Introduction

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Pulmonary surfactant is a mixture of phospholipids and surfactant proteins that is synthesized and secreted by the alveolar type II epithelial cell. Maintenance of a phospholipid-rich surfactant film at the alveolar air-liquid interface is essential to prevent alveolar collapse at end expiration. Surfactant insufficiency in premature infants results in respiratory distress syndrome (RDS), a leading cause of morbidity and mortality among neonates worldwide. Surfactant replacement preparations, particularly those containing surfactant protein (SP)-B or SP-C, have considerably improved survival of these infants. However, surfactant replacement therapy was not an effective treatment for RDS in a human infant with hereditary SP-B deficiency (1). Without exception, newborn infants with hereditary SP-B deficiency develop unremitting, lethal RDS despite intensive respiratory therapy (2, 3). Similarly, SP-B-deficient mice develop severe RDS and die shortly after birth (4). SP-B null mice have dramatically reduced pulmonary compliance, disorganized lamellar bodies and reduced levels of mature SP-C peptide in the alveolar space resulting from a block in processing of the SP-C proprotein; in contrast, SP-C (/) mice have normal levels of SP-B peptide, and survive the neonatal period with normal lung structure and surfactant pool sizes and only minor changes in lung function (5). This outcome is consistent with the concept that SP-B and SP-C are functionally redundant with respect to formation and maintenance of the surface film.

SP-C is an integral membrane protein expressed exclusively by alveolar type II cells in the postnatal lung (6). The SP-C proprotein is proteolytically cleaved in multivesicular bodies to remove N- and C-terminal peptides, thereby generating the 35 amino acid mature peptide (10- 12). The extremely hydrophobic SP-C mature peptide, consisting of a transmembrane and a short extramembrane domain, is stored in lamellar bodies with surfactant phospholipids until secretion into the alveolar space. Previous studies in wild-type mice infected with adenovirus encoding the SP-C mature peptide demonstrated that the peptide was secreted into the alveolar space in the absence of its flanking peptides and associated with the surface active large aggregate fraction of surfactant (13). This observation suggested that expression of SP-C mature peptide in SP-B (/) mice might bypass the block in SP-C processing and restore lung function by elevating cellular and alveolar levels of SP-C. To test this hypothesis, the SP-C mature peptide was expressed in type II cells of transgenic mice in the absence of its flanking peptides with the goal of crossing these mice into the SP-B (/) background. Surprisingly, expression of SP-C mature peptide transgene in the presence of endogenous SP-C disrupted lung development, resulting in lethal, neonatal RDS.

	Materials and Methods

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Generation of SP-C_24-57HA Transgenic Mice

Mouse SP-C mature peptide (SP-C_24-57) was amplified by polymerase chain reaction (PCR) using specific primers that included a kozak consensus sequence in the 5' primer and an hemagglutinin epitope (YPYDVPDYA) in the 3' primer. The hemagglutinin (HA) tag was included to differentiate the transgene from endogenous SP-C. This DNA fragment (SP-C_24-57HA) was cloned into a vector containing the 13-kb mouse SP-C promoter (a gift from Stephan Glasser, Children's Hospital, Cincinnati, OH), rabbit globin intron and a bovine growth hormone polyadenylation signal (14) and sequenced bi-directionally to verify the fidelity of the PCR product. To generate transgenic mice, the transgene was separated from vector DNA, purified, and microinjected into fertilized FVB/N oocytes by the Children's Hospital and University of Cincinnati Transgenic Core facilities. Founder mice (F₀) were identified by transgene-specific PCR and confirmed by Southern analyses.

Western Analysis of Lung Homogenate

Lung tissue was isolated from F₁ (18.5 d gestation), newborn F₁, or adult F₀ mice and homogenized in PBS with 1%/vol protease inhibitor cocktail (Sigma, St. Louis, MO). Equal amounts of protein from lung homogenates were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gels were electrophoretically transferred to nitrocellulose and probed with an antibody directed against the HA tag (Santa Cruz Biotech, Santa Cruz, CA), mature SP-C (15), mature SP-B (16), or SP-A (17). Quantitative Western analyses were performed on lung homogenates using enhanced chemifluorescence substrate, as described by the manufacturer (Amersham Pharmacia Biotech, Arlington Heights, IL), imaged by fluorescence scanning (Storm; Molecular Dynamics, Sunnydale, CA) and analyzed using ImageQuant software (Molecular Dynamics).

