close

Вход

Забыли?

вход по аккаунту

?

Glucocorticoid regulation of surfactant-associated proteins in rabbit fetal lung in vivo.

код для вставкиСкачать
THE ANATOMICAL RECORD 237:365-377 (1993)
Glucocorticoid Regulation of Surfactant-Associated Proteins in
Rabbit Fetal Lung In Vivo
PAUL L. DURHAM, CHRISTINE L. WOHLFORD-LENANE, AND JEANNE M. SNYDER
Department of Anatomy, University of Iowa College of Medicine, Iowa City, Iowa
ABSTRACT
The effects of a maternally administered synthetic glucocorticoid, betamethasone, on the levels of mRNA for the surfactant proteins
SP-A, SP-B, and SP-C and on the levels of SP-A protein were investigated
in day 27 gestational age rabbit fetal lung tissue. Betamethasone administration to the pregnant rabbit caused approximately a twofold increase in
the fetal lung level of SP-A protein and a threefold increase in fetal lung
SP-A mRNA levels when compared to levels in fetuses obtained from saline-treated or uninjected animals. SP-B mRNA was increased fourfold in
fetal lung tissue obtained from glucocorticoid-treated pregnant does when
compared to levels in fetuses of uninjected pregnant does. However, SP-B
mRNA levels in fetal lung tissue from saline-injected controls were also
significantly elevated, -twofold, when compared to fetal lung SP-B mRNA
levels in the uninjected control condition. SP-C mRNA levels in lung tissue
of fetuses from both saline-injected and betamethasone-injected pregnant
does were increased similarly, -twofold, over SP-C mRNA levels in fetal
lung tissue obtained from uninjected control does. These data are suggestive that betamethasone treatment increases fetal lung SP-A and SP-B
mRNA levels and that maternal stress alone can increase the expression of
SP-B and SP-C mRNA in rabbit fetal lung tissue. Using in situ hybridization, SP-A mRNA was shown to be present primarily in alveolar type I1
cells in fetuses of control and saline-injected does. However, SP-A mRNA
was easily detected in both alveolar type I1 cells and bronchiolar epithelial
cells of rabbit fetal lung tissue following maternal betamethasone treatment. In contrast, SP-B and SP-C mRNA were present only in alveolar type
I1 cells of lung tissue obtained from fetuses of control, saline, or betamethasone-treated does. Thus maternal administration of glucocorticoids increased SP-A protein as well as SP-A and SP-BmRNA levels in rabbit fetal
lung tissue. SP-A mRNA was localized to both alveolar type I1 cells and in
smaller amounts in bronchiolar epithelial cells of rabbit fetal lung tissue.
However, SP-B and SP-C mRNA were detected only in alveolar type I1
cells. 0 1993 Wiley-Liss, Inc.
Key words: Fetal lung, Surfactant proteins, Glucocorticoids, mRNA, In
situ hybridization
Alveolar type I1 cells synthesize and secrete surfactant, a lipoprotein substance that reduces surface tension at the air-alveolar interface. Surfactant is stored
within the alveolar type I1 cell in lamellar bodies, organelles that are released from the cell by exocytosis.
Surfactant is comprised of glycerophospholipids (-80%
by weight) and proteins (-10% by weight) (Weaver and
Whitsett, 1991). Dipalmitoylphosphatidylcholine, the
most abundant glycerophospholipid component of surfactant, is thought to be primarily responsible for
achieving the surface tension lowering properties of
surfactant. However, the surfactant-associated proteins greatly facilitate the spreading of surfactant glycerophospholipids on a n air-liquid interface (Weaver
and Whitsett, 1991).
0 1993 WILEY-LISS, INC
SP-A, a 35,000 Dalton sialoglycoprotein, is the most
abundant surfactant-associated protein (Weaver and
Whitsett, 1991; Ng et al., 1983). SP-A protein and
mRNA have been localized to alveolar type I1 cells and
bronchiolar epithelial cells in human, rat, and rabbit
lung tissue (Phelps and Floros, 1988; Auten et al.,
1990; Snyder, 1991). SP-A binds calcium and mediates
the transformation of secreted lamellar bodies into tubular myelin, a form of surfactant that is thought to be
Received October 22, 1992; accepted June 30, 1993.
Address reprint requests to Jeanne M. Snyder, Department of Anatomy, University of Iowa College of Medicine, Iowa City, IA 52245.
366
P.L. DURHAM ET AL.
the precursor of the monolayer of surfactant that covers the lung aqueous lining layer (Hawgood e t al.,
1985). It has been shown that exogenous SP-A regulates surfactant secretion and the synthesis of surfactant glycerophospholipids by alveolar type I1 cells, possibly via a specific receptor (Thakur et al., 1986;
Kuroki e t al., 1988a,b). SP-A has also been shown to
regulate phagocytosis by alveolar macrophages (Tenner et al., 1989). Two hydrophobic, lower molecular
weight surfactant-associated proteins, SP-B and SP-C,
have also been isolated and characterized (Glasser et
al., 1987, 1988). SP-B mRNA has been detected, using
in situ hybridization, in alveolar type I1 cells and bronchiolar epithelial cells in human, rat, and rabbit lung
tissue (Phelps and Floros, 1988, 1991; WohlfordLenane and Snyder, 1992). SP-C mRNA is only detected in alveolar type I1 cells in rabbit lung tissue
(Wohlford-Lenane et al., 1992). SP-B and SP-C have
been shown to greatly increase the rate of spreading of
surfactant glycerophospholipids; SP-A may interact
with SP-B and SP-C and further facilitate this function
(Takahashi and Fujiwara, 1986; Hawgood et al., 1987).
SP-D, a collagenous surfactant-associated protein with
structural similarity to SP-A, has also recently been
identified; however, its function is presently unknown
(Persson et al., 1988).
The maturation of the fetal lung alveolus is characterized by the differentiation of two cell types, alveolar
type I and alveolar type I1 epithelial cells (Snyder et
al., 1985). SP-A and its mRNA are induced coincident
with or just prior to alveolar type I1 cell differentiation
(Snyder 1991). SP-A mRNA has been shown by in situ
hybridization to be expressed in differentiated alveolar
type I1 cells and in bronchiolar epithelial cells in late
fetal and adult rabbit lung tissue (Auten et al., 1990;
Wohlford-Lenane and Snyder, 1992). In contrast, SP-B
and SP-C mRNA are detected in fetal lung tissue before type I1 cell differentiation and before the commencement of surfactant glycerophospholipid synthesis (Snyder, 1991). Using in situ hybridization, it has
been shown t ha t both SP-B and SP-C mRNA are expressed in prealveolar epithelial cells prior to type I1
cell differentiation (Wohlford-Lenane et al., 1992;
Wohlford-Lenane and Snyder, 1992). Whereas SP-B
mRNA is localized in both differentiated alveolar type
I1 cells and bronchiolar epithelial cells late in gestation, SP-C mRNA in late fetal and adult rabbit lung
tissue is restricted to differentiated alveolar type I1
cells.
It has been shown in intact animal models that maternally administered glucocorticoids increase fetal
lung phospholipid synthesis and secretion and accelerate the morphological development of the fetal lung
(Ballard, 1986).The induction of the surfactant-associated proteins in developing lung tissue is also hormonally regulated (Mendelson and Boggaram, 1989).
Phelps et al. (1987, 1991) found that dexamethasone
treatment of pregnant rats in vivo caused a n increase
in SP-A protein content as well as an increase in SP-A
mRNA and SP-B mRNA levels in fetal and postnatal
rat lung tissue. The effects of dexamethasone treatment of the pregnant rat on the levels of SP-A and
SP-B protein and SP-A, SP-B and SP-C mRNA in fetal
rat lung tissue have been described in two additional
studies (Schellhase and Shannon, 1991; Shimizu et al.,
1991). Connelly et al. (1991) recently reported that fetal lung mRNA levels for SP-A and SP-B were increased following betamethasone administration to the
pregnant rabbit but that SP-C mRNA levels were decreased 24 hours after glucocorticoid treatment.
