(Received for publication, September 22, 1995; and in revised form, December 18, 1995)
From the
Cell proliferation is in part regulated by extracellular matrix.
Therefore, it is possible that elevated O may indirectly
affect lung fibroblast growth via modulation of extracellular matrix.
In the present study, we investigated the effect of elevated O
on the synthesis of glycosaminoglycans (GAGs) and proteoglycans
(PGs) by fetal lung fibroblasts. A 48-h exposure to
50% O
reduced the incorporation of [
H]glucosamine
and
SO
into GAGs by fetal lung fibroblasts.
The relative proportion of the individual GAG molecules was not altered
by elevated O
. Fibroblasts exposed to 50% O
secreted less [
S]proteoglycans into the
medium than controls. Specifically, the synthesis of the small soluble
PG, biglycan, was decreased by exposure to 50% O
. Fetal
lung fibroblasts did not synthesize the small chondroitin/dermatan
sulfate PG, decorin. Elevated O
concentrations also reduced
the synthesis of membrane- and matrix-associated PGs. Furthermore,
exposure of fetal lung fibroblasts to
50% O
resulted in
a decreased mRNA expression for biglycan and versican core protein
sequences. In contrast, elevated O
increased the message
for type I collagen and fibronectin without affecting that of
-actin. The inhibitory effect of elevated O
on
biglycan mRNA and protein expression was overcome by incubating the
cells in 3% O
after the 48-h exposure to 50% O
.
The latter treatment also reversed the increased mRNA expression of
type I collagen associated with elevated O
but not that of
fibronectin. These results demonstrate that fetal lung fibroblasts, in
response to elevated oxygen concentrations, selectively down-regulate
their GAG and PG synthesis and that this O
effect is
reversible.
Disruption of cell-matrix interactions during lung development
may induce cellular responses, which can result in scar formation.
Administration of high concentrations of oxygen has been shown to
induce changes in extracellular matrix(1) . Glycosaminoglycans
(GAGs) ()are matrix components, which may mediate
matrix-dependent events during development, including branching, tissue
remodeling, and cell differentiation(2, 3) . GAGs are
long polyanionic carbohydrate chains composed of repeating disaccharide
units (uronic acid and hexosamine) carrying a negative charge, which
results in their binding to other matrix components and cell adhesion
molecules(4, 5) . Most GAGs are covalently linked to a
core protein as proteoglycans (PGs). PGs show a marked variation in the
size of the core protein and the number and size of GAG side chains
attached. Some proteoglycans are cell-associated, and others are
present in interstitial matrices(4, 5) . Increasing
evidence suggests that GAGs and PGs play an important role as
modulators of growth factor activities(6) . Several isoforms of
fibroblast growth factor bind to high affinity receptors and are active
when bound to specific cell surface heparan sulfate PGs(7) .
Similarly, the membrane proteoglycan betaglycan increases the binding
of TGF-
to the signaling TGF-
receptor(8) . The small interstitial proteoglycans biglycan,
decorin, and fibromodulin have been shown to bind TGF-
via their
core proteins(9, 10, 11) . Recent studies
have demonstrated that GAGs also directly affect cell proliferation.
Heparan sulfate molecules are potent inhibitors of smooth muscle cell
growth(12) . Additionally, hyaluronic acid inhibits fetal skin
fibroblast proliferation but stimulates collagen and noncollagen
protein synthesis(13) . These properties suggest that GAGs and
PGs may mediate, at least in part, cell adhesion, structural
organization, and cell proliferation during fetal lung development.
Thus, alterations in GAG and PG content in the interstitial matrix
following elevated oxygen may disrupt normal fibroblast adhesion and
affect fibroblast proliferation. It has been shown that the
distribution and density of PGs in alveolar basement membranes is
decreased in lung injury resulting from short term oxygen
exposure(14) . In contrast, exposure of newborn rats to 85%
oxygen for up to 6 weeks has been reported to result in an increase in
biglycan mRNA expression and immunoreactivity, specifically in alveolar
cells in areas of increased cellularity(15) . The effect of
oxygen on PG metabolism may be due to increased intracellular
generation of partially reduced oxygen species. Reactive oxygen species
have been demonstrated to affect GAG and PG synthesis in cultured
chondrocytes (16) . Physiological concentrations of reactive
oxygen species stimulate PG formation, while higher concentrations
decrease PG synthesis(16) . Additionally, high reactive oxygen
species concentrations damage existing cartilage PGs(16) .
