(Received for publication, November 13, 1996, and in revised form, January 23, 1997)
From the Division of Pulmonary Biology, Children's Hospital
Medical Center, Cincinnati, Ohio 45229-3039 and the
Department of Pediatrics, Harbor-UCLA Medical Center,
Torrance, California 90509
The function of the 102-amino acid C-terminal
propeptide of surfactant protein B (SP-B) was analyzed by
characterizing the phenotype associated with loss of expression of this
peptide domain in transgenic mice. A construct encoding the signal
peptide, N-terminal propeptide, and mature peptide of human SP-B
(hSP-Bc) was cloned under the control of the
3.7-kilobase human SP-C promoter and injected into fertilized eggs of
the FVB/N mouse strain. Founder mice expressing the
hSP-B
c transgene were bred with heterozygous SP-B
knockout mice (SP-B +/
). Offspring containing the transgene and one
allele of mouse SP-B were identified and subsequently crossed to
generate a transgenic line that expressed SP-B
c in a
null background (SP-B(
/
)/hSP-B
c(+/+)). Expression of
hSP-B
c in SP-B(
/
) mice was restricted to type II
cells and resulted in a 2-fold increase in mature SP-B relative to wild type littermates. These mice survived without any evidence of respiratory problems and had normal lung function, normal alveolar surfactant phospholipid pool sizes, and typical tubular myelin indicating that the 102-residue C-terminal propeptide of SP-B is not
required for normal structure and function of extracellular surfactant.
However, proteolytic processing of the SP-C proprotein was perturbed
resulting in the accumulation of a processing intermediate, Mr = 11,000, similar to the phenotype detected
in SP-B(
/
) mice; furthermore, lamellar bodies in type II cells of
SP-B(
/
)/hSP-B
c(+/+) mice were much larger than in
the wild type animal and saturated phosphatidylcholine content in lung
tissue was significantly increased although the incorporation of
choline into saturated phosphatidylcholine was normal. Collectively,
these results demonstrate a role for the C-terminal propeptide of SP-B
in SP-C proprotein processing and the maintenance of lamellar body
size. The C-terminal propeptide may be an important determinant of
intracellular surfactant pool size.
Pulmonary surfactant is a complex mixture of lipids and proteins that is synthesized exclusively by the alveolar type II epithelial cell. Surfactant is stored in large inclusions (lamellar bodies) that are secreted into the alveolar airspace by exocytosis. Newly secreted lamellar bodies unravel into a tubular network (tubular myelin) that subsequently adsorbs and spreads as a phospholipid-rich film that reduces surface tension at the air-liquid interface. Rapid adsorption and spreading of the phospholipid film are critical and require the presence of specific proteins, in particular surfactant proteins B and C (1-3).
Surfactant protein B (SP-B)1 is a hydrophobic peptide of 79 amino acids that avidly associates with surfactant phospholipids in the alveolar airspace (reviewed in Ref. 4). Homozygous mutations leading to the complete absence of SP-B in newborn human infants result in the rapid onset of respiratory distress syndrome which is refractory to mechanical ventilation and surfactant replacement (5, 6). The latter observation suggests that simple addition of mature peptide to the SP-B-deficient airway is not sufficient to restore lung function. Therefore, despite an obvious requirement for SP-B in normal lung function, the precise role(s) of SP-B in the structure, function, and metabolism of surfactant remains unclear.
Human SP-B is synthesized by the type II epithelial cell as a
preproprotein of 381 amino acids. The mature peptide is generated by
sequential cleavage of the signal peptide (23 amino acids), the
N-terminal propeptide (177 residues), and the C-terminal propeptide (102 residues) (7, 8). Propeptide cleavage occurs within the late
endosome/multivesicular body which subsequently directs newly
synthesized SP-B to the lamellar body (9); mature SP-B is also recycled
from the alveolar airspace, via the endocytic pathway to the
multivesicular body (10). Previous in vitro evidence supports the concept that trafficking of the mature peptide through the
biosynthetic pathway requires the N-terminal but not the C-terminal domain of the propeptide (11, 12). In the absence of both propeptide
domains the mature peptide is degraded within the endoplasmic reticulum
(11); however, an SP-B construct encoding both the N-terminal
propeptide and the mature peptide (hSP-Bc) produces a
truncated proprotein that is sorted to the multivesicular body where
the propeptide is appropriately cleaved to liberate mature SP-B (12).