The goal of the present study was to characterize the
effects of a maternally administered synthetic glucocorticoid, betamethasone, on SP-A protein levels and
on the induction and localization of mRNA for SP-A,
SP-B, and SP-C in rabbit fetal lung tissue. Rather than
pooling fetuses from each litter, we performed these
analyses on individual fetuses in order to ascertain the
individual variation in these developmental events.
We also included two control conditions in our study,
i.e., saline injection (vehicle control) as well as a n uninjected, unhandled control in order to determine the
effects of maternal stress on fetal lung development.
We have shown in a previous study that maternal glucocorticoid administration accelerates the formation of
alveolar airspaces within rabbit fetal lung tissue and
has a modest stimulatory effect on surfactant phospholipid synthesis (Snyder et al., 1992). In the present
study, we report that maternally administered glucocorticoids significantly increase the levels of mRNA for
SP-A and SP-B, as well a s the levels of SP-A protein, in
rabbit fetal lung tissue. Lung SP-C mRNA levels were
not significantly increased in fetuses of betamethasone-treated does when compared to levels in fetuses of
saline-injected does (vehicle controls). Using in situ hybridization, SP-A mRNA was observed in alveolar type
I1 cells and in very small amounts in bronchiolar epithelial cells of fetal lung tissue. Following betamethasone treatment of the pregnant doe, SP-A mRNA in
bronchiolar epithelial cells was more easily and consistently detectable. However, SP-B and SP-C mRNA
were detected only in alveolar type I1 cells of rabbit
fetal lung tissue obtained from either control, salineinjected, or betamethasone-treated pregnant does.
MATERIAL AND METHODS
Animals
Pregnant New Zealand white rabbits obtained from
a local rabbitry (Knapp Creek, Amana, IA) were maintained in a n animal care facility for several days. To
examine the effects of maternal administration of glucocorticoids on fetal lung tissue, pregnant does were
injected on days 25 and 26 of pregnancy with betamethasone (200 pg/kg, Celestone Soluspan, Schering
Corporation, Kenilworth, NJ). Controls were left either
untreated or were injected with a n equal volume of the
vehicle (saline control) on days 25 and 26 of pregnancy.
All animals were sacrificed on day 27 of pregnancy by
the rapid administration of sodium pentobarbital (500
mg) into a n ear vein. After removal of the fetuses from
the uterus, their lungs were dissected free, and the
upper, middle, and lower lobes of the right lung were
excised and immediately frozen in liquid nitrogen. The
betamethasone experiments were repeated at least
four times for each analysis. Tissues were stored a t
- 70°C until utilized for biochemical and morphological
analyses. All analyses, biochemical and morphological,
were performed on lung tissues from individual fetuses. A total of 15 fetuses from control does, 14 fetuses
from saline-treated does, and 30 fetuses from be-
GLUCOCORTICOID REGULATION OF SP-A, SP-B, AND SP-C mRNA
tamethasone-treated does were used to perform this
study.
lrnrnunoblot Analyses
Fragments of rabbit fetal lung tissue were homogenized in distilled water containing a protease inhibitor,
phenylmethylsulfonyl fluoride (1 mM), and a 600 x g
supernatant was prepared. Protein concentrations
were measured in the supernatant using the method of
Lowry et al. (1951). Seventy-five pg of lung homogenate protein were separated on a sodium dodecyl sulfate (SDS)-polyacrylamide gel (lo%), then transferred
to Immobilon membranes (Millipore Corporation, Bedford, MA) as described previously (Snyder and Mendelson, 1987). Unoccupied sites on the membranes were
blocked by incubating in buffer A [bovine serum albumin (BSA, 5% wiv), sodium chloride (NaCl, 0.15 M),
and Nonidet P-40 (0.2%, v/v) in Tris-HC1 (0.01 M, pH
7.4)] overnight at 4°C. The membranes were then incubated in goat antirabbit SP-A (1 pg/ml) in buffer A
for 1 hour at room temperature. The membranes were
rinsed in buffer B [Tris-HC1 (0.01 M, pH 7.4), NaCl
(0.15 M), Nonidet P-40 (0.2%, v/v), sodium deoxycholate (0.25%, w/v), and SDS (0.1%,w/v)], then incubated in rabbit antigoat IgG (1 pg/ml, Organon
Teknika-Cappel, West Chester, PA) for 1hour at room
temperature. Membranes were rinsed in buffer B, then
incubated in buffer A containing [ 1251]-labeledProtein
A, 1 x lo6 cpndml (Amersham Corporation, Bedford,
MA) for 1 hour at room temperature. The membranes
were rinsed in buffer B, then incubated in Tris-HC1
(0.01 M, pH 7.4) and NaCl (0.15 M) overnight at 4°C.
Immunoreactive bands were detected by exposing the
membrane to Kodak XAR-2 film with a n intensifier
screen (Lighting Plus; Du Pont, Wilmington, DE) at
-70°C and quantified using densitometry. Densitometric data were normalized such that the mean of the
control values was equal to one.
RNA Isolation
Fetal lung tissue, -50 mg, was homogenized in 1.2
ml RNAzol (Cinna/Biotecx Laboratories International,
Friendswood, TX) (Chomczynski and Sacchi, 1987).
The homogenate was transferred to a microfuge tube
containing 120 pl of chloroform, shaken vigorously for
10 seconds, and then incubated on ice for 5 minutes.
After centrifugation at 4”C, the resultant upper aqueous phase was transferred to a fresh microfuge tube,
600 p1 of ice-cold isopropyl alcohol was added, the solution mixed by inversion, and then stored at -20°C for
45 minutes. Following centrifugation in a microfuge (>
10,000 x g) for 15 minutes, the precipitated total RNA
was washed two times with 75% ethanol and pelleted
by centrifugation after each wash. The pellet was
dried, then resuspended in diethylpropylcarbonate
(DEPCbtreated water, and stored at -20°C. RNA was
quantitated by determining the absorbance at 260 nm.
Ten pg of total RNA from each sample were separated
by electrophoresis on a 1.2% agarose-5% formaldehyde
gel (Maniatis et al., 1982). The RNA was then transferred to a Nytran membrane (Schleicher & Schuell,
Keene, NH) by capillary transfer in sodium phosphate
buffer (25 mM, pH 6.5). RNA was baked onto the membrane by heating a t 80°C for 1 hour.
367
DNA Probes
The 1.9 kb rabbit SP-A cDNA probe was a kind gift
of Drs. C.R. Mendelson and V. Boggaram (Dallas, TX;
Boggaram et al., 1988). The 1.8 kb rabbit SP-B cDNA
probe was a kind gift of Dr. F. Possmayer (London,
Ontario; Xu et al., 1989). The 0.5 kb rabbit SP-C cDNA
probe was generated in our laboratory using the reverse transcriptase-polymerase
chain
reaction
(Durham et al., 1991). The rabbit mitochondrial cytochrome oxidase subunit I1 (CO 11) cDNA utilized as
a n internal control in our Northern blots was a kind
gift of Dr. S. Horowitz (Rochester, NY; Horowitz et al.,
1989). Bacterial cells containing the surfactant-associated protein cDNAs in Bluescript (SP-B and SP-C,
Stratagene, La Jolla, CA) or pGEM (SP-A and CO 11,
Promega Corporation, Madison, WI) vectors were
grown overnight in 50 ml of LB medium containing
ampicillin (50 pg/ml). Plasmid DNA was isolated utilizing the Qiagen plasmid DNA preparation method
(Qiagen, Studio City, CA). After digestion of the purified plasmid DNA with the appropriate restriction enzymes, the isolated cDNAs were separated on a n agarose gel (1%) and purified from the gel using a
Geneclean DNA purification kit (BIO 101, La Jolla,
CA).