Immunohistochemistry, Immunogold Labeling, and Electron Microscopy in Transgenic Mice

Lungs from fetuses (18.5 d gestation) or newborn pups were isolated and fixed for light microscopy as previously described (18). Immunohistochemistry was performed with antisera directed against the HA tag or thyroid transcription factor 1 (TTF-1) (19). Fixed, cryoprotected, frozen lung tissue from SP-C_24-57HA transgenic and wild-type mice was processed and immunogold labeling was performed with HA antiserum, as described previously (20). Morphometric analyses were performed by counting gold particles localized to specific subcellular compartments, as previously described (20).

	Results

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SP-C proprotein is incompletely processed to the mature peptide in SP-B (/) mice; consequently, SP-B-deficient mice have reduced levels of SP-C mature peptide in the alveolar space. To test the hypothesis that expression of fully processed SP-C mature peptide could rescue the neonatal lethal phenotype in SP-B (/) mice, expression of a transgene encoding mouse SP-C mature peptide (SP-C_24-57HA) was directed to type II cells in transgenic mice using the mouse 13 kb SP-C promoter (Figure 1A). Twelve potential founder mice died in the immediate postnatal period; although only two pups were recovered for genotyping, both of these animals carried the transgene, suggesting that expression of SP-C_24-57HA may result in neonatal lethality. Of the mice that survived the neonatal period, five potential founder mice were identified by PCR and Southern blot analyses of tail DNA (data not shown). Only two of these mice, F₀A and F₀C, transmitted the transgene to their offspring; however, although these founder mice survived without any overt evidence of lung disease, all transgenic offspring died of RDS shortly after birth. These results suggested that mosaicism in the founder mice resulted in low-level expression of the transgene in F₀A and F₀C and higher expression in F₁ offspring.

View larger version (38K): [in this window] [in a new window]   Figure 1.   Transgenic progeny and a dead founder mouse express high levels of SP-C24-57HA peptide. (A) A construct consisting of the mouse SP-C mature peptide and a hemagglutinin tag under the control of the mouse 13 kb promoter was used to generate several transgenic mouse lines. (B) Western analysis was performed on 1 or 10 µg of lung homogenate from F1A and wild-type fetuses (18.5 d of gestation), newborn (F1C and F0X) pups, or adult (F0A and F0C) mice using an HA antibody. F1A, F1C and F0X express high levels of the transgene, whereas F0A and F0C (founder mice) have much lower levels of expression. No HA immunoreactivity was detected in wild-type mice. (C) F1A pups expressed twice as much SP-C24-57HA peptide as F1C pups by quantitative Western analysis. Transgenic offspring expressed significantly higher levels of SP-C24-57HA peptide compared with their founder parents. Units on the y-axis are arbitrary.

To determine the level of SP-C_24-57HA expression in founder mice and transgenic progeny, Western analyses were performed on lung homogenates from fetal, newborn, and adult transgenic (i.e., F₀A and F₀C) or wild-type mice. High levels of SP-C_24-57HA peptide were detected in F₁ offspring from both transgenic lines (Figure 1B) with F₁A pups expressing twice as much of SP-C_24-57HA peptide as F₁C pups (Figure 1C). Both F₁A and F₁C transgenic pups expressed dramatically higher levels of SP-C_24-57HA peptide than their founder parents (Figure 1) consistent with the hypothesis that the founder mice were mosaic for transgene expression. In addition, high levels of SP-C_24-57HA peptide were detected in a founder pup (F₀X) that died shortly after birth (Figure 1B). These data indicate that elevated levels of SP-C_24-57HA peptide correlated with neonatal lethality in three independent transgenic lines.