These results clearly demonstrate that the N-terminal propeptide is
required for the intracellular trafficking of SP-B; the function of the
C-terminal propeptide, however, remains unknown.
Lung morphogenesis and surfactant phospholipid synthesis in SP-B(/
)
mice proceed normally prior to birth (13). However, lamellar body
formation is disrupted, resulting in abnormal inclusions consisting of
multivesicular bodies and disorganized lamellae. Neither mature
lamellar bodies nor tubular myelin were detected in the SP-B(
/
)
mice. In addition to effects on lamellar body biogenesis, SP-B
deficiency resulted in aberrant processing of the SP-C proprotein
leading to accumulation of an Mr = 11,000 processing intermediate and a decrease in mature SP-C peptide levels.
The purpose of the present study was to assess the function of the
C-terminal propeptide of SP-B by expressing hSP-B
c in
SP-B(
/
) mice and determining which aspects of the SP-B(
/
) phenotype were not corrected by transgene expression.
FVB/N transgenic mice
expressing a transgene construct encoding the signal peptide,
N-terminal propeptide, and mature peptide of human SP-B
(hSP-Bc) under the control of the 3.7-kilobase human
SP-C promoter were generated as described previously (12). Two of four
transgenic mouse lines were used in the present study. Mice from
transgenic Lines 6.1 and 1.4 were bred with hemizygous 129J × Swiss Black SP-B mice generated by gene targeting (13). Offspring
bearing one wild type mouse SP-B allele and the hSP-B
c transgene were identified as described below, and siblings crossed to
generate mice expressing hSP-B
c in a null background
(SP-B(
/
)/hSP-B
c(+/+)).
hSP-Bc transgenic mice were identified by PCR
amplification of a 600-base pair fragment, spanning the junction of the human SP-C promoter and human SP-B cDNA, as described previously (12). SP-B(+/
) and SP-B(
/
) genotypes were identified by
amplification of a 1.3-kilobase fragment, spanning the neomycin
resistance gene and intron 4 of the targeted SP-B gene. One
µM each of a 5
primer specific for the neomycin
resistance gene (5
ATTGCCTCTGTGGGTGTGTATGTG) and a 3
primer specific
for the SP-B gene (5
GGTGGAGAGGCTATTCGGCTATGA) were incubated with 100 ng of genomic DNA in 2.5 mM MgCl2, 50 mM Tris-HCl (pH 7.6), 50 mM KCl, 100 µM dNTPs, and 0.5 units of Amplitaq (Perkin-Elmer) for 2 min at 94 °C, followed by a 30-cycle PCR amplification comprised of
94 °C denaturation for 30 s, 63 °C annealing for 30 s
and 2 min of elongation at 72 °C. The SP-B(
/
) genotype was
identified by the absence of a 260-base pair fragment spanning introns
3 and 4 of the mouse SP-B gene following amplification with 1 µM each of 5
primer (5
CTTCCTTGGTCATCTTTGTGAGGAGGTGGA) and 3
primer (5
CGGTGTCGCCAAGTGCTTGATGTCTACCTG) using the PCR conditions described above. The SP-B(
/
) genotype was confirmed by
Southern blotting of BamHI-restricted genomic DNA with a
species-specific mouse SP-B cDNA and neomycin resistance probes as
described previously (13).