Northern Blot Analysis
The membranes that contained RNA were prehybridized in a plastic bag for 4 hours at 42°C in 12 ml of
hybridization buffer [BSA (0.2%, w/v), polyvinylpyrrolidone (0.2%, w/v), ficoll (MW 400,000, 0.2%, w/v),
Tris-HC1 (0.05 M, pH 7.41, sodium pyrophosphate
(0.1%, w/v), SDS (l.O%, w/v), dextran sulfate (10%)
w/v), formamide (50%, v/v), NaCl (1M), and heat-denatured, herring sperm DNA (0.1%, w/v)]. cDNA
probes were radiolabeled to a specific activity of > 1 x
lo9 c p d p g using [32P]-CTP (Amersham, 3,000 Ci/
mmol) and a random primer labeling kit (Boehringer
Mannheim Biochemicals, Indianapolis, IN). Radiolabeled cDNA probe at a concentration of 1 x lo6 cpndml
was mixed with hybridization buffer, drawn into a syringe, and injected into the bag. Hybridization was performed overnight at 42°C with gentle agitation. The
blots were then washed two times in 250 ml of 2 x SSC
[NaCl (0.3 M) and sodium citrate (0.03 M)] at room
temperature for 5 minutes, two times in 250 ml of 2 x
SSC and SDS (1%) w/v) at 65°C for 30 minutes, and
finally in 250 mlO.1 x SSC a t room temperature for 15
minutes. The blots were exposed to X-ray film with a n
intensifier screen at - 70°C for 4-24 hours. Radioactive
bands were quantitated using densitometry. Following
hybridization and exposure to X-ray film, the [32P]-labeled cDNA probe was removed from the membrane by
incubating the membrane in 250 ml of Tris-HCl(5 mM,
pH 8.01, EDTA (0.2 mM), sodium pyrophosphate
(0.05%,w/v), polyvinyl pyrrolidone (0.002%, w/v, MW
40,000),BSA (0.002%, w/v), and ficoll(0.002%, w/v) for
2-4 hours a t 65°C. The membranes were then hybridized with another [32Pl-labeledsurfactant cDNA probe.
Membranes were stripped and reprobed up to three
times without appreciable loss of total RNA from the
membrane. To compensate for RNA gel loading artifacts, all RNA-containing membranes were probed
with the [32P]-radiolabeledrabbit mitochondrial CO I1
cDNA. The levels of CO I1 mRNA in the fetal lung
368
P.L. DURHAM ET AL.
tissue were not altered by saline-injection or betamethasone-injection of the pregnant doe. The densometric data for the surfactant-associated proteins were
first expressed as the ratio of the absorbance of the
mRNA band for surfactant-associated protein to the
absorbance for the CO I1 mRNA band in the same sample and on the same membrane. These adjusted densometric data were then normalized such that the mean
of the control values was equal to one.
In Situ Hybridization
Purified SP-A cDNA in a pGEM plasmid that had
been digested with Puu I1 and Hind I11 was used a s a
template for the synthesis of the sense or antisense
SP-A cRNA transcripts, respectively. The Bluescript
SK vector containing the SP-B cDNA insert was purified, then linearized with CZa I1 and Not I and utilized
to synthesize the sense and antisense SP-B cRNA transcripts, respectively. A BssH I1 digest of the purified
SP-C cDNA in a Bluescript plasmid was utilized in the
synthesis of the sense and antisense SP-C cRNA
strands. RNA transcripts were synthesized using [3H]CTP (4.82 x lo7 dpm/pg) and [3H]-UTP (5.83 x lo7
d p d p g , both New England Nuclear Corp., Boston,
MA). Labeling the cRNA was performed using a RNA
transcription kit (Gemini Riboprobe System 11,
Promega, Madison, WI), 1pg of digested plasmid DNA,
and the appropriate RNA polymerase. The radiolabeled cRNA probes were hydrolyzed to -200 bp fragments as described by Cox et al. (1984).
The methods used for in situ hybridization were performed as described previously (Wohlford-Lenane et
al., 1992; Wohlford-Lenane and Snyder, 1992). Briefly,
6-pm-thick frozen sections of the fetal lung tissue were
prepared, then mounted on glass slides. Sections of
lung tissue from two fetuses from the same litter per
condition and from the control, saline-injected, and betamethasone condition were placed on the same slide.
Sections were fixed for 20 minutes in freshly prepared
paraformaldehyde (4%, w/v, pH 7.4), rinsed three times
in phosphate-buffered saline (PBS), pH 7.4, and dehydrated through a graded ethanol series. The sections
were then digested in Pronase E (0.125 mg/ml, Sigma,
St. Louis, MO) in Tris-HC1 (50 mM, pH 7.5), EDTA (5
mM) buffer. The slides were rinsed in PBS, pH 7.4,
followed by a brief immersion in triethanolamine
buffer (TEA, 0.1 M, pH 8.0), incubated for 10 minutes
in TEA buffer that contained acetic acid (0.25%, v/v)
and rinsed in 2 x SSC before being dehydrated
through a n ethanol series.
The sections were hybridized overnight at 50°C in 35
pl of hybridization buffer [NaCI (300 mM), Tris-HC1
(10 mM, pH 8.0), EDTA (1mM), formamide (50% v/v),
1 x Denhardt’s solution, dextran sulfate (lo%, w/v),
and yeast tRNA (0.28 mg/ml)] that contained the appropriate [3H]-probe. The hybridization buffer for SP-A
and SP-C contained 4.6 x lo3 cpm/ml, whereas that for
SP-B contained 11.4 x lo3 cpm/pl. Following hybridization, the slides were rinsed in 4 x SSC, then incubated for 30 minutes at 37°C in Tris-HC1 (10 mM, pH
8.0), EDTA (1 mM), NaCl (500 mM) buffer that contained RNase A (20.0 pg/ml) and RNase T1 (3.0 U/ml).
After dehydration, the slides were coated with NTB-2
liquid emulsion (Kodak, Rochester, NY) diluted 1:l
with distilled water, air dried, sealed in a black slide
*
2.501
2.00
-
u
c
3
O
$
1.50-
al
.-
1.00-
?!
0.50 0.00
control
saline
betarnethasone
n=15
n=14
n=30
Fig. 1. The effect of betamethasone treatment of the pregnant doe on
the relative amount of SP-A protein in rabbit fetal lung tissue. SP-A
protein content was evaluated by Western blot analysis and densitometry of the reactive bands. The data, which are expressed relative
to control levels (mean = l), represent mean 2 the standard error of
the mean. Betamethasone treatment of the pregnant doe caused a
significant increase in the content of fetal lung SP-A protein when
compared to saline-injected or uninjected control levels (asterisk, P <
0.05). The levels of SP-A protein in fetal lung tissue obtained from
saline-injected pregnant does were not different from control levels.
box containing dessicant, and exposed for 1-5 weeks at
4°C. The slides were then immersed in D-19 developer
(Kodak) for 3 minutes, rinsed in distilled water, immersed in Rapid Fixer (Kodak) diluted 1 : l with distilled water for 3 minutes, and rinsed in tap water for
5 minutes. The slides were then briefly stained in hematoxylin, dehydrated, mounted, and examined by two
investigators.
Data Analysis
*
Data are expressed as the mean the standard error
of the mean. Statistical comparisons were made using
Student’s t-test (unpaired) and a n Epistat software
package.
RESULTS
SP-A
The injection of betamethasone into the pregnant doe
on days 25 and 26 of gestation resulted in a doubling of
the relative amount of SP-A protein present in the fetal
lung tissue at day 27 of gestation (Fig. 1).Lung SP-A
protein levels in fetuses from the vehicle control (saline-injected) condition were not changed when compared to the lung SP-A protein levels in fetuses from
the uninjected controls. Betamethasone injection also
resulted in a n induction of SP-A mRNA in fetal lung
tissue, a n approximately threefold increase when compared to the levels of SP-A mRNA in fetal lung tissue
obtained from control pregnant does (Figs. 2,3). Saline
injection had no significant effect on fetal lung SP-A
mRNA levels when compared to levels in fetuses of
uninjected controls.
