, Phillips J III, Prince M, Markin C, Speer M, Schwartz DA, Gaddipati R, Marney A, Johnson J, Roberts R, Haines J, Stahlman M, and Loyd JE. Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Am J Respir Crit Care Med 165: 1322–1328, 2002.[Abstract/Free Full Text]
  24. Wang Z, Gurel O, Baatz JE, and Notter RH. Acylation of pulmonary surfactant protein-C is required for its optimal surface active interactions with phospholipids. J Biol Chem 271: 19104–19109, 1996.[Abstract/Free Full Text]
  25. Weaver TE and Conkright JJ. Functions of surfactant proteins B and C. Annu Rev Physiol 63: 555–578, 2001.[CrossRef][ISI][Medline]
  26. Wert SE, Glasser SW, Korfhagen TR, and Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 156: 426–443, 1993.[CrossRef][ISI][Medline]
  27. Yang L, Naltner A, Kreiner A, Yan D, Cowen A, Du H, and Yan C. An enhancer region determines hSP-B gene expression in bronchiolar and ATII epithelial cells in transgenic mice. Am J Physiol Lung Cell Mol Physiol 284: L481–L488, 2003.[Abstract/Free Full Text]