SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and
Plasticity
Leptin mediates the parathyroid
hormone-related protein paracrine stimulation of fetal lung
maturation
J. S.
Torday,
H.
Sun,
L.
Wang, and
E.
Torres
Department of Pediatrics and Obstetrics and Gynecology,
Harbor-University of California Los Angeles Research and Education
Institute, Torrance, California 90502
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ABSTRACT |
Developing rat
lung lipofibroblasts express leptin beginning on embryonic day (E) 17, increasing 7- to 10-fold by E20. Leptin and its receptor are expressed
mutually exclusively by fetal lung fibroblasts and type II cells,
suggesting a paracrine signaling "loop." This hypothesized
mechanism is supported by the following experimental data:
1) leptin stimulates the de novo synthesis of surfactant
phospholipid by both fetal rat type II cells (400% · 100 ng
1 · ml
1 · 24 h
1) and adult human airway epithelial cells
(85% · 100 ng
1 · 24 h
1);
2) leptin is secreted by lipofibroblasts in amounts that
stimulate type II cell surfactant phospholipid synthesis in vitro;
3) epithelial cell secretions such as parathyroid
hormone-related protein (PTHrP), PGE2, and dexamethasone
stimulate leptin expression by fetal rat lung fibroblasts;
4) PTHrP or leptin stimulate the de novo synthesis of
surfactant phospholipid (2- to 2.5-fold/24 h) and the expression of
surfactant protein B (SP-B; >25-fold/24 h) by fetal rat lung explants,
an effect that is blocked by a leptin antibody; and 5) a
PTHrP receptor antagonist inhibits the expression of leptin mRNA by
explants but does not inhibit leptin stimulation of surfactant phospholipid or SP-B expression, indicating that PTHrP paracrine stimulation of type II cell maturation requires leptin expression by
lipofibroblasts. This is the first demonstration of a paracrine loop
that functionally cooperates to induce alveolar acinar lung development.
lung development; surfactant; type II cell; lipofibroblast
 |
INTRODUCTION |
LIPID-LADEN
LIPOFIBROBLASTS were first identified as a morphologically
distinct cell type in fetal and neonatal rat lung in 1970 (8). Both lipofibroblasts and adipocytes originate from mesoderm and are physically distinguished by the presence of large pools of triglyceride stores in their cytoplasm (11). The
metabolism of lipid by these two cell types is also strikingly similar
with respect to their lipogenic pathways, which are regulated by the same signal transduction pathway (11). Mature adipocytes
express leptin, a 16-kDa cytokine product of the obesity
(ob) gene (30). Fetal lung lipofibroblasts
produce fibroblast pneumonocyte factor (FPF), a 10,000 to 20,000 molecular weight peptide that stimulates surfactant phospholipid
synthesis by fetal type II cells (17). Differentiation of both cell types is stimulated by glucocorticoids (17, 33) and downregulated by androgens (10,
12). Furthermore, previous studies from our laboratory had
suggested that lung epithelial cell secretions such as PGE2
(28) and parathyroid hormone-related protein (PTHrP; see
Ref. 29) stimulate type II cell-fibroblast interactions
via a soluble fibroblast growth factor of unknown identity. These
observations, combined with the similarities between adipocytes and
lipofibroblasts, and the recent reports that leptin is expressed in
developing lung (9, 30) prompted us to investigate the
possible role of leptin as a lipofibroblast growth factor that might
stimulate fetal lung development.
It has long been recognized that lung development is mediated by
soluble paracrine growth factors that mediate epithelial-mesenchymal interactions (15). These signals are bidirectional
(1, 23), and the earliest known signals originate from the
epithelium (22). We had previously identified PTHrP as a
fetal rat alveolar type II cell product that is expressed beginning in
the glandular phase of fetal lung development (16) and
stimulates type II cell differentiation indirectly by stimulating fetal
lung fibroblast factors (14). PTHrP stimulates fibroblast
maturation through a receptor-mediated signal transduction pathway
involving the production of cAMP and inositol phosphate
(14), both of which induce fibroblast differentiation into
adipocytes (34). Based on these observations, we
hypothesized that leptin mediates the paracrine effect of PTHrP on
alveolar type II cell surfactant synthesis.