We found a significant, positive correlation (P <
0.01) between the relative amount of SP-A mRNA and
SP-A protein in lung tissue from individual fetuses in
the control and saline-injected conditions (Fig. 4A). In
contrast, no correlation was found between the relative
amount of SP-A mRNA and SP-A protein in individual
369
GLUCOCORTICOID REGULATION OF SP-A, SP-B, AND SP-C mRNA
SP-A
SP-B
SP-c
co 11
C S S B B B C S S B
Fig. 2. Representative autoradiographs of Northern blot analyses of rabbit fetal lung mRNA levels of
the surfactant-associated proteins, SP-A, SP-B, and SP-C and of a control mRNA, mitochondria1 cytochrome oxidase subunit I1 (CO 11).Ten pg of total lung RNA from day 27 gestational age rabbit fetuses
obtained from control (C), saline-injected (S), or betamethasone-injected (B) pregnant does were separated on a 1.5%agarose gel, transferred to nylon membranes, and hybridized with [32P]-cDNAprobes for
SP-A, SP-B, SP-C, and the internal standard, CO 11, as indicated.
fetuses from the betamethasone condition (P < 0.64)
(Fig.4B). For example, in several fetuses obtained from
betamethasone-treated does, SP-A mRNA levels were
increased as much as eightfold, whereas SP-A protein
levels were less than doubled (Fig. 4B).
Using in situ hybridization, SP-A mRNA was detected in control, day 27 gestational age fetal lung tissue in epithelial cells that had the morphological characteristics of alveolar type I1 cells (Fig. 5A, B). A small
amount of SP-A mRNA hybridization was detected in
some of the bronchiolar epithelial cells in the control
tissue (Fig. 5A,B). Similarly, SP-A mRNA was also localized in alveolar type I1 cells and in smaller amounts
in bronchiolar epithelial cells in day 27 gestational age
fetal lung tissue obtained from saline-injected pregnant does (Fig. 5C,D). SP-A mRNA was always observed in both alveolar type I1 cells and in bronchiolar
epithelial cells of the day 27 gestational age fetal lung
tissue obtained from betamethasone-treated pregnant
does (Fig. 5E,F). The fetal lung tissue in the betamethasone condition was also morphologically more
mature, i.e., the tissue had thinner alveolar walls and
more lumenal space than fetal lung tissue from the
control or saline conditions, as reported previously
(compare Fig. 5A,C,E) (Snyder et al., 1992). When the
day 27 gestational age fetal lung tissue was hybridized
with a r3H]-SP-A sense cRNA probe, no hybridization
was observed (Fig. 5G,H).
5.001
*t
4.00
L
C
3
0
k
T
3.00 -
W
.c
0
-
2.00 -
P
T
1.00-
1
0.00
control
saline
betamethasone
n=14
n=10
n=26
Fig. 3. The effects of betamethasone treatment of the pregnant doe
on the relative amount of SP-A mRNA in rabbit fetal lung tissue.
SP-A mRNA levels were evaluated by Northern blot analysis and
densitometry of the reactive bands. To control for RNA-loading artifacts, each Northern blot was also analyzed using a rabbit CO I1
cDNA probe and the data expressed as the ratio of the absorbance of
the surfactant-associated protein mRNA band to the absorbance of
the CO I1 mRNA band in the same lane. The data, which are expressed relative to control levels (mean = I), represent mean _i the
standard error of the mean. Betamethasone treatment of the pregnant
doe resulted in a significant increase in the relative amount of fetal
lung SP-A mRNA when compared to the control values (asterisk, P <
0.05) and the saline-injected controls (dagger, P < 0.05). The fetal
lung SP-A mRNA levels in the vehicle controls (saline-injected) were
not different from levels in the uninjected control condition.
370
A
P.L. DURHAM ET AL.
tamethasone-treated pregnant does, SP-B mRNA was
only detected in alveolar type I1 cells (Fig. 7E,F). When
the day 27 gestational age fetal lung tissue was hybridized with a [3Hl-SP-B sense cRNA probe, no hybridization was detected (Fig. 7G,H).
8.00
A = control
a
z
E
0
= saline
6.00
.c
SP-c
c
5
4.001
~
0.004
0.00
I
1.00
2.00
3.00
4.00
5.00
relative amount of protein
IY
2
-F
%
"'""1
'
W
I
Y
c
5
4.00-
w
0
5
0
.>
c
0
-
e
2.00-
I
0.00 -I
0.00
W
W
6.001
W
~
=
1.
=m
W
W .
W
W
W
B
W
W
1
1.00
2.00
3.00
4.00
5.00
relative amount of protein
The relative amount of SP-C mRNA in the day 27
gestational age fetal lung tissue was increased -twofold by betamethasone injection of the pregnant doe
when compared to the levels in lung tissue from fetuses
of the uninjected, control does (Figs. 2, 8). However,
lung SP-C mRNA levels in fetuses obtained from the
saline-injected control does were also increased twofold
when compared to the lung SP-C mRNA levels in fetuses of the uninjected control does. The stimulatory
effect of either saline injection or betamethasone injection of the pregnant doe on fetal lung SP-C mRNA
levels was statistically significant ( P < 0.05).
SP-C mRNA was detected using in situ hybridization
in alveolar type I1 cells of day 27 gestational age fetal
lung tissue obtained from control pregnant does (Fig.
9A,B). SP-C mRNA was not detected in airway epithelial cells in the control tissue (Fig. 9A,B). A similar
pattern of SP-C mRNA localization was observed in the
day 27 gestational age fetal rabbit lung tissue obtained
from both saline and betamethasone-treated pregnant
does (Fig. 9C, D, E, F). When the fetal lung tissue was
hybridized with a L3H1-SP-C sense cRNA probe, no hybridization was detected (Fig. 9G,H).
DISCUSSION
Fig. 4. Correlation between the relative amount of SP-A protein and
SP-A mRNA in day 27 gestational age rabbit fetal lung tissue. A.
Correlation between lung SP-A mRNA and SP-A protein levels in
individual fetuses of control (A)and saline-injected ( 0 )pregnant does.
There was a significant correlation ( P < 0.01)between SP-A protein
and SP-A mRNA levels in these tissues. B. Correlation between SP-A
mRNA and SP-A protein levels in individual fetuses of betamethasone-treated pregnant does. No significant correlation between SP-A
mRNA and SP-A protein levels was found in these tissues (P< 0.64).
SP-8
The relative amount of SP-B mRNA in the lung tissue from day 27 gestational age fetuses of betamethasone-treated pregnant does was increased fourfold
when compared to lung SP-B mRNA levels in fetuses
from the uninjected control does and was also significantly increased over lung SP-B mRNA levels in fetuses of the saline-injected control does (-twofold)
(Figs. 2, 6). SP-B mRNA levels in the lung tissue of
fetuses obtained from the saline-injected does (vehicle
control) were increased significantly over SP-B mRNA
levels in lung tissue obtained from fetuses of uninjected
control does (-twofold).
SP-B mRNA, localized by in situ hybridization, was
present in lung tissue obtained from day 27 gestational
age fetuses of control pregnant does in epithelial cells
with the morphological characteristics of alveolar type
I1 cells (Fig. 7A, B). No SP-B mRNA was detected in
bronchiolar epithelial cells of the control fetal lung tissue (Fig. 7A,B). SP-B mRNA had a similar localization
in lung tissue obtained from day 27 gestational age
fetuses of saline-injected pregnant does (Fig. 7C,D).
