Department of Veterans Affairs Research Service and The University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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During the alveolar stage of lung development,
lipid droplet-laden interstitial cells are present at the base of
elongating alveolar septa. These cells that have been named lipid
interstitial cells or lipofibroblasts (LFs) may supply lipids for
surfactant production, the synthesis of membrane phospholipids,
and/or energy metabolism. They also have myofibroblastic
characteristics and participate in the generation of the interstitial
elastic fiber network, that is, in the pulmonary alveolar septum. To
understand how this cell regulates its lipid-storing and
elastin-producing properties, we have examined the effects of
peroxisome proliferators on the expression of the genes that are
associated with an elastin-producing myofibroblastic phenotype or an
adipocyte-like phenotype. Two known ligands for peroxisome
proliferator-activated receptors, 5,8,11,14-eicosatetraynoic acid
(ETYA) and 15-deoxy--12,14-prostaglandin J2
(15-dPGJ2), decrease elastin
gene transcription and the steady-state levels of tropoelastin (TE) and
-smooth muscle actin mRNAs in cultured LFs. Concurrently, cultured
LFs increase the expression of adipocyte lipid binding protein, which
is regarded as an adipocyte-specific protein, and accumulate lipid
droplets. Their abilities to store lipids and express desmin
intermediate filaments,
-smooth muscle actin, and smooth muscle
myosin heavy chain in contractile filaments in vitro illustrate
similarities among the pulmonary LF, the hepatic lipocyte, and the
contractile interstitial cell, which contribute to the repair reaction
in the lung after pulmonary injury.
myofibroblast; lung development; adipocyte; lipid storage; surfactant
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INTRODUCTION |
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DURING PERINATAL LUNG DEVELOPMENT, interstitial cells
that contain lipid droplets can be readily identified and have been termed lipid interstitial cells or pulmonary lipofibroblasts (LFs) (18,
21). These cells are first visible in rats after gestational day 15, remain abundant until
postnatal day 11, and afterward decrease in number (18). The droplets contain primarily neutral lipids,
triglycerides, and cholesterol esters as well as retinyl esters (18,
19). The disposition of the lipids and the reasons for their abundance
during this phase of lung development remain incompletely understood.
During this same period, these lipid-laden cells produce elastin and
other extracellular matrix proteins in vivo and in vitro (2, 19). Their
elastin production in vivo is linked to the myofibroblastic phenotype
because elastin is produced by skin myofibroblasts during keloid
formation and -smooth muscle-containing cells in the fetal bovine
lung (24, 28). Although the plastic phenotype of the myofibroblast has made it more difficult to define, recent progress in the biochemical and biophysical properties of these cells that have been reviewed has
produced a better and more defined understanding of their phenotype
(28). Culturing the lipid interstitial cells on a plastic
surface in the presence of fetal bovine serum is accompanied by a decrease in the volume density of lipid droplets and an increase in elastin production with increasing duration of the primary cultures
(2, 17). The features of lipid storage and extracellular matrix production are also shared by the hepatic stellate cells, also
known as hepatic lipocytes (4). These cells are the major storage depot
for retinol (vitamin A) in the adult animal and assume the
characteristics of myofibroblasts, producing
-smooth muscle actin
(
-SMA) and abundant collagen, during liver fibrosis (30). Culturing
the hepatic lipocyte on plastic is accompanied by a decrease in the
volume density of lipid droplets and retinyl esters and an increase in
-SMA and collagen production (26). In both the hepatic lipocyte and
the pulmonary LF, the factors that control the change from the lipid
storage to the myofibroblastic phenotype have not been completely
identified.
Although the LF and the hepatic lipocyte accumulate lipid droplets,
this does not appear to reflect a state of terminal differentiation as
it does for adipocytes (9). In the context of developmental and/or
inflammatory mediators (such as transforming growth factor-), these
cells, at least temporarily, lose their lipid-storing capacity and more
vigorously produce extracellular matrix proteins (9, 28). We desired to
determine whether mediators such as peroxisome proliferators
and their receptors that promote preadipocyte differentiation also
influence the phenotype of the LF and how they influence the
production of extracellular matrix proteins by the LF. Others have
hypothesized that the LF can be a progenitor of the contractile interstitial cell, which is a major contributor to the exuberant production of collagen and other extracellular matrix proteins, that
occurs during the reparative reaction to pulmonary injury (1).
Therefore, understanding the regulatory mechanisms that control the
transition from an adipocyte-like to a myofibroblastic phenotype could
ultimately allow one to direct the adult interstitial fibroblast back
to more of a lipid-storage phenotype and potentially reduce the
fibrotic response that may become excessive during repair
reactions after pulmonary injury (28).