RNAs encoding mouse and human SP-B were analyzed by S1 nuclease assays using 32P-end-labeled, species-specific probes and 2 µg of total RNA as described previously (14). Total SP-B protein was analyzed by ELISA (15) and Western blotting (11, 12), using equal amounts of protein from lung homogenates of 6-7-week-old mice of each genotype. Mature SP-B, Mr ~18,000, and SP-C, Mr ~4,000, were separated by SDS-polyacrylamide gel electrophoresis in the absence of sulfhydryl reducing agents and detected by blotting with antiserum 28031 (11), which detects only SP-B, or antiserum 559 (13), which detects both SP-B and SP-C; SP-C proprotein was detected by antiserum 68514 (16) which recognizes antigenic determinants in the 20-amino acid N-terminal propeptide of SP-C.
Surfactant Metabolic MeasurementsSP-B(/
)/hSP-B
c(+/+) mice and
FVB/N mice (wild type) at 7-8 weeks of age were used for measurements
of saturated phosphatidylcholine (Sat PC) pool size and choline
incorporation in vivo. Wild type and
SP-B(
/
)/hSP-B
c(+/+) mice were given 8 µl/g body
weight intraperitoneal injections of 0.2 µCi/µl
[3H]choline chloride (DuPont NEN). Groups of 5-7 wild
type and SP-B(
/
)/hSP-B
c(+/+) mice were killed, and
an alveolar lavage was recovered from each animal at five preselected
times from 3 to 48 h after isotope injection based on our previous
experiences with precursor labeling in mice (17). Mice were given
intraperitoneal pentobarbital to achieve deep anesthesia, and the
distal aorta was cut to exsanguinate each animal. The chest of the
animal was opened; a 20-gauge blunt needle was tied into the proximal
trachea; 0.9% NaCl was instilled into the airways until the lungs were
inflated, and the fluid was withdrawn by syringe. The lavage procedure
was repeated four times, and samples from each lung were pooled for
analysis. Lung tissue after alveolar lavage was weighed and homogenized
in 2 ml of 0.9% NaCl. Alveolar lavage and aliquots of the lung
homogenates were extracted with chloroform:methanol (2:1) and Sat PC
was isolated (18). The Sat PC was divided for measurement of
phosphorous (19) and radioactivity. The total radioactivity recovered
in Sat PC for the alveolar lavage, lung tissue, and total lung
(alveolar and tissue) of each animal was calculated. Percent secretion
of labeled Sat PC was calculated as the radioactivity in alveolar Sat
PC divided by the total radioactivity in the alveolar lavage plus lung
tissue × 100. Pool sizes of Sat PC in alveolar lavage, lung
tissue, and total lung were calculated as µmol per kg body weight.
The DNA measurements for lung tissue were according to Hill and Whatley
(20). For measurements of phospholipid composition, chloroform/methanol
extracts of pooled alveolar lavages from 2 SP-B(
/
)/hSP-B
c(+/+) mice and 2 wild type mice each
were used for two-dimensional thin layer chromatography (21). The spots
were visualized with iodine vapor, scraped, and assayed for phosphorous
content (19).
Static lung compliance was
measured in 12 SP-B(+/+)/hSP-Bc(+/+), 8 SP-B(+/
)/hSP-B
c(+/+), and 10 SP-B(
/
)/hSP-B
c(+/+) mice at 6-7 weeks of age. Mice
were injected intraperitoneally with sodium pentobarbital (200 mg/kg)
and placed in a chamber containing 100% O2. In this
manner, the lungs were completely deflated at the time of death (22). A
catheter (20-gauge angiocatheter) was inserted into the trachea and
connected to a silicon pressure sensor (X-ducerTM,
Motorola, Phoenix, AZ). The chest wall and diaphragm were opened carefully to avoid injuring the lungs. The lungs were inflated with air
in small increments (0.1 ml every 5-8 s) to a maximal pressure of 40 cm H2O. Lungs were deflated in stepwise fashion. Airway
opening pressure and lung volume were recorded at each inflation and
deflation increment. All measurements were completed within 15 min
after death. Pressure-volume curves were generated for each animal.
Lung compliance was determined by calculating the slopes and intercepts
of the straight portion of the deflation curve where pressure ranged
from
10 to +10 cm H2O (23). Specific lung compliance was
calculated as lung compliance divided by the lung weight.