Likewise, in lung tissue obtained from fetuses of be-
We found that betamethasone treatment of the pregnant doe caused a significant increase in the SP-A protein content (twofold) and SP-A mRNA content (threefold) of day 27 gestational age rabbit fetal lung tissue
when compared to the SP-A protein and mRNA content
of fetal lung tissue obtained from either untreated or
saline-injected pregnant does. Connelly et al. (1991)
recently reported a n eightfold increase in SP-A mRNA
levels in day 27 day gestational age pooled rabbit fetal
lung tissue following maternal treatment of the pregnant doe with betamethasone (150 pglkg, administered
on day 26 of gestation) when compared to vehicle controls. Phelps et al. (1987) have observed increased levels of SP-A protein and translatable SP-A mRNA in
pooled fetal lung tissues obtained from dexamethasone-treated (200 pglkg, 24 hours) pregnant rats when
compared to vehicle controls, although these effects
were not quantitated. Shimizu et al. (1991) reported
that both pre- and postnatal lung SP-A protein content
were increased following dexamethasone administration (200 pglkg, 24 hours) to the pregnant rat, although
the effects were less than twofold when compared to
vehicle controls. Schellhase and Shannon (1991) demonstrated that SP-A protein levels were significantly
increased (-two- to threefold when compared to vehicle
controls) in day 17 and day 19 gestational age fetal
lung tissue from pooled litters following 3 days of dexamethasone treatment (1,000 pg/kg, 72 hours) of the
pregnant rat. Stimulatory effects of dexamethasone on
SP-A mRNA levels were observed in the day 17 gestational age fetal lung tissue; however, no consistent positive effect of dexamethasone on the expression of SP-A
mRNA was observed in day 19 gestational age r at fetal
GLUCOCORTICOID REGULATION OF SP-A, SP-B, AND SP-C mRNA
Fig. 5. In situ hybridization of SP-A mRNA in day 27 gestational
age rabbit fetal lung tissue. Asterisks indicate the lumen of a bronchiole; arrows indicate representative alveolar type I1 cells. All micrographs are of sections that were exposed autoradiographically for
the same interval and are the same magnification. Bar, 100 p. A, B.
Brightfield (A) and corresponding darkfield (B) micrographs of fetal
lung tissue, obtained from a control pregnant doe, that was hybridized
with a 13H]-SP-A antisense cRNA probe. SP-A mRNA was detected
primarily in alveolar type I1 cells but a small amount of SP-A mRNA
was also present in bronchiolar epithelial cells. C, D. Brightfield (C)
and corresponding darkfield (D) micrographs of fetal lung tissue, obtained from a saline-injected pregnant doe, that was hybridized
371
with a [3Hl-SP-A antisense cRNA probe. SP-A mRNA was detected
primarily in alveolar type I1 cells but was also present in small
amounts in bronchiolar epithelial cells. E, F. Brightfield (E) and corresponding darkfield (F) micrographs of fetal lung tissue, obtained
from a betamethasone-treated pregnant doe, that was hybridized with
a [3Hl-SP-Aantisense cRNA probe. SP-A mRNA was detected in both
alveolar type I1 cells and bronchiolar epithelial cells. Arrowheads
indicate SP-A mRNA hybridization present in bronchiolar epithelial
cells. G , H. Brightfield ( G ) and corresponding darkfield (H) micrographs of fetal lung tissue, obtained from a saline-treated pregnant
doe, that was hybridized with a r3H1-SP-A sense cRNA. No hybridization was detected.
372
P.L. DURHAM ET AL.
*t
5.00 7
4.00 c
c
3
*
3.00-
*
0
:
2.00-
._
T
?!
1 .oo-
T
0.00
control
saline
betarnethasone
n=14
n=10
n=26
Fig. 6. Effects of betamethasone treatment of the pregnant doe on
the relative amount of SP-B mRNA in rabbit fetal lung tissue. SP-B
mRNA levels were evaluated by Northern blot analysis and densitometry of the reactive bands. To control for RNA loading artifacts,
each Northern blot was also analyzed using a rabbit CO I1 cDNA
probe and the data expressed as the ratio of the absorbance of the
surfactant-associated protein mRNA band to the absorbance of the
CO I1 mRNA band in the same lane. The data, which are expressed
relative to control levels (mean = l), represent mean the standard
error of the mean. Betamethasone treatment of the pregnant doe resulted in a significant increase in the relative amount of fetal lung
SP-B mRNA when compared to the uninjected control (asterisk, P <
0.01) and saline-injected condition values (dagger, P < 0.01). Saline
injection of the pregnant doe also resulted in a significant increase in
fetal lung SP-B mRNA levels when compared to levels in fetuses of
uninjected control pregnant does (asterisk, P < 0.01).
*
lung tissue (Schellhase and Shannon, 1991). In in vitro
studies, it has been observed that glucocorticoids dramatically increase the SP-A protein and SP-A mRNA
content of day 21 rabbit fetal lung explants after 3 days
in culture (Mendelson et al., 1986). Thus, together
these data are suggestive that glucocorticoids administered to pregnant rats or rabbits or the in vitro exposure of fetal lung tissue to glucocorticoids results in a
significant increase in fetal lung SP-A mRNA and protein content.
SP-A mRNA was easily detected in alveolar type I1
cells and a small amount of SP-A mRNA was also consistently present in bronchiolar epithelial cells of day
27 gestational age fetuses obtained from betamethasone-treated pregnant does. In fetal tissue obtained
from saline injected or control fetuses, SP-A mRNA
was primarily restricted to alveolar type I1 cells, although small amounts of SP-A mRNA were detected in
bronchiolar epithelial cells. SP-A mRNA has previously been detected using in situ hybridization in alveolar type I1 cells of human and rabbit adult lung
tissue (Phelps and Floros, 1988; Auten et al., 1990). In
addition, Auten et al. (1990) have also reported that
SP-A mRNA is present in bronchiolar epithelial cells of
human and rabbit lung tissue and is first detectable in
bronchiolar epithelial cells on day 31 of gestation in the
rabbit fetus. We recently have reported similar observations (Wohlford-Lenane and Snyder, 1992). In the
present study, we observed that the SP-A mRNA in
bronchiolar epithelial cells of day 27 gestational age
fetal lung tissue obtained from betamethasone-treated
pregnant does was consistently and more easily detectable than the SP-A mRNA from the control tissues.
Our in situ hybridization data are suggestive that the
increase in SP-A mRNA levels observed in rabbit fetal
lung tissue in response to maternal glucocorticoid administration may be due to an increase in the number
of alveolar type I1 cells and/or an increase in the cellular levels of SP-A mRNA per alveolar type I1 cell. Because the levels of SP-A mRNA in bronchiolar epithelial cells appear to be much lower than in alveolar type
I1 cells and because the number of alveolar type I1 cells
in the lung is much greater than the number of bronchiolar epithelial cells, we speculate that the effect of
glucocorticoids on SP-A mRNA levels are manifested
primarily in the alveolar type I1 cell. However, in order
to directly address this issue, it will be necessary to
perform quantitative in situ hybridization to determine the relative amounts of SP-A mRNA in the two
lung epithelial cell types.
Interestingly, we found that betamethasone treatment of the pregnant doe resulted in a threefold induction of SP-A mRNA levels but only a twofold increase
in the SP-A protein content of fetal lung tissue when
compared to levels in fetal lung tissue obtained from
either untreated or saline-injected control does. In contrast to previous studies that addressed glucocorticoid
regulation of surfactant-associated protein mRNA levels using pooled fetal lung tissues from a litter, we
analyzed SP-A mRNA and SP-A protein levels in individual fetuses. Therefore, we were able to further examine the discrepancy between the increase in SP-A
protein levels (-twofold) and the increase in SP-A
mRNA levels (-threefold) by performing a regression
analysis of these parameters in individual fetuses.