Several transcription factors that regulate lipid accumulation in
adipocytes have been identified and recently reviewed (16). The
peroxisome proliferator-activated receptor- (PPAR-
), a member of
a family of receptors that utilizes the retinoid-X receptor (RXR) as a
DNA binding partner, has been shown to be both necessary and sufficient
to confer a lipid-storage phenotype to mesenchymal cells in culture
(32). Two members of the PPAR family have been studied rather
extensively, namely, PPAR-
and PPAR-
. The ligands for these
nuclear proteins are small amphipathic molecules such as fatty acids
and prostaglandins. The prostanoid 5,8,11,14-eicosatetraynoic acid
(ETYA) transactivates through both PPAR-
and PPAR-
. More specific
ligands for PPAR-
have been identified and include its apparent
natural ligand 15-deoxy-
-12,14-prostaglandin (PG)
J2 (15-dPGJ2) as well as a class of
drugs used for the treatment of diabetes mellitus, the
thiazolidinediones. Both ETYA and
15-dPGJ2 promote the
differentiation of preadipocytes to adipocytes and the accumulation of
lipid droplets (8, 34).
Elastin gene expression in cultured pulmonary LFs is increased by retinoic acid, a process that likely involves the RXR (15). We hypothesized that PPARs and RXR may influence lipid accumulation and elastin gene expression in the pulmonary LFs because this mesenchymal cell shares several morphological and biochemical characteristics with adipocytes and contributes to extracellular matrix accumulation during lung development and disease (21). Therefore, we examined the effects of ligands for the PPARs on elastin, type I collagen, and actin gene expression and on lipid accumulation in cultured pulmonary LFs.
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MATERIALS AND METHODS |
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Isolation and culturing lung fibroblasts. A lipid-laden subpopulation of fibroblasts was isolated from rat lungs at postnatal day 8 with a previously described procedure (5). Postconfluent cells were maintained in 25-cm2 tissue culture flasks in a mixture containing 50% Dulbecco's modified Eagle's medium and 50% Ham's F-12 medium supplemented with 2.5% bovine calf serum, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.5% Eagle's minimum essential medium nonessential amino acids (Sigma Chemical, St. Louis, MO), 100 µg/ml of streptomycin, and 100 U/ml of penicillin. At 7 or 8 days after subcultivation, some LF cultures were supplemented with various concentrations of ETYA or 15-dPGJ2 or the diluent dimethyl sulfoxide (DMSO). ETYA and 15-dPGJ2 were dissolved in DMSO, and the maximal concentration (vol/vol) of DMSO added was 0.015% in control and PPAR-ligand exposed cultures. Other LF cultures were supplemented with 75 µM ETYA or 15 µM 15-dPGJ2 for varying periods of time. A similar procedure was used to evaluate the effects of a 48-h exposure to 10 µM indomethacin and 20 µM 6,7-hydroxycoumarin, also known as esculatin (inhibitors of cyclooxygenase- and lipoxygenase-mediated PG metabolism, respectively). To evaluate the mechanism of the 15-dPGJ2-mediated decrease in tropoelastin (TE) mRNA, 5 µM cycloheximide was added to cultures of LFs for 12 h in either the presence or absence of 15 µM 15-dPGJ2. Preliminary studies showed that this concentration of cycloheximide inhibited total protein synthesis by 90%, in agreement with the findings of others (3).
Isolation of RNA and Northern
analysis. After the period of exposure to ETYA or
15-dPGJ2, total cellular RNA was
isolated with guanidinium isocyanate (6). Northern analyses were
performed with 1.2% agarose gels containing 6% formaldehyde as
previously described (19). The cationic nylon membranes were probed
with 32P-labeled cDNAs for rat TE,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal
phosphoprotein P-0, adipocyte lipid binding protein (ALBP or aP2;
provided by Dr. Bruce Spiegelman), rat actin, and mouse
1(I)-procollagen (19). The cDNA
for procollagen was provided by Dr. Benoit DeCrumbrugge. Ribosomal
phosphoprotein P-0 was used to normalize for differences in RNA loading
because this mRNA is not altered by PPAR ligands (32). The cDNA probe for rat ribosomal phosphoprotein P-0 was obtained with a reverse transcriptase-polymerase chain reaction with rat liver RNA and primers
that were designed with the mouse cDNA sequence (12, 19). The cDNA was
sequenced after subcloning into pBluescript SK(
) with the
dideoxy method to confirm that the clone was ribosomal phosphoprotein
P-0. After hybridization and washing, the membranes were exposed to
Kodak X-OMAT-AR5 film (Eastman Kodak, Rochester, NY) with an
intensifying screen. Exposure times were varied to achieve bands with
densities that were within the detection limits of the film, and the
resultant autoradiograms were subjected to densitometry with a Shimadzu
CS9000U densitometer in the zigzag mode.
Analysis of elastin gene
transcription. For these experiments, LFs were cultured
in 100-mm culture dishes for 10 days after the first subcultivation.