Murine sense and antisense riboprobes used were synthesized as described previously (24); human riboprobes were synthesized from full-length SP-B cDNA (25) subcloned from pkc4 as an EcoRI fragment into the EcoRI site of pGEM3Z (f+). In situ hybridization was performed under stringent conditions as reported by Damore-Bruno et al. (24) with the addition of an extensive post-hybridization washing schedule (26) to minimize possible cross-reactivity between the murine and human probes.
Electron MicroscopyTransgenic and control lungs from 6-week old mice were fixed by tracheal infusion (27) with 1.5% paraformaldehyde, 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Following overnight immersion in the same fixative, lungs were minced into 1-mm cubes and processed for electron microscopy as described by Fehrenbach et al. (28). Briefly, after fixation, blocks were rinsed for 1 h in 4-6 changes of 0.1 M sodium cacodylate buffer (pH 7.3) (SCB), post-fixed 2-3 h in 1% OsO4 in SCB, rinsed several times in SCB and several times in distilled H2O, and incubated overnight in 4% aqueous uranyl acetate. After several rinses in distilled H2O, blocks were dehydrated through a graded series of ethanols and transferred to Epon 812 (Shell Chemical Co.) via propylene oxide. Ultrathin sections (60-90 nm) were prepared, counterstained with lead citrate, and evaluated and photographed using a JEOL-100-CXII transmission electron microscope.
Statistical AnalysisFor the lung function studies, differences among animals were evaluated by comparing lung compliances, specific lung compliances, intercepts, and inflation lung volumes at 30 cm of H2O by two-way analysis of variance; significant differences between mean values was determined by Bonferroni/Dunn test. Differences between groups of animals in surfactant metabolic studies and ELISA analysis were tested by two-tailed Student's t test. Values are expressed as mean ± S.E.
Rescue of SP-B(/
) Mice by hSP-B
c
Generation of transgenic mouse lines expressing truncated human
SP-B (hSP-Bc, in which the sequence encoding the
102-residue C-terminal propeptide was deleted) in type II epithelial
cells has been previously described (12). Since these mice expressed both endogenous SP-B and the human SP-B
c
transgene, there was a 3-4-fold increase of mature SP-B peptide
in lung homogenates of animals that were homozygous for the transgene.
Surfactant composition and phospholipid pool sizes, static compliance,
and type II ultrastructure were all normal in adult
SP-B
c transgenic mice, indicating that increased levels
of SP-B did not perturb surfactant homeostasis or lung function.
hSP-B
c transgenic mice (line 6.1) were bred with
SP-B(+/
) mice and offspring that were homozygous for
hSP-B
c and heterozygous for the mouse SP-B allele
(SP-B(+/
)/hSP-B
c(+/+)) were identified by PCR. These mice were subsequently interbred to achieve expression of hSP-B
c in the null background
(SP-B(
/
)/hSP-B
c(+/+)) (Fig. 1). Of
105 offspring screened, 21 were identified as both mSP-B(
/
) and
hSP-B
c(+/+), consistent with Mendelian inheritance of a
recessive allele and postnatal survival of all animals carrying the
hSP-B
c transgene. In the absence of the transgene no SP-B(
/
) offspring were detected confirming the perinatal lethality associated with ablation of the SP-B locus (13) and the ability of
hSP-B
c to rescue these animals. "Rescued"
SP-B(
/
) mice were indistinguishable from wild type littermates with
respect to body weight, lung weight, reproductive capacity, and
longevity, indicating that the C-terminal propeptide of SP-B is not
essential for survival.
A separate transgenic line (line 1.4) in which the
hSP-Bc transgene was expressed at approximately 1% of
wild type SP-B was also bred into the SP-B knockout background. Of 60 offspring from 6 litters no rescued SP-B(
/
) survivors were detected
by PCR analyses. To confirm transgene transmission in this line, 2 litters were delivered on day 19 of gestation and analyzed for genotype
by PCR. Of 15 offspring, 5 were both transgene-positive and homozygous
for the disrupted SP-B allele. In contrast to wild type littermates,
these animals rapidly succumbed to respiratory failure, as described
previously for SP-B knockout mice (13), indicating that the level of
hSP-B
c transgene expression in the 1.4 line was not
sufficient to rescue SP-B(
/
) offspring.