Lung SP-A mRNA and protein levels were very well
correlated (P < 0.01) in fetuses of untreated control
pregnant does or in fetuses of saline-injected pregnant
does. In contrast, SP-A mRNA and protein levels in
fetuses of betamethasone-treated pregnant does were
not significantly correlated (P < 0.64). O’Reilly et al.
(1989) observed a similar discrepancy between the effects of glucocorticoids on SP-A mRNA and protein levels in a human lung adenocarcinoma cell line that has
type I1 cell-like properties. These investigators found
that although SP-A mRNA levels were increased in the
dexamethasone-treated cells, intracellular SP-A protein content remained unaltered or was decreased
(O’Reilly et al., 1989). It has previously been shown
that glucocorticoids increase the rate of transcription of
the SP-A gene and also regulate SP-A mRNA half-life
in human fetal lung tissue (Boggaram et al., 1989). Our
results are suggestive that glucocorticoids may regulate fetal lung SP-A content at yet another level, i.e.,
by altering the translatability of the SP-A mRNA in
fetal lung tissue.
We found that the level of SP-B mRNA was increased -fourfold in fetal lung tissue obtained from
betamethasone-treated does when compared to SP-B
mRNA levels in control fetal lung tissue but was increased -twofold when compared to lung SP-B mRNA
levels in fetuses of saline-injected pregnant does (the
vehicle control). Using a solution hybridization assay,
SP-B mRNA levels were reported to be increased -twofold in day 27 gestational age rabbit fetal lung tissue
when compared to levels in control fetuses (Connelly et
al., 1992). Shimizu et al. (1991) have recently reported
that maternal administration of glucocorticoids to the
GLUCOCORTICOID REGULATION OF SP-A, SP-B, AND SP-C mRNA
Fig. 7. In situ hybridization of SP-B mRNA in day 27 gestational
age rabbit fetal lung tissue. The asterisks indicate the lumen of a
bronchiole; arrows indicate representative alveolar type I1 cells. All of
the presented micrographs are from sections that were exposed autoradiographically for the same interval and are the same magnification. Bar, 100pm. A, B. Brightfield (A) and corresponding darkfield
(B) micrographs of fetal lung tissue, obtained from a control pregnant
doe, that was hybridized with a t3H1-SP-B antisense cRNA probe.
SP-B mRNA was detected in alveolar type I1 cells. C, D. Brightfield
( C )and corresponding darkfield (D) micrographs of fetal lung tissue,
obtained from a saline-injected pregnant doe, that was hybridized
373
with a [3Hl-SP-B antisense cRNA probe. SP-B mRNA was only detected in alveolar type I1 cells. E, F. Brightfield (E) and corresponding
darkfield (F) micrographs of fetal lung tissue, obtained from a betamethasone-treated pregnant doe, that was hybridized with a t3H]SP-B antisense cRNA probe. SP-B mRNA was detected in alveolar
type I1 cells but was not present in bronchiolar epithelial cells. G , H.
Brightfield ( G ) and corresponding darkfield (H) micrographs of fetal
lung tissue, obtained from a betamethasone-treated pregnant doe,
that was hybridized with a [3Hl-SP-B sense cRNA probe. No hybridization was detected.
374
P.L. DURHAM ET AL
5.00 4.00 -
+-
C
3
i
._
-
?!
tent in rat fetal lung tissue following dexamethasone
administration to the pregnant rat. Fisher et al. (1991)
have recently reported that adult rat lung steady-state
SP-B mRNA levels are more markedly increased than
are SP-A mRNA levels following exogenously administered glucocorticoids to adult rats. Thus it appears
that in the fetal rat lung expression of the SP-B gene is
more sensitive to glucocorticoids than the SP-A gene,
whereas in rabbit fetal lung tissue, the SP-A and SP-B
genes are affected in a relatively similar manner by
maternal glucocorticoid administration.
We found that saline-injection of the pregnant doe
caused a statistically significant increase in the relative amount of fetal lung SP-B mRNA when compared
to lung SP-B mRNA levels in fetuses of uninjected control does. We observed in a previous study (Snyder et
al., 1992) that saline injection of the pregnant doe
caused a significant increase in the volume density of
type I1 cells in day 27 gestational age fetal lung tissue.
We hypothesized that the increase in type I1 cell differentiation might be the result of a n elevation in endogenous glucocorticoid levels since Ballard et al.
(1978) have reported that plasma glucocorticoid levels
were increased -70% following saline injection of the
pregnant rabbit. However, it is also possible that other
hormones or regulatory factors that are altered by maternal stress may act to accelerate fetal lung differentiation. Nonetheless, factors associated with maternal
stress significantly increase the expression of SP-B
mRNA in rabbit fetal lung tissue.
SP-B mRNA was detected in alveolar type I1 cells in
day 27 gestational age rabbit fetal lung tissue by use of
in situ hybridization. SP-B mRNA was essentially undetectable in bronchiolar epithelial cells of fetal lung
tissue obtained from control, saline-injected, or betamethasone-injected pregnant does. These data are
suggestive that bronchiolar epithelial cells do not increase their levels of SP-B mRNA in response to maternal administration of glucocorticoids in the fetal
rabbit lung. In contrast, Phelps and Floros (1991) have
recently reported that maternal glucocorticoid treatment increases the level of SP-B mRNA to the same
degree in both alveolar type I1 cells and bronchiolar
epithelial cells of fetal rat lung tissue.
Saline-injection of the pregnant doe caused a twofold
induction of SP-C mRNA in fetal lung tissue. Betamethasone treatment of the pregnant doe also caused
a twofold increase in fetal lung SP-C mRNA content
when compared to lung levels in fetuses of uninjected
controls. However, there was no difference between the
fetal lung SP-C mRNA levels from the betamethasonetreated pregnant does and the levels in the fetal lung
tissue from saline-injected does (vehicle controls).
Schellhase and Shannon (1991) have reported that maternally administered dexamethasone increased the
levels of SP-C mRNA in rat fetal lung tissue when
compared to levels in vehicle controls. In contrast, Connelly et al. (1991) reported that maternal administration of betamethasone to a pregnant doe caused a significant decrease (to -half of control levels) in the level
of SP-C mRNA in day 27 gestational age rabbit fetal
lung tissue. Whitsett and coworkers (OReilly et al.,
1989; Whitsett et al., 1987) have shown that glucocorticoids markedly enhance SP-C mRNA synthesis in human fetal lung tissue explants and in a n adenocarci-
2~ooL13ism
3.00-
0.00
1 .oo
control
saline
betarnethasone
n=14
n=10
n=26
Fig. 8. The effect of betamethasone treatment of the pregnant doe on
the relative amount of SP-C mRNA in rabbit fetal lung tissue. SP-C
mRNA levels were evaluated by Northern blot analysis and densitometry of the reactive bands. To control for RNA loading artifacts,
each Northern blot was also analyzed using a rabbit CO I1 cDNA
probe. The data are expressed as the ratio of the absorbance of the
surfactant-associated protein mRNA band to the absorbance of the
CO I1 mRNA band in the same lane. The data, which are expressed
relative to control levels (mean = l), represent mean -t the standard
error of the mean. Betamethasone treatment of the pregnant doe resulted in a significant increase in the relative amount of fetal lung
SP-C mRNA when compared to control values (asterisk, P < 0.01).
Saline-injection of the pregnant doe also resulted in a significant increase in fetal lung SP-C mRNA levels when compared to uninjected
controls (asterisk, P < 0.01).Levels of mRNA in fetal lung tissue
obtained from saline-injected and betamethasone-injected pregnant
does were not different from each other.
pregnant rat increases fetal lung SP-B protein levels
10-fold on day 19 of gestation but has no effect at day
21 of gestation or postnatally. Phelps e t al. (1991) have
reported a greater than eightfold induction in the level
of SP-B mRNA in lung tissue from day 18 to day 22
gestational age fetuses of dexamethasone-treated
(2,000 pgkg, 24 hours) pregnant rats when compared
to vehicle controls. Similar to the results of Shimizu et
al. (1991), they observed that the effects of dexamethasone on the expression of SP-B mRNA are greatest
early in gestation when the control levels of SP-B
mRNA are very low (Phelps and Floros, 1991). Schellhase and Shannon (1991) also observed a marked increase in the content of SP-B mRNA in fetal lung tissue
following dexamethasone treatment of pregnant rats
early in gestation when compared to vehicle controls.