The pattern of SP-C_24-57HA expression was analyzed by immunohistochemistry of lung sections from fetal F₁A and newborn F₁C pups using an antibody directed against the HA antigen. HA immunoreactivity was detected in the distal lung epithelium in transgenic progeny from both transgenic lines consistent with the well-characterized expression pattern directed by the SP-C promoter (Figures 2B and 2C). HA immunoreactivity was not detected in lung sections from wild-type littermates (Figures 2A and 2D). In F₁A and F₁C pups, lung development was delayed/ arrested compared with wild type littermate controls (Figures 2A-2C). Lung tissue from F₁A pups contained large cystic structures, showed little branching morphogenesis, and resembled lung structures found at 13-14 d of gestation in wild-type mice (Figure 2B). In addition, detached HA positive epithelial cells were present in the lumen of the cystic structures (Figure 2E). F₁C lung tissue showed considerable branching morphogenesis but little sacculation, similar to lung morphogenesis at 16-16.5 d of gestation (Figure 2C). Consistent with the results from Western analyses (Figure 1B), HA staining was more intense in epithelial cells of lung tissue from F₁A pups than that from F₁C pups (Figures 2E and 2F). These data suggest that expression of SP-C_24-57HA peptide resulted in delayed/arrested lung development with the severity of lung immaturity correlating with the level of SP-C_24-57HA expression.

View larger version (60K): [in this window] [in a new window]   Figure 2.   Expression of SP-C24-57HA peptide results in developmental arrest and lung immaturity. Immunohistochemistry was performed on paraffin-embedded lung tissue from F1A and wild-type fetuses (18.5 d of gestation) or newborn F1C pups using an HA antibody. F1A (B) and F1C (C) pups show delayed lung development compared with wild-type littermates (A). HA immunoreactivity was detected in the majority of distal lung epithelial cells in F1A (B and E) and F1C (C and F) pups, whereas no HA reactivity was detected in wild-type lung sections (A and D). Detached HA positive epithelial cells (arrow) were present in the lumen of cystic alveolar spaces (E).

To determine the intracellular localization of SP-C_24-57HA peptide, immunogold labeling of F₁A and F₁C lung tissue was performed with HA antisera. In F₁A and F₁C transgenic pups, HA immunoreactivity was detected throughout the regulated secretory pathway, including the endoplasmic reticulum, Golgi, multivesicular bodies, and extracted lamellar bodies at significantly higher levels than control regions (mitochondria and the nucleus) (Figure 3A); immunogold labeling was most prominent in the endoplasmic reticulum and Golgi. Detection of SP-C_24-57HA on the limiting membrane and inner vesicles (Figure 3B, inset) of multivesicular bodies and in the lumen of extracted lamellar bodies (Figure 3B) indicated that some of the peptide reached the distal secretory pathway and may have been secreted; however, HA immunoreactivity was not detected in the cystic alveolar space (data not shown). Evaluation of type II cell ultrastructure by transmission electron microscopy did not detect any abnormalities such as aggresome formation or grossly distended endoplasmic reticulum.

View larger version (151K): [in this window] [in a new window]   Figure 3.   SP-C24-57HA peptide is localized predominantly to the early secretory pathway in type II cells. Immunogold labeling of fixed, cryoprotected, frozen lung sections from F1A (18.5 d gestation) and newborn F1C or wild-type pups was performed using an HA antibody. (A) Morphometric analyses indicated that HA labeling was present throughout the regulated secretory pathway of type II cells, including the endoplasmic reticulum (ER), Golgi, multivesicular bodies (MVB), and lamellar bodies (LB) at significantly higher levels than control regions. (B) In both F1A (not shown) and F1C transgenic pups, HA immunoreactivity was detected on the limiting membrane and inner vesicles (arrows) of multivesicular bodies (inset) and in the lumen of lamellar bodies (arrowheads). (C) HA immunoreactivity in the ER.