Characterization of hSP-Bc Transgene Expression in
Rescued SP-B(
/
) Mice
S1 nuclease analyses of lung tissues from
SP-B(/
)/hSP-B
c(+/+) mice detected only human SP-B
RNA confirming the complete absence of mouse SP-B in these animals
(Fig. 2). These results were corroborated by in
situ hybridization studies (Fig. 3) in which only
human SP-B RNA was detected in SP-B(
/
)/hSP-B
c(+/+) mice. Expression of hSP-B
c RNA was restricted to
alveolar type II epithelial cells in contrast to wild type littermates in which endogenous SP-B RNA was expressed in both type II cells and
nonciliated bronchiolar cells (Clara cells). The expression of hSP-B
mRNA in type II cells was consistently observed in 12 SP-B(
/
)/hSP-B
c(+/+) animals of different ages from
six separate litters. Quantitation of SP-B protein by ELISA indicated that the level of mature peptide in bronchoalveolar lavage fluid was
elevated approximately 2-fold in SP-B(
/
)/hSP-B
c(+/+) mice relative to wild type littermates (Fig.
4A). Western blot analyses of lung
homogenates from SP-B(
/
)/hSP-B
c(+/+) demonstrated that all of the hSP-B
c precursor was fully processed to
the mature 8-kDa peptide (Fig. 4B). In contrast to normal
processing of hSP-B
c, the SP-C proprotein was aberrantly
processed resulting in the accumulation of an 11-kDa intermediate as
previously reported in SP-B(
/
) mice and human infants (5, 13) (Fig.
5B). However, unlike SP-B(
/
) mice, levels
of the mature SP-C peptide, Mr = 5000, did not
appear to be decreased.
Characterization of Lung Structure in Rescued SP-B(/
)
Mice
Previous ultrastructural analyses of lung tissue from
hSP-Bc(+/+) transgenic mice (line 6.1) indicated that
type II cell structure was not affected by expression of
hSP-B
c or increased levels of SP-B mature peptide (12).
In contrast, expression of hSP-B
c in SP-B(
/
) mice
resulted in the formation of greatly enlarged lamellar bodies (Fig.
6C) containing normal concentric lamellar
membranes in approximately 20% of type II cells. Closely apposed
lamellar bodies were observed, and in many instances, lamellar bodies
appeared to be coalescing, suggesting that the increased size of these
inclusions was related in part to increased fusion (Fig.
6D). Enlarged and/or fusing lamellar bodies were detected in
all type II cells of SP-B(
/
)/hSP-B
c(+/+) mice but
never observed in wild type littermates or hSP-B
c
transgenic mice. Typical tubular myelin profiles were readily detected
in SP-B(
/
)/hSP-B
c(+/+) mice indicating that
SP-B
c was sufficient to restore this ultrastructural
feature of extracellular surfactant in SP-B(
/
) mice (not shown).
Other ultrastructural abnormalities were not detected in the lungs of
SP-B(
/
)/hSP-B
c(+/+) mice, and ultrastructural
features of nonciliated bronchiolar cells were indistinguishable from
Clara cells in wild type mice.
Characterization of Lung Function in Rescued SP-B(/
)
Mice
Intracellular and extracellular pool sizes of Sat PC
were estimated in 32 SP-B(/
)/SP-B
c(+/+) mice and 36 wild type mice from precursor incorporation studies (Fig.