Thus fetal lung SP-B mRNA levels are increased by
maternal glucocorticoid treatment in both the rat and
rabbit species.
We found that the stimulatory effect of glucocorticoids (relative to vehicle controls) on SP-B mRNA levels in rabbit fetal lung tissue was similar in magnitude
to its effect on SP-A mRNA levels. Connelly et al.
(1991) found that fetal rabbit lung SP-A mRNA was
more sensitive to the stimulatory effect of glucocorticoids. In contrast, Phelps et al. (1991) and Shellhase
and Shannon (1991) found a significantly greater effect
of maternal glucocorticoids on rat lung SP-B mRNA
levels than on SP-A mRNA levels in day 19 gestational
age fetuses when compared to vehicle controls. Shimizu
et al. (1991) also reported that the SP-B protein content
increased to a greater degree than SP-A protein con-
GLUCOCORTICOID REGULATION OF SP-A, SP-B, AND SP-C mRNA
Fig. 9. In situ hybridization of SP-C mRNA in day 27 gestational
age rabbit fetal lung tissue. Asterisks indicate the lumen of a bronchiole; arrows indicate alveolar type I1 cells. All of the micrographs
are from sections that were exposed autoradiographically for 5 weeks,
except G and H, which were exposed for 3 weeks. All micrographs are
the same magnification. Bar, 100 Fm. A, B. Brightfield (A) and the
corresponding darkfield (B) micrographs of fetal lung tissue, obtained
from a control pregnant doe, that was hybridized with a 13Hl-SP-C
antisense cRNA probe. SP-C mRNA was present in alveolar type I1
cells. C, D. Brightfield (C) and corresponding darkfield (D) micro-
375
graphs of fetal lung tissue, obtained from a saline-injected pregnant
doe, that was hybridized with a f3HI-SP-C antisense cRNA probe.
SP-C mRNA was only present in alveolar type I1 cells. E, F. Brightfield (El and corresponding darkfield (F) micrographs of fetal lung
tissue, obtained from a betamethasone-treated pregnant doe, that was
hybridized with a [3H]-SP-C-antisense cRNA. SP-C mRNA was only
detected in alveolar type I1 cells. G, H. Brightfield (G)and corresponding darkfield (H) micrographs of fetal lung tissue, obtained from a
control pregnant doe, that was hybridized with a [3Hl-SP-C sense
cRNA probe. No hybridization was detected.
376
P.L. DURHAM ET AL.
noma cell line. Likewise, Liley et al. (1989) also found
a stimulatory effect of glucocorticoids on SP-C mRNA
levels in human fetal lung explants. The effect of glucocorticoids on lung SP-C mRNA levels in the rabbit
fetus differ considerably from the effects of glucocorticoids in rat and human fetal lung. SP-C mRNA is apparently not increased by maternal glucocorticoids in
the rabbit fetal lung, although we did observe that fetal
lung SP-C mRNA levels are sensitive to factors related
to maternal stress.
SP-C mRNA was only detected in alveolar type I1
cells of lung tissue of day 27 gestational age fetuses
obtained from control, saline-injected, or betamethasone-injected pregnant does. No SP-C mRNA was detected in bronchiolar epithelial cells in fetal lung tissue
obtained from any condition. Thus the effects of glucocorticoids and saline-injection on SP-C mRNA induction in the rabbit fetal lung tissue were restricted to
effects in alveolar type I1 cells.
In a previous study, we have shown that betamethasone treatment of the pregnant doe increases phosphatidylcholine synthesis in the fetal lung tissue modestly, i.e., by -20% (Snyder et al., 1992). Thus the
stimulatory effects of maternally administered glucocorticoids on fetal lung surfactant SP-A protein and on
SP-A and SP-B mRNA levels are much greater in magnitude than are the stimulatory effects of glucocorticoids on surfactant phospholipid synthesis. We suggest,
therefore, that the beneficial effects of glucocorticoids
on fetal lung surfactant metabolism may be manifested
more prominently by increases in the levels of the surfactant-associated proteins than by increases in the
levels of surfactant phospholipids.
tection of mRNA's in sea urchin embryos by in situ hybridization
using asymmetric RNA probes. Dev. Biol., 101:485-502.
Durham, P.L., E.K. Davis-Nanthakumar, and J.M. Snyder 1992 Developmental regulation of surfactant-associated proteins in rabbit fetal lung in vitro. Exp. Lung Res. 18t775-793.
Fisher, J.H., F. McCormack, S.S.Park, T. Stelzner, J.M. Shannon, and
T. Hofmann 1991 In vivo regulation of surfactant proteins by
glucocorticoids. Am. J . Respir. Cell Mol. Biol., 5t63-70.
Glasser, S.W., T.R. Korfhagen, T.E. Weaver, T. Pilot-Matias, J.L. Fox,
and J.A. Whitsett 1987 cDNA and deduced amino acid sequence
of human pulmonary surfactant-associated proteolipid SPL (Phe).
Proc. Natl. Acad. Sci. U.S.A., 84.4007-4011.
Glasser, S.W., T.R. Korfhagen, T.E. Weaver, J.C. Clark, T. Pilot-Matias, J . Meuth, J.L. Fox, and J.A. Whitsett 1988 cDNA, deduced
polypeptide structure and chromosomal assignment of human
pulmonary surfactant proteolipid: SPL (pVal). J. Biol. Chem.,
263r9-12.
Hawgood, S., B.J. Benson, and R.L. Hamilton, Jr. 1985 Effects of a
surfactant-associated protein and calcium ions on the structure
and surface activity of lung surfactant lipids. Biochem., 24t184190.
Hawgood, S., B.J. Benson, J. Schilling, D. Damm, J.A. Clements, and
R.T. White 1987 Nucleotide and amino acid sequences of pulmonary surfactant protein SP18 and evidence for cooperation between SP18 and SP26-36 in surfactant lipid adsorption. Proc.
Natl. Acad. Sci., U.S.A., 84336-70.
Horowitz, S., N. Dafni, D.L. Shapiro, D.A. Holm, R.H. Notter, and D.J.
Quible 1989 Hyperoxic exposure alters gene expression in the
lung. J. Biol. Chem., 264:7092-7095.
Kuroki, Y., R.J. Mason, and D.R. Voelker 1988a Chemical modification of surfactant protein A alters high affinity binding to rat
alveolar type I1 cells and regulation of phospholipid secretion. J.
Biol. Chem., 263t17596-17602.
Kuroki, Y., R.J. Mason, and D.R. Voelker 1988b Alveolar type I1 cells
express a high-affinity receptor for pulmonary surfactant protein
A. Proc. Natl. Acad. Sci., U.S.A., 85r5566-5570.
Liley, H.G., R.T. Tyler-White, R.G. Warr, B.J. Benson, S. Hawgood,
and P.L. Ballard 1989 Regulation of messenger RNA's for the
hydrophobic surfactant proteins in human lung. J. Clin. Invest.,
83r1991-1197.
Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall 1951 Protein measurement with Folin phenol reagent. J . Biol. Chem., 193:
265-225.