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MATERIALS AND METHODS |
Reagents.
Leptin (rat, human) and leptin antibody (rat, polyclonal) were
acquired from Linco (St. Charles, MO). The leptin RIA kit was obtained
from DSL (Webster, TX). PTHrP-(1---34) and PTHrP-(7---34) amide were
obtained from Bachem (Torrance, CA). Dexamethasone and PGE2
were purchased from Sigma Biochemicals (St. Louis, MO).
Animals.
Time-mated Sprague-Dawley rats [embryonic day (E) 0, the day of
mating] were obtained from Charles River Breeders (Hollister, CA).
These experiments were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals.
Cell culture.
WI-38 and H441 cells were obtained from the American Type Culture
Collection (Rockville, MD).
Isolation of lipofibroblasts and type II cells from fetal lung.
The following methods have been used extensively in our
laboratory (6). Three to five dams were used per
preparation. The dams were killed with an overdose of pentobarbital
sodium (100 mg/ml ip), and the pups were removed from the uterus by
laparotomy and kept on ice. The lungs were removed en bloc in a laminar
flow hood using sterile technique and put into ice-cold sterile Hanks' balanced salt solution without calcium or magnesium. The solution was
decanted, and 5 vol of 0.05% trypsin (Worthington) were added to the
lung preparation.
The lungs were dissociated in a 37°C water bath using a Teflon
stirring bar to physically disrupt the tissue. When the tissue had been
completely dispersed in a unicellular suspension (~20 min), the cells
were spun down at 500 g for 10 min at room temperature in a
50-ml polystyrene centrifuge tube. The supernatant was decanted, and
the cell pellet was resuspended in MEM (GIBCO) containing 10% FBS to
yield a mixed cell suspension of ~3 × 108 cells as
determined by a Coulter particle counter (Hialeah, FL). The cell
suspension was added to 75-cm2 culture flasks (Corning
Glass Works, Corning, NY) for 30-60 min at 37°C in a
CO2 incubator to allow for differential adherence of the
fibroblasts (20). Fibroblasts were maintained in MEM until
further processing.
Preparation of type II cell cultures.
The unattached cells from the above-described cell preparation were
transferred to another 75-cm2 culture flask for an
additional 60-min period. After this second culture period, the medium
and nonadherent cells were removed from the flask and diluted with 1 vol of culture medium. This diluted suspension was cultured overnight
in a 75-cm2 flask at 37°C in a CO2 incubator
to allow the type II cells to adhere (2). Type II cells
were identified by their appearance in culture under phase-contrast
microscopy, lamellar body content and microvillar processes, or by
cytokeratin-positive staining.
Explant culture.
Fetal rat lung tissue was cut into 1-mm cubes with a McIlwain tissue
chopper (Brinkmann Instruments, Westbury, NY) and incubated in 0.5 ml
Waymouth's MB-252/l medium containing penicillin (100 U/ml),
streptomycin (100 U/ml), and fungizone (2.5 µg/ml) while rocking on
an oscillating platform (3 cycles/min) in an atmosphere of 5%
CO2-95% air at 37°C (25).
mRNA extraction.
Total cellular RNA was isolated using previously described methods
(5).
Semiquantitative RT-PCR.
The appropriate cDNA fragments were amplified using 400 ng of
total RNA from lung tissues or cells, avian myeloblastosis virus RT,
and random hexamers and deoxyribonucleotides. The reactions were run at
42°C for 75 min and terminated by heating at 95°C for 5 min.