7). The alveolar Sat PC pool size, estimated from
alveolar lavage samples, was similar in the two groups. The lung tissue
pool of Sat PC was increased 30% in
SP-B(
/
)/hSP-B
c(+/+) mice compared with wild type
mice, and the amount of Sat PC in total lung was increased 23%
(calculated as the sum of the alveolar and tissue Sat PC). The percent
of the total lung Sat PC recovered by alveolar lavage was 20.9 ± 0.4% in SP-B(
/
)/hSP-B
c(+/+) mice and 27.7 ± 0.5% in wild type mice (p < 0.0001) consistent with
increased size and number of lamellar bodies noted in the
ultrastructural studies. There were no differences between the two
groups of mice in body weight (SP-B(
/
)/hSP-B
c(+/+):
23.8 ± 0.5 g, wild type: 22.4 ± 0.2 g) and total
lung DNA (SP-B(
/
)/hSP-B
c(+/+): 997 ± 37 µg,
wild type: 923 ± 49 µg). The compositions of the phospholipids
recovered by alveolar lavage were similar in the two groups of mice
(Table I).
|
The incorporation of
radiolabeled choline (Fig. 8), into lung phospholipid,
was measured as total incorporation into Sat PC. [3H]Choline incorporation into Sat PC was similar in
SP-B(/
)/hSP-B
c(+/+) and control mice. The loss of
labeled Sat PC after precursor injection, measured at 24 and 48 h,
indicated that [3H]choline-labeled Sat PC was
significantly higher in lung tissue of
SP-B(
/
)/hSP-B
c(+/+) mice. The percent
choline-labeled Sat PC secreted to the alveolar space was decreased by
about 20% in SP-B(
/
)/hSP-B
c(+/+) mice relative to
control mice, and the difference was significant at 3 and 15 h
(p < 0.01).
Pulmonary Function
Static lung compliance was
determined by analyzing pressure-volume curves. There were no
differences in static lung compliance corrected for body weight when
SP-B(/
)/hSP-B
c(+/+),
SP-B(+/minus])/hSP-B
c(+/+), and
SP-B(+/+)/hSP-B
c(+/+) mice were compared (data not
shown). There were also no differences in y intercepts,
inflation lung volumes at 30 cm of H2O, or lung compliances
normalized to lung wet weight or volume. The values for static lung
compliance/g body weight were not significantly different from values
obtained in wild type FVB/N mice (3.54 ± 0.34 µl/cm
H2O per g body weight), indicating that the Swiss Black
genetic background had no influence on these values. Additionally, lung
compliance in hSP-B
c(+/+) transgenic mice (line 6.1) was
not significantly different from that in wild type control mice,
indicating that hSP-B overexpression also did not influence lung
compliance (data not shown).
Recent studies from this laboratory demonstrated that the
102-residue C-terminal propeptide of SP-B was not required for
intracellular trafficking of the hydrophobic, mature peptide (11), for
targeting of the proprotein to lamellar bodies (12), or for proteolytic processing of the proprotein (12). The present study was therefore undertaken to identify the function of the C-terminal propeptide of
SP-B by analyzing the phenotype associated with loss of expression of
this peptide domain in transgenic mice. Expression of an
hSP-Bc transgene in type II cells of SP-B(
/
) mice
resulted in postnatal survival, normal lung function, and normal
surfactant composition indicating that the C-terminal propeptide was
not required for reversal of neonatal lethality associated with SP-B
deficiency (13); however, these "rescued" mice
(SP-B(
/
)/hSP-B
c(+/+)) contained abnormally large
lamellar bodies with increased tissue pools of saturated
phosphatidylcholine and accumulated an SP-C processing intermediate
consistent with a role for the C-terminal propeptide in SP-C processing
and lamellar body biogenesis.
Several observations argue for a specific role for the C-terminal
propeptide in lamellar body biogenesis and SP-C processing in type II
epithelial cells. First, the phenotype observed in SP-B(/
)/hSP-B
c(+/+) mice is not related to the site
of transgene integration; both the 1.6 and 1.4 transgenic lines express the hSP-B
c transgene in the wild type background without any detectable effect on lung structure or function (12). Second, although the genetic background of the knockout mouse can clearly influence the phenotype (29, 30), this does not appear to be the case
for the SP-B(
/
)/hSP-B
c(+/+) mice; both F1 and F6
SP-B(
/
) offspring of SP-B knockout mice crossed into the FVB/N
background (the strain used to generate the 1.6 and 1.4 transgenic
lines) exhibit the same phenotype as that originally reported for the
SP-B knockout in the 129J/Swiss Black background (data not shown) (13).