Whether elevated oxygen concentrations directly affect GAG and PG
synthesis or destroy existing PGs has yet not been determined. In the
present study, we examined the effect of hyperoxia on GAG and PG
synthesis by fetal lung fibroblasts. The data suggest that in vitro fetal lung fibroblasts selectively down-regulate their GAG and PG
synthesis as an early response to elevated oxygen concentration.
To
determine the effect of O on cell number, parallel culture
wells were incubated with serum-free MEM. After the 48-h incubation
period, the cells were trypsinized and counted electronically using a
Coulter particle counter (Coulter Electronics, Hialeah, FL).
Aliquots (100 µl) of dialyzed media samples,
cell membrane fraction, and extracellular matrix fraction were then
incubated in PBS with or without chondroitinase ABC (100 milliunits/ml)
or heparitinase (20 milliunits/ml) at 37 °C. After a 24-h
incubation, samples were boiled in SDS-sample buffer and fractionated
on 5% (w/v) SDS-polyacrylamide gel. Gels were fixed in 10% (v/v) acetic
acid, prepared for fluorography by soaking in EnHance
(DuPont NEN), dried, and exposed to Kodak XAR-5 film using Dupont
Cronex intensifying screens. The films were quantified using an
Ultroscan XL laser densitometer (Pharmacia Biotech Inc.).
Figure 1:
Effect of elevated
O concentrations on cell number and GAG synthesis by fetal
rat lung fibroblasts. The cells were exposed to various concentrations
of O
. After a 48-h incubation, detached cells were removed
by aspiration, the wells of 24-well plates were rinsed, and the adhered
cells were trypsinized and then counted electronically. The
glycosaminoglycan synthesis was assessed by the incorporation of
[
H]glucosamine into GAGs as described under
``Experimental Procedures.'' Values are means ± S.E., n = 3 independent experiments each carried out in
triplicate. *, p < 0.05 compared with 3%
O
.
Figure 2:
Effect of elevated O
concentrations on synthesis of individual GAG molecules by fetal rat
lung fibroblasts. Glucosamine-labeled GAGs were separated by HPLC using
a DEAE column. HA, hyaluronan; HS, heparan sulfate; CS, chondroitin sulfate. Values are means ± S.E., n = 3 independent experiments each carried out in
triplicate.
Figure 3:
Effect of elevated O
concentrations on PGs in the culture media of fetal rat lung
fibroblasts. The cells were exposed for 24 h to 3 or 50% O
prior to a 24-h incubation with
SO
in
the same O
concentration. The media samples were dialyzed
against distilled water, lyophilized, reconstituted in PBS, and
incubated with either chondroitinase ABC (ABC) or heparitinase (H) or left untreated (C). Proteoglycans were
separated on 5% SDS-polyacrylamide gels. A representative autoradiogram
is shown. The positions of undigested PGs (biglycan, versican, and
heparan sulfate proteoglycans (HSPGs)) are shown on the left. Molecular mass marker positions are displayed on the right. Similar results were obtained in three separate
experiments.
Figure 4:
Reversibility of the effect of elevated
O concentrations on PG synthesis by fetal lung fibroblasts.
The cells were exposed for 24 h to 3 or 50% O
prior to a
24-h incubation with
SO
in the same O
concentration. To determine reversibility, another set of cells
was exposed for 48 h to 50% followed by a 48-h incubation in a gas
phase of 3% O
. The cells were labeled with
SO
during the last 24-h incubation in 3%
O
. The media samples were dialyzed against distilled water,
lyophilized, reconstituted in PBS, and incubated with either
chondroitinase ABC (ABC) or heparitinase (H) or left
untreated (C). A representative autoradiogram is shown. The
position of undigested biglycan is shown on the left.
Molecular mass marker positions are displayed on the right.
Similar results were obtained in a second
experiment.