Coamplification with glyceraldehyde-3-phosphate dehydrogenase cDNA was
used as an internal standard. PCR was initiated by Taq DNA
polymerase and allowed to proceed for 30 cycles with an annealing temperature of 50°C. The following primers were used in the RT-PCR assay: rat surfactant protein (SP) B (sense, 5'-TACACAGTACTTCTACTAGATG; antisense, 3'-ATAGGCTGTTCACTGGTGTTCC); human leptin (sense,
5'-CCTATCTTTTCTATGTCCAAGC; antisense, 3'-GTGAGGATCTGTTGGTAGACTG);
human leptin receptor designed to detect all of the known isoforms
(sense, 5'-TACTTTGGAAGCCCCTGATG; antisense, 3'-AAGCACTGAGTGACTGCACG);
rat leptin (sense, 5'-TTATGTTCAAGCAGTGCCTATC; antisense,
3'-CATCCAACTGTTGAAGAATGTC); and rat leptin receptor(sense, 5'-ACCTTCAGTTCCAGATTCGA; antisense, 3'-TGAGATTGGTCTGATTTCCC). The
identities of all RT-PCR products were confirmed by Southern blotting.
mRNA expression was quantitated by densitometry (Eagle Eye; Stratagene).
Phospholipid assay.
The rate of saturated phosphatidylcholine synthesis was determined as
previously described (6) with modifications. Briefly, confluent monolayer cultures of fetal rat type II cells, H441 cells, or
fetal rat lung explants were treated with leptin, PTHrP, leptin
antibody, or PTHrP receptor antagonist and subsequently incubated with
[3H]choline chloride (1 µCi/ml) for 4 h at 37°C
in 5% CO2-95% air. Lamellar body fractions were prepared
from the cells and tissues as described by Snyder et al.
(21). To correct for procedural losses, fetal rat lung
tissue was incubated with [U-14C]glycerol (10 µCi/ml,
10 mCi/mmol; NEN, Boston, MA) for 24 h, and lamellar bodies were
prepared as previously described (21). This lamellar body
preparation (10,000 dpm) was added to each sample before processing for
[3H]phosphatidylcholine. Phospholipids were extracted
from the lamellar body fractions by the method of Bligh and Dyer
(3) and were dried under a stream of nitrogen at 50°C.
The phospholipid extracts were mixed with 0.5 ml of an osmium tetroxide
solution (70 mg/100 ml carbon tetrachloride) and reacted for 15 min at
room temperature (26). The reaction mixture was dried
under a stream of nitrogen at 60°C, and the dried extract was
chromatographically separated by TLC in chloroform-methanol-water
(65:25:4 vol/vol/vol; see Ref. 26). The phospholipids were
subsequently scraped from the chromatography plates and analyzed for
their radioactive content by liquid scintillation spectrometry.
Protein determination.
Protein determination was made using the Bradford (4)
dye-binding method.
Statistical analyses.
ANOVA was used to compare data within experimental groups. The null
hypothesis was rejected when P < 0.05 was not obtained.
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RESULTS |
Initially, we examined the expression and ontogeny of leptin by
fetal rat lung in the last third of gestation, during the phase of lung
development when the fibroblast and type II cell mature. As can be seen
in Fig. 1, the expression of leptin
increases progressively between E17 and E21, which corresponds to the
transition from the glandular (
E17) to the canalicular (E18 and E19)
and saccular (
E20) stage of fetal rat lung development. The apparent decrease in leptin expression on E22 was observed consistently (n = 5). Leptin expression by E19 fetal rat lung
fibroblasts (Fig. 2, top) was
significantly increased by dexamethasone (8-fold), PTHrP (6-fold), and
PGE2 (6-fold), which are endocrine and paracrine factors
known to stimulate lipofibroblast development. Similar effects on
leptin expression were obtained (Fig. 2, bottom) by incubating human embryonic lung fibroblasts (WI-38) with these same
agonists. H441 human small cell carcinoma cells (Fig.
3) and E18 fetal rat type cells expressed
the leptin receptor, whereas expression of the leptin receptor was
faint in fetal rat lung fibroblasts and WI-38 cells, consistent with
the hypothesized paracrine nature of the leptin mechanism within the
alveolar acinus. Fetal rat lung fibroblasts cumulatively secrete leptin
in the culture medium over a 24-h period (Fig.
4), increasing from 23 ± 8 ng · ml
1 · 4 h
1 · 106 cells to 57 ± 12 ng · ml
1 · 8 h
1 · 106 cells and 97 ± 58 ng · ml
1 · 24 h
1 · 106 cells, equivalent to ~5
ng · ml
1 · h
1 · 106
cells; incubation of these cells with PTHrP increased secretion of leptin by greater than twofold more than the 6-h rate. Incubation of
fetal rat type II cells with leptin concentrations comparable to the
amounts we had observed being produced by the fetal rat lung
fibroblasts over a 24-h period stimulated the incorporation of
[3H]choline into lamellar body-saturated
phosphatidylcholine at 10, 50, and 100 ng/ml by 150-400% (Fig.