Third, it is unlikely that the loss of SP-B expression in Clara cells
of SP-B(
/
)/hSP-B
c(+/+) mice contributed to the
enlarged lamellar bodies detected in type II cells of these animals.
Although lamellar body morphology was dramatically altered in type II
cells, Clara cell ultrastructure was not perturbed; furthermore, the
C-terminal propeptide is not secreted into the airway in healthy
animals (7, 9) making it unlikely that the Clara cell is a source of
C-terminal propeptide for lamellar body biogenesis or SP-C processing
in type II cells. Finally, it is unlikely that elevated levels of
mature SP-B peptide in SP-B(
/
)/hSP-B
c(+/+)
mice contribute to increased lamellar body size and accumulation of the
SP-C processing intermediate since overexpression of SP-B in the
hSP-B
c(+/+) transgenic line produced no alterations in
type II cell ultrastructure, tubular myelin formation, or SP-C
processing. Therefore, the C-terminal propeptide is either directly or
indirectly involved in processing of the SP-C proprotein in type II
cells. Effects on lamellar body size may be secondary to SP-C
misprocessing or may represent a separate function of this peptide
domain.
As evident from the study of SP-B(/
) mice (13), the formation of
lamellar bodies is clearly aberrant in the absence of SP-B, resulting
in large inclusions containing multivesicular bodies and occasional
loosely organized membrane lamellae. Expression of
hSP-B
c in SP-B(
/
) mice leads to formation of
inclusion bodies with tightly organized concentric lamellae, similar to lamellar bodies detected in wild type littermates. In
SP-B(
/
)/hSP-B
c(+/+) mice, lamellar body formation
proceeds in the absence of the C-terminal propeptide and in the
presence of misprocessed SP-C demonstrating that the mature SP-B
peptide and/or the N-terminal propeptide are sufficient for lamellar
body formation. While the formation of lamellar bodies in
SP-B(
/
)/hSP-B
c(+/+) mice appears normal, the
regulation of lamellar body size is clearly compromised. Accumulation
of tissue PC and dramatically enlarged lamellar bodies are similar to
findings in the lipid storage disease seen in beige mice, an animal
model of Chediak-Higashi syndrome (31). Although the biochemical basis
of Chediak-Higashi syndrome is not known, other lipid storage diseases
result from an accumulation of substrate in lysosomes. It is now clear
that several of these storage diseases are related to a deficiency of
one or more saposins, small 80-amino acid peptides that act as
co-enzymes for lysosomal glycolipid hydrolysis (reviewed in Ref. 32).
Interestingly, the C-terminal propeptide of SP-B bears strong homology
to the saposins, including conservation of six cysteine residues, a
site for Asn-linked oligosaccharide, and various hydrophobic amino
acids; furthermore, the lamellar body is a lysosomal-like compartment
that contains hydrolases and membrane glycoproteins common to lysosomes
(9, 33). Collectively, these observations are consistent with the
hypothesis that the C-terminal propeptide plays an important role in
regulating lamellar body size and intracellular surfactant pool size.
The mechanism leading to increased lamellar body contents and size is
unclear but may include increased fusion of individual lamellar bodies and/or reduced catabolism of surfactant phospholipids.
The metabolism measurements complement the cellular abnormalities and
provide some insight into the effects of the hSP-Bc transgene. The increased lung tissue pools of Sat PC are consistent with the large and numerous lamellar bodies because a large percentage of Sat PC is associated with lamellar bodies in the normal lung. Radiolabeled choline incorporation into Sat PC is the best indicator of
de novo synthesis for in vivo measurements, and
no consistent differences between wild type and
SP-B(
/
)/hSP-B
c(+/+) mice were found. This result is
consistent with the finding of normal Sat PC synthesis in term
SP-B(
/
) mice that have a complete disruption of lamellar body
formation.2 The synthetic machinery for Sat
PC seems to be insensitive to alterations in surfactant packaging and
secretory pathways. The slower rate of secretion of de novo
synthesized Sat PC and the lower percent total secretion of Sat PC
indicate a processing defect. Of interest, Sat PC metabolism also was
abnormal in the beige mouse model of Chediak-Higashi syndrome (34).