Figure 5:
Effect of elevated O
concentrations on PGs in cell membrane and matrix fractions of fetal
rat lung fibroblasts. The cells were exposed for 24 h to 3 or 50%
O
prior to a 24-h incubation with
SO
in the same O
concentration. After the media was
removed, cells were rinsed and the membrane (cell-associated) fraction
was isolated with 2% Triton X-100 (see ``Experimental
Procedures''). The remaining extracellular matrix fraction was
rinsed and extracted by scraping in SDS sample buffer. The fractions
were treated with either chondroitinase ABC (ABC) or
heparitinase (H) or left untreated (C) and then
analyzed by 5% SDS-polyacrylamide gel electrophoresis. Two
representative autoradiograms are shown. The positions of undigested
PGs (biglycan, and heparan sulfate proteoglycans (HSPGs)) are
shown on the left. Molecular mass marker positions are
displayed in the middle. Similar results were obtained in a
second experiment.
Figure 6:
Expression of biglycan, decorin, versican,
and -actin mRNAs by fetal rat lung fibroblasts. Total cellular RNA
(10 µg) was analyzed by Northern hybridizations using cDNAs for
human biglycan (lane 1), human decorin (lane 2),
human vesican (lane 3), and rat
-actin (lane 4).
The sizes of the mRNAs are indicated in kb.
Figure 7:
The effect of elevated O
concentrations on biglycan, versican, and
-actin mRNA levels in
fetal rat lung fibroblasts. The cells were exposed to various
concentrations of O
for 0-72 h. Total cellular RNA
(10 µg) was analyzed by successive Northern hybridizations of the
same filter (ethidium bromide-stained gel) using cDNA probes indicated. A, a representative autoradiogram is shown. B,
autoradiograms of Northern blots were quantified by laser scanning
densitometry. Open bar, cells exposed to 3% O
; dark gray bar, cells exposed to 50% O
. Similar
results were obtained in a second
experiment.
Figure 8:
The effect of elevated O
concentrations on biglycan, type I collagen, fibronectin, and
-actin mRNA levels in fetal rat lung fibroblasts. The cells were
exposed for 48 h to either 3 or 50% O
. To examine
reversibility, cells exposed to 50% O
were incubated for 48
h in 3% O
. Total cellular RNA (10 µg) was analyzed by
successive Northern hybridizations of the same filter using cDNA probes
indicated. A representative autoradiogram is shown. Autoradiograms of
Northern blots were quantified by laser scanning densitometry. Filled bars, cells exposed to 3% O
; gray
bars, cells exposed to 50% O
; black bars,
cells exposed to 50% O
followed by 3% O
.
Similar results were obtained in a second
experiment.
Increasing evidence suggests that cell-matrix interactions
play an important role in lung morphogenesis. Several studies suggest
that matrix molecules can alter the growth of cells, with some
components increasing and others decreasing proliferation(29) .
Thus, changes in matrix molecules following an insult may modulate cell
interaction with these matrix molecules, thereby disrupting normal
proliferation. This is of particular importance in the preterm human
infant, exposed to elevated oxygen concentrations for weeks or months,
since a major portion of lung growth and development occurs over these
first weeks or months of life. Chronic exposure to elevated
concentrations of O is known to reduce lung growth and to
cause pulmonary fibrosis in these infants(30, 31) .
In the present study, we report that elevated O concentrations decrease the synthesis of GAGs and PGs by fetal
lung fibroblasts. Using metabolic labeling with
Na
SO
, we showed that biglycan was
the most abundant proteoglycan secreted into the medium by fetal lung
fibroblasts. In contrast to several different types of cells, including
human skin and gingival lung fibroblasts(32) , fetal rat lung
fibroblasts did not synthesize the small soluble proteoglycan, decorin.
Cultured bovine aortic endothelial cells(27) , human umbical
vein endothelial cells(27) , and rat pleural mesothelial cells (33) have also been found to express biglycan but not decorin.