5). Leptin also stimulated choline
incorporation into lamellar body-saturated phosphatidylcholine by H441
cells, although the effect was lower than that of leptin on the fetal
type II cells, i.e., there was no effect at 10 ng/ml, 40% at 50 ng/ml,
and 85% at 100 ng/ml (Fig. 6). The
stimulation of surfactant synthesis by these cells of human adult lung
origin is evidence for the presence of functional leptin receptors on adult type II cells, which is consistent with the observed expression of leptin receptor mRNA by these cells (see Fig. 3).

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Fig. 1.
Leptin ontogeny in fetal rat lung. Semiquantitative
RT-PCR was performed using RNA from fetal rat lung at the indicated
gestational ages [embryonic day (E) 17-E22]. Coamplification
with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was used as
an internal standard for normalization of aliquot volumes for leptin
mRNA expression.
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Fig. 2.
Semiquantitative RT-PCR of RNA extracts from fetal rat
lung fibroblasts and WI-38 human embryonic lung fibroblasts. Primary
fetal rat lung fibroblasts (E18) and WI-38 human embryonic lung
fibroblasts were treated with either dexamethasone (Dex, 1 × 10 8 M), parathyroid hormone-related protein (PTHrP,
5 × 10 7 M), or PGE2 (5 × 10 7 M) for 24 h and subsequently analyzed for leptin
mRNA expression using semiquantitative RT-PCR. Coamplification with
GAPDH cDNA was used as an internal standard for normalization of the
aliquot volumes for leptin mRNA expression. Center:
representative blots for RT-PCR of leptin mRNA. Top and
bottom, densitometric quantitation of the corresponding
blots. Each bar represents the mean ± SD; n = 5 experiments. C, control; OD, optical density. ***P < 0.0001 by ANOVA.
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Fig. 3.
Semiquantitative RT-PCR for leptin receptor mRNA. Primary
fetal rat lung fibroblasts (FrLf) and type II cells (TII), WI-38 human
embryonic lung fibroblasts, and H441 adult lung epithelial cells were
assayed for leptin receptor mRNA expression. Coamplification with GAPDH
cDNA was used as an internal standard for the normalization of the
aliquot volumes for leptin receptor mRNA expression.
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Fig. 4.
Fetal rat lung fibroblasts secrete leptin under the
influence of PTHrP. E21 fetal rat lung fibroblasts were cultured in
2-cm2 wells in serum-free DMEM containing 0.1% fatty
acid-free BSA for up to 6 h with or without PTHrP (5 × 10 7 M). Leptin concentrations were determined by an ELISA
for rat leptin. Each bar represents the mean ± SD;
n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA.
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Fig. 5.
Leptin stimulates surfactant phospholipid synthesis by
fetal lung type II cells. E19 fetal rat lung type II cells were
incubated with leptin for 24 h and exposed to
[3H]choline chloride for the last 4 h. The type II
cell lamellar body fractions were subsequently assayed for
[3H]phosphatidylcholine content. Each bar represents the
mean ± SD; n = 5. *P < 0.05 and
**P < 0.01, control (C) vs. leptin treated by ANOVA.
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Fig. 6.
Leptin stimulates surfactant phospholipid synthesis by
adult lung epithelial cells. H441 adult lung epithelial cells were
incubated with leptin for 24 h and exposed to
[3H]choline chloride for the last 4 h. The H441 cell
lamellar body fractions were subsequently assayed for
[3H]phosphatidylcholine content. Each bar represents the
mean ± SD; n = 5. *P < 0.05 and
**P < 0.01, control vs. leptin treated by ANOVA.
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In subsequent experiments, we examined the interaction of PTHrP
and leptin in fetal rat lung explant culture. Incubation of E18 fetal
rat lung explants with leptin (200 ng · ml
1 · 24 h
1) or PTHrP
(5 × 10
7 M/24 h) significantly increased the
incorporation of [3H]choline into
[3H]phosphatidylcholine (>2-fold; Fig.