To determine if expression of other surfactant proteins was altered in F₁A and F₁C pups, equal amounts of lung homogenate were immunoblotted with antibodies directed against SP-A, SP-B mature peptide, or SP-C mature peptide. Levels of SP-A and mature SP-B peptide were dramatically reduced in lung homogenates from F₁A and F₁C transgenic pups compared with wild type littermate controls (Figure 4). Mature SP-C peptide was not detected in lung tissue from F₁A or F₁C pups, indicating that the SP-C_24-57HA peptide was not recognized by an antibody specific for the SP-C mature peptide. SP-A protein is first detected at low levels on Day 14 of gestation in the mouse (21), whereas processing of SP-B and SP-C to the mature peptides commences on Day 17 of gestation (22). Consistent with the results from morphologic studies (Figure 2), these data suggested that lung development in F₁A and F₁C pups did not progress beyond the stage characterized by SP-A expression and processing of SP-B and SP-C. To determine if the lung structures in F₁A and F₁C mice expressed an earlier marker of the distal lung epithelium, immunohistochemistry was performed on lung sections from wild-type, F₁A, and F₁C pups using a TTF-1 antibody. TTF-1 was detected in the nucleus of epithelial cells from wild type, F₁A (Figure 5), and F₁C (not shown) lung tissue. Taken together, the results of histologic, immunohistochemical, and Western analyses suggest that expression of SP-C_24-57HA peptide during development results in delay/arrest of lung development during the pseudoglandular stage.

View larger version (76K): [in this window] [in a new window]   Figure 4.   Surfactant protein expression is dramatically reduced in mice expressing high levels of SP-C24-57HA peptide. Equal amounts of lung homogenate from F1A and wild-type fetuses (18.5 d gestation) or newborn F1C and F0X pups were immunoblotted with antibodies directed against SP-A, mature SP-B peptide, and mature SP-C peptide. SP-A and mature SP-B peptide levels were profoundly reduced in F1A, F1C, and F0X pups compared with wild-type littermates. SP-C mature peptide was detected in wild-type but not transgenic mice. Molecular weight markers (× 103) are indicated to the right of the figure.

View larger version (137K): [in this window] [in a new window]   Figure 5.   TTF-1 expression is unchanged in SP-C24-57HA transgenic mice. Immunohistochemistry was performed on paraffin-embedded lung sections from SP-C24-57HA and wild-type fetuses (16.5 d gestation) using a TTF-1 antibody. In SP-C24-57HA (B and D) and wild-type (A and C) pups, TTF-1 immunoreactivity was detected in the nucleus of epithelial cells in the distal lung and proximal airways (upper panels, low magnification; lower panels, high magnification).

	Discussion

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SP-B null mice have reduced levels of SP-C mature peptide in the alveolar spaces resulting in both SP-B and SP-C deficiency (4). In wild-type mice, the SP-C mature peptide was sorted and secreted into the alveolar space in the absence of the flanking peptides (13). Previous studies indicated that administration of surfactant preparations containing either SP-B or SP-C mature peptide restored lung function in surfactant-depleted animals, suggesting that SP-B and SP-C are functionally redundant with respect to surface film dynamics. The current study was undertaken to test the hypothesis that expression of the mature SP-C peptide would rescue the neonatal lethal phenotype in SP-B null mice. Unexpectedly, expression of SP-C_24-57HA peptide in the lungs of transgenic mice perturbed fetal lung structure, resulting in neonatal, lethal respiratory distress syndrome.

Of more than 1,200 oocytes injected with the transgene, only two transgenic founder mice were identified. Both founders were mosaic for the transgene and expressed significantly less SP-C_24-57HA peptide than their progeny, which likely contributed to their survival. The unexpectedly low number of surviving transgenic founders was likely related to neonatal death of mice expressing high levels of the transgene. Due to the tendency of mice to cannibalize dead offspring, we were only able to recover enough tissue to genotype two dead pups and confirm that both were indeed transgenic.

Elevated levels of SP-C_24-57HA peptide were associated with altered lung structure in progeny from both transgenic lines. The more severely affected F₁A mice expressed higher levels of SP-C_24-57HA peptide and the effect on lung structure was more severe than in F₁C mice. Consistent with lung immaturity, levels of SP-A, SP-B, and SP-C were dramatically decreased relative to wild-type littermates in offspring from both transgenic lines. In contrast, TTF-1 expression was not altered, suggesting that early epithelial markers were not affected by expression of the SP-C_24-57HA transgene. Taken together, these data suggest that expression of the SP-C_24-57HA transgene significantly delayed lung maturation, resulting in lethal, neonatal RDS.