The lack of decorin synthesis may be due to the use of primary cell
cultures, because the human embryonic lung fibroblast cell line, HFL-1,
has been shown to synthesize decorin(34) . Although cell
culture findings should be interpreted with caution, it is likely that
the absence of decorin synthesis has a functional role in late fetal
lung development. It should be noted that under similar experimental
conditions as used for fetal rat lung fibroblasts, fetal rat skin
fibroblasts expressed decorin. Thus, it is unlikely that the decorin
gene is silenced by methylation of the control regions in isolated
fetal lung fibroblasts. The exact role of soluble PGs is unknown, but
they have been implicated in cell adhesion. Soluble PGs may inhibit
cell adhesion to fibronectin and collagen by binding to the GAG binding
site of these matrix molecules, thus making it inaccessible to the cell
surface PGs(4, 5) . Soluble as well as membrane- and
matrix-bound small PGs may also play a role in the control of cell
proliferation. Increasing evidence suggests that small PGs control
TGF-
bioactivity by sequestering TGF-
in the extracellular
matrix(9, 11) . The larger membrane- and
matrix-associated PGs have also been implicated in binding and
regulating the bioactivity of several growth
factors(7, 35, 36) . It has been suggested
that the binding of growth factors to extracellular matrices is of
general significance and may explain the growth-promoting or inhibiting
activities of extracellular matrices (5) .
We have
previously reported growth inhibition of cultured fetal lung
fibroblasts in response to elevated oxygen exposure(37) . It is
possible that altered PG production may contribute to this growth
inhibition. We found that elevated O mainly decreased
biglycan synthesis of fetal lung fibroblasts. However,
cell-matrix-associated PGs were also affected by elevated O
concentrations. The results obtained at the protein level were
confirmed by mRNA observations. Whether similar changes in proteoglycan
production by fetal lung fibroblasts in response to elevated
concentrations of O
occur in vivo remains to be
elucidated. Short term exposure of newborn rats to 95% O
has been reported to decrease the density of PGs in alveolar
basement membranes(14) . In contrast, an increase in biglycan
synthesis has been reported in a newborn rat model of chronic
hyperoxia-induced lung injury(15) . Increases in hyaluronan and
PGs have also been described in animal models of pulmonary fibrosis
using N-nitroso-N-methylurethane (38) or
bleomycin (39, 40) and in humans with
fibroproliferative lung diseases(41, 42) . These
animal models of pulmonary fibrosis showed that changes in hyaluronan
and PGs occurred prior to collagen accumulation, suggesting that
hyaluronan and PG may modulate the later fibrotic
response(43) . Exposure of animals to elevated O
concentrations has also been shown to increase the amount of lung
collagen as well as elastin and
fibronectin(44, 45, 46) . To our knowledge,
no studies have reported a temporal relationship of PG and collagen in
an O
toxicity model of pulmonary fibrosis. In the present
study, we found that fetal lung fibroblasts increased type I collagen
and fibronectin mRNA expression in response to elevated concentrations
of O
, suggesting that fetal lung fibroblasts selectively
up-regulate and down-regulate gene expression of individual
extracellular matrix molecules. The observation that the effect of
elevated O
concentrations on PG and type I collagen gene
expression was reversible but that on fibronectin mRNA expression was
not strongly supports such selective regulation of extracellular matrix
expression by fetal lung fibroblasts in response to O
.
The exact mechanism by which oxygen may modulate GAG and PG
synthesis of fetal lung fibroblasts is not known. The inhibition of GAG
and PG synthesis by 50% O is not due to the loss of cell
viability, and the cell number remained constant. Furthermore, we have
previously demonstrated that fetal lung fibroblasts developed tolerance
to 50% O
, as measured by LDH release(37) .
Similarly, an alteration of
SO
incorporation
induced by elevated oxygen is not due to the changes in
sulfotransferase activities because
[
H]glucosamine incorporation into GAGs was also
altered. The toxic effects of oxygen are believed to be initiated
through increased intracellular generation of partially reduced oxygen
species. Superoxide, generated by the action of xanthine oxidase on
hypoxanthine, has been reported to decrease the synthesis of PGs in
cultured bovine chondrocytes(16) , bovine articular cartilage
explants(47) , and isolated perfused rat kidneys(48) .
Superoxide also damaged PG molecules at the level of the core protein,
while GAG side chains were resistant to free radical
attack(16) . In the present study, we did not investigate
whether elevated concentrations of O
damaged intact PGs
synthesized by fetal lung fibroblasts. However, the parallel decreases
in PG mRNA and
SO
incorporation into PGs make
it unlikely that the observed decrease in PGs is due to such an action
by reactive oxygen species. Consistent with our findings, superoxide
has been shown to increase collagen synthesis in the human fetal
fibroblast cell line, IMR-90(49) .