ACKNOWLEDGMENTS
Maniatis, T., E.F. Fritsch, and J. Sambrook 1982 Extraction, purifiWe appreciate the technical assistance of Troy Mccation, and analysis of mRNA from eukaryotic cells. In: Molecular Cloning: A Laboratory Manual. Cold Springs Harbor LaboraCarthy. We thank the secretarial staff of the Departtory, Cold Springs Harbor, NY, pp. 187-210.
ment of Anatomy for typing this manuscript. This re- Mendelson,
C.R., and V. Boggaram 1989 Regulation of pulmonary
search was supported in part by grants from the
surfactant protein synthesis in fetal lung: A major role of glucoNational Institutes of Health, NIH-HD-13912 and
corticoids and cyclic AMP. TEM, 1.20-25.
Mendelson, C.R., C. Chen, V. Boggaram, C. Zacharias, and J.M. SnyNIH-DERC-DK 25295.
der 1986 Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue. J . Biol. Chem., 261r9938LITERATURE CITED
9943.
Auten, R.L., R.H. Watkins, D.L. Shapiro, and S. Horowitz 1990 Sur- Ng, V.L., V.L. Herndon, C.R. Mendelson, and J.M. Snyder 1983 Characterization of rabbit surfactant-associated proteins. Biochim.
factant apoprotein A (SP-A) is synthesized in airway cells. Am. J .
Biophys. Acta., 754r218-226.
Respir. Cell Mol. Biol., 3.491-496.
OReilly, M.A., A.F. Gazdar, J.C. Clark, T.J. Pilot-Matias, S.E. Wert,
Ballard, P.L. 1986 Glucocorticoid effects in vivo. In: Hormones and
W.M. Hull, and J.A. Whitsett 1989 Glucocorticoids regulate surLung Maturation. Springer-Verlag, New York, pp. 24-64.
factant protein synthesis in a pulmonary adenocarcinoma cell
Ballard, P.L., P.D. Gluckman, A. Brehier, J.A. Kitterman, S.L. Kapline. Am. J. Physiol., 257tL385-L392.
lan, A.M. Rudolph, and M.M. Grumbach 1978 Failure to detect an
effect of prolactin on pulmonary surfactant and adrenal steroids Persson, A,, K. Rust, D. Chang, M. Moxley, W. Longmore, and E.
Crouch 1988 CP4: A pneumocyte-derived collagenous surfactantin fetal sheep and rabbits. J. Clin. Invest., 62.879-883.
associated protein. Evidence for heterogeneity of collagenous surBoggaram, V., K. Qing, and C.R. Mendelson 1988 The major apoprofactant proteins. Biochem., 27t8576-8584.
tein of rabbit pulmonary surfactant: Elucidation of primary sePhelps, D.S., and J. Floros 1988 Localization of surfactant protein
quence and cyclic AMP and developmental regulation. J. Biol.
synthesis in human lung by in situ hybridization. Am. Rev.
Chem., 263t2939-2947.
Respir. Dis., 137t939-942.
Boggaram, V., M.E. Smith, and C.R. Mendelson 1989 Regulation of
Phelps, D.S., and J. Floros 1991 Dexamethasone in vivo raises surexpression of the gene encoding the major surfactant protein
factant protein B mRNA in alveolar and bronchiolar epithelium.
(SP-A) in human fetal lung in vitro. Disparate effects of glucoAm. J. Physiol., 26OrL146-Ll52.
corticoids on transcription and on mRNA stability. J . Biol.
Chem., 264r11421-11427.
Phelps, D.S., S. Church, S. Kourembanas, H.W. Taeusch, and J . Floros
1987 Increases in the 35 kDa surfactant-associated protein and
Chomczynski, P., and N. Sacchi 1987 Single-step method of RNA isoits mRNA following in vivo dexamethasone treatment of fetal
lation by acid guanidinum thiocyanate-phenol-chloroformextracand neonatal rats. Electrophoresis, 8r235-238.
tion. Anal. Biochem., 162:156-159.
Connelly, I.H., G.L. Hammond, P.G.R. Harding, and F. Possmayer Schellhase, D.E., and J.M. Shannon 1991 Effects of maternal dexamethasone on expression of SP-A, SP-B, and SP-C in the rat fetal
1991 Levels of surfactant-associated protein messenger ribonulung. Am. J. Respir. Cell Mol. Biol., 4t304-312.
cleic acids in rabbit lung during perinatal development and after
hormonal treatment. Endocrinology, 129t2583-2591.
Shimizu, H., K. Miyamura, and Y. Kuroki 1991 Appearance of surfactant proteins, SP-A and SP-B, in developing rat lung and the
Cox, K.H., D.V. DeLeon, L.M. Angerer, and R.C. Angerer 1984 De-
GLUCOCORTICOID REGULATION OF SP-A, SP-B, AND SP-C mRNA
effects of in uiuo dexamethasone treatment. Biochim. Biophys.
Acta, 1081r53-60.
Snyder, J.M., and C.R. Mendelson 1987 Induction and characterization of the major surfactant apoprotein during rabbit fetal lung
development. Biochim. Biophys. Acta, 920.226-236.
Snyder, J.M., C.R. Mendelson, and J.M. Johnston 1985 The morphology of lung development in the human fetus. In: Pulmonary Development. Transition from Intrauterine to Extrauterine Life. G.
Nelson, ed. Marcel Dekker, New York, pp. 19-46.
Snyder, J.M., H.F. Rodgers, J.A. O’Brien, N. Mahli, S.A. Magliato,
and P.L. Durham 1992 Glucocorticoid effects on rabbit fetal lung
maturation in vivo: An ultrastructural morphometric study.
Anat. Rec., 232.133-140.
Snyder, J.S. 1991 The Biology of the Surfactant-Associated Proteins.
In: Pulmonary Surfactant: Biochemical, Functional, Regulatory
and Clinical Concepts. J.R. Bourbon, ed. CRC Press, Boca Raton,
pp. 105-126.
Takahashi, A,, and T. Fujiwara 1986 Proteolipid in bovine lung surfactant: its role in surfactant function. Biochem. Biophys. Res.
Commun., 135527-532.
Tenner, A.J., S.L. Robinson, J. Borchelt, and J.R. Wright 1989 Human
pulmonary surfactant protein (SP-A), a protein structurally homologous to Clq, can enhance FcR- and CR1-mediated phagocytosis. J. Biol. Chem., 264.13923-13928.
377
Thakur, N.R., M. Tesan, N.E. Tyler, and J.E. Bleasdale 1986 Altered
lipid synthesis in type I1 pneumonocytes exposed to lung surfactant. Biochem. J., 240.579-690.
Weaver, T.E., and J.A. Whitsett 1991 Function and regulation of expression of pulmonary surfactant-associated proteins. Biochem.
J., 273:249-264.
Whitsett, J.A., T.E. Weaver, J.C. Clark, N. Sawtell, S.W. Glasser, T.R.
Korfhagen, and W.M. Hull 1987 Glucocorticoid enhances surfactant proteolipid Phe and pVal synthesis and RNA in fetal lung. J.
Biol. Chem., 262:15618-15623.
Wohlford-Lenane, C.L., and J.M. Snyder 1992 Localization of surfactant-associated proteins SP-A and SP-B mRNA in rabbit fetal
lung tissue by in situ hybridization. Am. J. Respir. Cell Mol.
Biol., 7.335-343.
Wohlford-Lenane, C.L., P.L. Durham, and J.M. Snyder 1992 Localization of surfactant-associated protein-C (SP-C) mRNA in fetal rabbit tissue by in situ hybridization. Am. J. Respir. Cell Mol. Biol.,
6r225-234.
Xu, J., C. Richardson, C. Ford, T. Spencer, Y. LiJuan, G. Mackie, G.
Hammond, and F. Possmayer 1989 Isolation and characterization
of the cDNA for pulmonary surfactant associated protein B
(SP-B)in the rabbit. Biochem. Biophys. Res. Commun., I60:325332.
Документ
Категория
Без категории
Просмотров
0
Размер файла
1 749 Кб
Теги
associates, rabbits, glucocorticoid, lung, protein, vivo, regulation, surfactants, fetal
1/--страниц
Пожаловаться на содержимое документа