7). The concentration of leptin was
chosen based on its maximally effective dose in monolayer culture [see
effect of leptin on type II cells in monolayer culture (Fig. 4)] and
was doubled to compensate for tissue penetration. The PTHrP dose was
based on previous studies of its effect on surfactant synthesis in
explant culture (14). The stimulatory effect of PTHrP was
blocked by coincubation with leptin antibody (1:100 dilution). However,
the stimulatory effect of leptin was not blocked by coincubation with
the PTHrP receptor antagonist. Maintaining E18 fetal rat lung tissue in
explant culture for 3 days resulted in a 10-fold increase in leptin
mRNA expression (Fig. 8), consistent with
its developmental upregulation during this phase of lung maturation.
This increase in leptin mRNA expression was blocked by coincubation
with the PTHrP receptor antagonist [PTHrP-(7---34) amide, 5 × 10
6 M]. Incubation of E18 fetal rat lung explants with
leptin (200 ng · ml
1 · 24 h
1) or PTHrP (5 × 10
7 M/24 h) also
stimulated the expression of SP-B (Fig.
9); coincubation of PTHrP-treated E18
explants with leptin antibody (1:100 dilution) blocked the PTHrP
stimulation of SP-B expression. The same effect was observed when
leptin-treated explants were coincubated with leptin antibody (data not
shown), as expected. In contrast to this, coincubation of
leptin-treated explants with the PTHrP receptor antagonist (5 × 10
7 M/24 h) had no effect on leptin stimulation of SP-B
expression.

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Fig. 7.
Leptin antibody inhibits PTHrP stimulation of surfactant
phospholipid. E18 fetal rat lung explants were maintained in culture
for 24 h. Explants were incubated with leptin during the 24-h
culture period and were exposed to [3H]choline chloride
for the last 4 h. Leptin and PTHrP significantly increased
[3H]phosphatidylcholine synthesis (n = 5). *P < 0.01, controls vs. treated, by ANOVA.
Coincubation of PTHrP-treated explants with leptin antibody (LeptinAb,
1:100 dilution) blocked the stimulatory effect of PTHrP on saturated
phosphatidylcholine synthesis; coincubation of leptin-treated explants
with PTHrP receptor antagonist (PTHrP Ant, 5 × 10 6
M) had no effect on saturated phosphatidylcholine synthesis.
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Fig. 8.
Leptin mRNA expression is PTHrP dependent. E18 fetal rat
lung explants were maintained in culture for 3 days with or without
PTHrP receptor antagonist (1 × 10 6 M). Explants
were subsequently analyzed for leptin mRNA expression by
semiquantitative RT-PCR. Coamplification with GAPDH cDNA was used as an
internal standard for normalization of the aliquot volumes for leptin
mRNA expression. Bar graph represents densitometric quantitation of the
leptin mRNA bands; each bar is the mean ± SD; n = 5. ***P < 0.0001 by ANOVA.
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Fig. 9.
PTHrP paracrine stimulation of surfactant protein-B
(SP-B) is leptin dependent. E18 fetal rat lung explants were maintained
in culture for 24 h with or without leptin (200 ng/ml), PTHrP
(5 × 10 7 M), PTHrP plus leptin antibody
(PTHrP/LeptinAb; 1:100 dilution), or leptin plus PTHrP receptor
antagonist (Leptin/PTHrP Ant, 5 × 10 6 M). SP-B mRNA
expression was determined using semiquantitative RT-PCR as shown in the
representative blot. Coamplification with GAPDH cDNA was used as an
internal standard for normalization of the aliquot volumes for leptin
mRNA expression. Bar graph represents the mean ± SD for control
vs. treated groups; n = 5. ***P < 0.0001 by ANOVA.