The mechanism by which SP-C_24-57HA disrupted lung development is not clear. Intratracheal infection with adenovirus encoding SP-C_24-57HA resulted in secretion of the peptide by wild-type mice but not SP-C (/) mice (13). This result suggested that endogenous SP-C facilitated intracellular trafficking and secretion of SP-C_24-57HA in wild-type mice. In the present study, transgenic mice expressed SP-C_24-57HA at much higher levels than adenovirus-infected mice, but very little peptide was detected in the airway despite the presence of endogenous SP-C. The most likely explanation for this outcome is that high levels of SP-C_24-57HA overwhelmed endogenous SP-C and accumulated in the early secretory pathway. Consistent with this hypothesis, SP-C_24-57HA was detected predominantly in the endoplasmic reticulum (ER) and Golgi of transgenic mice, whereas endogenous SP-B and SP-C were very difficult to detect in the endoplasmic reticulum of wild-type mice (10, 11, 20).

Polypeptides enter the ER in an unfolded state and interact transiently and sequentially with multiple chaperones (e.g., immunoglobulin binding protein [BiP], clanexin, calreticulin) and folding catalysts (e.g., protein disulfide isomerase) to produce a functional conformation. Missense mutations and inframe deletions and insertions often impair the ability of the affected polypeptide to fold to a functional conformation and/or decrease the stability of the functional conformation (23, 24). Quality control mechanisms, predominantly in the ER but also in the Golgi and plasma membrane, identify misfolded proteins and target these proteins for degradation by the ubiquitin-proteasome pathway following retrotranslocation into the cytosol (25- 28). Class I mutations prevent transport of the affected protein out of the ER but do not interfere with its ability to be efficiently degraded. Class II mutations inhibit both intracellular transport and turnover, leading to accumulation of undegraded, abnormal protein in the early secretory pathway which, in turn, can lead to apoptosis, abnormal differentiation or altered proliferation (25, 29). Class II mutations have been linked to a number of diseases, including osteogenesis imperfecta, Charcot-Marie Tooth syndrome, and hereditary emphysema. Given that the SP-C mature peptide is extremely hydrophobic and prone to aggregation (30, 31), we propose that SP-C_24-57HA behaves as a Class II mutation such that SP-C aggregation leads to cytotoxicity and, ultimately, altered lung development.

Fetal lung development was not altered in SP-C (/) mice (5). This observation is consistent with the hypothesis that the toxic effects of SP-C aggregation, rather than diminished levels of SP-C in type II cells or the airway, underlies altered lung development in SP-C_24-57HA transgenic mice. This hypothesis further predicts that mutations that promote misfolding and/or aggregation of the SP-C proprotein may contribute to pathogenesis. Consistent with this prediction, transient transfection of A549 cells with an SP-C construct containing a point mutation, resulted in accumulation of SP-C protein in aggresomes (32). Further, a mutation in the human SP-C gene was recently identified in a patient with a family history of interstitial lung disease (33). This mutation occurred in only one allele of the SP-C gene, suggesting that, as in the case of the SP-C_24-57HA transgenic mice, lung disease can occur in the presence of normal SP-C protein. Whether this or other mutations in the human SP-C gene result in aggregation of SP-C protein, cytotoxicity or, ultimately, lung disease, remains to be directly tested.

Although we cannot exclude the possibility that inclusion of the nine amino acid HA epitope (YPYDVPDYA) in the transgene contributed to the severity of lung disease in transgenic mice, this seems unlikely. Expression of the entire hemagglutinin protein, under control of the human SP-C promoter, in transgenic mice was not associated with altered lung development (34). Similarly, expression of HA epitope-tagged, constitutively active G protein q subunit in embryonic heart did not perturb cardiac development in transgenic mice (35). It is possible that the HA tag caused misfolding and accumulation of the SP-C transgene in the ER; however, it is more likely that the absence of the 23 amino acid NH₂-terminal propeptide and/or the 139 amino acid COOH-terminal peptide domain resulted in misfolding and/or aggregation of the extremely hydrophobic mature peptide. Regardless of the underlying cause of aggregation, expression of SP-C_24-57HA in type II cells ultimately resulted in lung immaturity and neonatal death. Given this outcome, it is conceivable that aggregation of any protein in type II cells during lung development may lead to pathogenesis. The severity of the phenotype may be dictated more by the extent of aggregation and the timing of the insult during development rather than the specific protein.