However, in that mouse [3H]glycerol incorporation into
Sat PC was decreased to about 30% that in wild type mice, and the
alveolar pool of Sat PC was decreased by about 30%.
SP-B(
/
)/hSP-B
c(+/+) mice have remarkable cellular
and metabolic abnormalities in the surfactant processing pathways, but
they achieve a normal alveolar pool size for surfactant. The net effect
of the hSP-B
c transgene correction in SP-B(
/
) mice
likely reflects regulation of the alveolar pool to maintain normal
surfactant homeostasis. Many mechanisms that promote surfactant secretion have been described (35); however, very little is known about
pool size regulation. Other transgenic models indicate that cytokines
such as granulocyte-macrophage colony-stimulating factor are produced
by type II cells and can modulate surfactant component clearance
kinetics (17, 36). The hSP-B
c transgene correction
results in a healthy animal because the net effect is the production of
normal amounts of surfactant with a normal composition and adequate
content of mature SP-B.
Although surfactant function was restored in the
SP-B(/
)/hSP-B
c(+/+) mice, the accumulation of an
SP-C processing intermediate seen in SP-B(
/
) mice was not corrected
by the hSP-B
c transgene. Restoration of normal lung
function in SP-B(
/
)/hSP-B
c(+/+) mice in the
continued presence of the Mr = 11,000 SP-C
processing intermediate indicates that the primary reason for neonatal
lethality in SP-B(
/
) mice is deficiency of mature SP-B and/or
mature SP-C, not accumulation of the Mr = 11,000 protein. Although the functional consequences of SP-C deficiency are
not yet known, it is clear that reduced levels of SP-B protein are
associated with altered lung function. A human infant with partial SP-B
deficiency was recently reported to have severe respiratory distress
syndrome and chronic lung disease (37). Mice that are hemizygous for the SP-B allele and express SP-B at 50% of the wild type level with
normal levels of mature SP-C exhibit air trapping at low lung volumes
(38). Further decreases in SP-B levels to approximately 1% of wild
type result in neonatal lethality as evidenced by the hSP-B
c 1.4 transgenic line; however, these animals also
had decreased levels of mature SP-C peptide similar to SP-B(
/
)
mice. Taken together, these observations suggest that decreased SP-B concentration, secondary to premature birth, infection, or other causes, may contribute to the pathogenesis of lung disease.
SP-B is expressed in nonciliated bronchiolar Clara cells as well as
alveolar type II epithelial cells. Although the function of SP-B in
type II cells is likely related to packaging of the surfactant complex,
the role of SP-B in the Clara cell is not known. In
SP-B(/
)/hSP-B
c(+/+) mice, SP-B expression was restricted to type II cells. The observation that tubular myelin formation, dynamic compliance, and airway pool size were normal in
SP-B(
/
)/hSP-B
c(+/+) mice indicates that Clara cell
SP-B is not required for surfactant function and metabolism. During hyperoxic lung injury, SP-B expression increases in Clara cells and
decreases in type II cells (39, 40); however, it is not known if SP-B
proprotein in Clara cells is processed to the mature peptide and
secreted or if the Clara cell contributes SP-B to the alveolar
surfactant pool following lung injury. It is therefore unclear if SP-B
expression in Clara cells is related to surfactant function or some
novel function.
In summary, the C-terminal propeptide of SP-B likely plays an important
role in regulating SP-C proprotein processing and lamellar body size.
Increased lamellar body size in SP-B(/
)/hSP-B
c(+/+) mice was accompanied by increased levels of tissue Sat PC suggesting that the C-terminal propeptide may be an important determinant of
intracellular surfactant pool size.