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DISCUSSION |
The hypothesized role of leptin as a paracrine mediator of the
lung type II cell-fibroblast interaction resulting in surfactant synthesis was supported by our experimental results. Leptin and its
receptor are expressed by fetal rat lung fibroblasts and type II cells,
respectively, before the onset of type II cell maturation, beginning on
E19-E20, and leptin expression is under both endocrine and paracrine
control. Leptin stimulates both surfactant phospholipid and protein
synthesis by type II cells by a PTHrP-dependent signaling pathway,
based on the observation that the PTHrP receptor antagonist inhibits
the developmental upregulation of leptin expression in explant culture;
also, the leptin antibody blocks the PTHrP stimulation of surfactant
phospholipid and protein expression, although the PTHrP receptor
antagonist has no effect on (downstream) leptin-stimulated SP-B expression.
In the present series of experiments, we have observed that leptin mRNA
expression in the developing fetal rat lung increases during the period
of alveolar lung differentiation when pulmonary surfactant phospholipid
synthesis is induced (E18-E20). Leptin expression appears to peak
at E20 and then progressively declines on E21 and E22. This phenomenon
has also been observed for FPF (18) and may be due to the
thinning of the alveolar wall, which occurs during the process of
alveolarization. The process of alveolar differentiation is a
well-recognized hormonally regulated paracrine mechanism
(17) that depends upon interactions between the type II
epithelial cell and mesenchymal fibroblast. Type II cells elaborate PTHrP (29), which promotes the differentiation of the
mesenchyme into lipofibroblasts in a manner similar to adipocyte
differentiation, including the expression of key adipocytic enzymes,
triglyceride uptake, storage, and secretion (11). These
PTHrP-induced lipofibroblasts then apparently produce a soluble growth
factor that stimulates type II cell surfactant phospholipid synthesis
(14, 29) and SP-B expression, as shown in this study; the
other surfactant proteins (SP-A, -C, and -D) were not examined,
although they also play an essential role in surfactant metabolism.
With these principles in mind, we hypothesized that the mature
lipofibroblast, like the mature adipocyte, would express leptin, a
secreted product of the mature adipocyte. Because leptin belongs to the
interleukin-6 family of cytokines (32), and interleukin-6
stimulates the synthesis of pulmonary surfactant by type II cells
(13), PTHrP-stimulated expression of leptin by
lipofibroblasts provides a closed paracrine loop from the type II cell
to the lipofibroblast and back to the type II cell.
It has long been recognized that alveolar type II cell differentiation
is dependent on mesenchymal-epithelial interactions (15),
which are mediated by low-molecular-weight soluble factors (7). Smith (18) was the first investigator to
identify a specific hormonally induced mesenchymal factor that
stimulated surfactant phospholipid synthesis over 20 years ago
(19), although the identity of this factor has remained
undetermined. He termed this differentiation factor FPF. We
subsequently discovered that FPF was downregulated by androgens
(6, 12), providing further evidence for a biological role
of FPF in the timing of lung development, since there is a spontaneous
sex difference in the rate of lung development and pulmonary surfactant
synthesis (6, 27) that is androgen dependent
(24). Leptin has many of the same characteristics as FPF:
1) its molecular weight (16,000) is within the
range previously reported for FPF (19); 2) it
is expressed by lung mesenchymal cells during fetal development in a
pattern like that reported for FPF (i.e., beginning in the canalicular
phase, peaking on E21, and then declining on E22; see Ref.
19); and 3) its expression is stimulated by
glucocorticoids (19) and inhibited by both androgens
(9) and transforming growth factor-
(25,
27). These similarities between leptin and FPF strongly support
their common identity. Their functional similarities are further
reinforced by their common cellular origins; leptin is produced by the
ob gene of the mature adipocyte (31), which
lung lipofibroblasts bear a strong resemblance to, both structurally
and functionally (11). FPF is also produced by
adepithelial lung fibroblasts (M. Post, personal communication). In
summary, leptin may be the long-sought-after soluble factor that
mediates the hormonal effects on fetal lung development, which govern
surfactant production.
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ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-55268.
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FOOTNOTES |
Address for reprint requests and other correspondence: J. S. Torday, Dept. of Pediatrics/OB-GYN, Center for Developmental Biology, RB-1, 1124 W. Carson St., Torrance, CA 90502 (E-mail: jtorday{at}prl.humc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 August 2000; accepted in final form 10 January 2001.
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