During the last 12 h, groups of four plates were supplemented with 75 µM ETYA or 15 µM 15-dPGJ2 or remained unsupplemented (control). After this 12-h period, nuclei were
isolated as previously described (15). The nuclear RNA was extended by
in vitro transcription following a procedure described previously (15).
Cationic nylon (Nytran, Schleicher and Scheull, Keene, NH) filters were
prepared containing 5 µg of plasmid DNA containing a cDNA insert for
rat GAPDH, rat elastin, ribosomal phosphoprotein P-0, or 5 µg of the
parent plasmids pPBR322 or pBluescript SK(), respectively.
Autoradiograms were exposed for 72-120 h, and densitometry was
performed. When the slot containing the plasmid without the insert
yielded a detectable signal, the densitometric reading was subtracted
from the densitometric reading for the slots that contained the plasmid
plus the insert to obtain the specific density. The specific densities
of the slots containing elastin cDNA for the control and RNA-exposed
cells were divided by the specific densities of the slots containing
the ribosomal phosphoprotein P-0 insert for the respective treatment
condition. This normalization compensated for differences in overall
transcription related to minor inequalities in the number of
nuclei used.
To examine whether 15-dPGJ2
promotes a direct interaction between a PPAR and the 5'-flanking
region of the elastin gene, LFs were transiently transfected with a
reporter construct containing 3.1 kb of the rat elastin promoter
regulatory region. In some instances, the LFs were cotransfected with
2 µg of an expression plasmid containing the mouse
PPAR-1 cDNA in pSVSport (32). Some cultures were exposed to 10 µM
15-dPGJ2, whereas others were exposed only to the vehicle DMSO. The transfections and chloramphenicol acetyltransferase (CAT) enzymatic assays were preformed as previously described, except that the reaction with
[14C]chloramphenicol
proceeded for 16 h rather than for 2 h (5). In all instances, the LFs
were cotransfected with the pTK-
GAL expression plasmid that contains
-galactosidase cDNA under the control of the thymidine kinase
promoter. The CAT enzymatic activity was normalized for the
-galactosidase enzymatic activity to account for differences in
transfection efficiency among the cultures.
Analyses of TE protein. After LFs were incubated with ETYA or 15-dPGJ2, the media were collected for analysis of soluble elastin by an enzyme-linked immunosorbent assay (ELISA) (16). The ELISA detects 75-kDa TE and other smaller immunoreactive elastin peptides that will collectively be referred to as soluble elastin. To normalize the data to the quantity of DNA per flask or well, the DNA content in an aliquot of the cell layer was assayed (27).
Because the ELISA detects other soluble elastin moieties besides 75-kDa
TE, additional studies were done to specifically quantify TE. Lung
fibroblasts were pretreated for 12 h in the presence or
absence of 15-dPGJ2, then exposed
for 60 min to 25 µg/ml of -aminoproprionitrile, and pulse labeled
for 6 h with
L-[3,4-3H]N-proline
(DuPont NEN, Boston, MA) with or without
15-dPGJ2.
-Aminoproprionitrile
was required to recover enough
3H-labeled soluble elastin in the
culture medium to produce a detectable fluorographic signal. Medium
samples containing equal quantities of radioactivity were used to
immunoprecipitate the newly labeled soluble elastin moieties with goat
anti-rat
-elastin, and the 75-kDa TE and other smaller
immunoreactive soluble elastin moieties were resolved by sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and identified
by fluorography (19). The quantities of 75-kDa TE in the presence and
absence of 15-dPGJ2 were analyzed
by densitometry.
The effects of ETYA and 15-dPGJ2 on total protein synthesis were evaluated by quantifying [3H]leucine incorporation into trichloroacetic acid-insoluble material. Various concentrations of ETYA or 15-dPGJ2 were added to some of the wells of 12-well plates, and 14 h later, the medium was replaced with leucine-free Dulbecco's modified Eagle's medium containing 2.5 µCi of L-[4,5-3H]leucine/ml. After 4 h, the incorporation of [3H]leucine into trichloroacetic acid-insoluble protein was quantified as described previously (15).
Analysis of cytoskeletal proteins using
immunoblotting. LFs were isolated from rats
on postnatal day 8 and used without
culturing or were cultured as previously described. Cultured LFs were
recovered by gentle scraping in 0.145 M NaCl, 0.0015 M
KH2PO4,
0.0027 M KCl, and 0.0086 M
Na2HPO4
[phosphate-buffered saline (PBS)], pH 7.4. After
centrifugation, cellular pellets were heated at 100°C for 5 min in
SDS-PAGE sample buffer and clarified by centrifugation (20). An aliquot
was removed for protein assay, and a portion of the remainder was
subjected to SDS-PAGE and immunoblotting with a gel containing 5%
bis-acrylamide for myosin heavy chain or a gel containing 7.5%
bis-acrylamide for vimentin, desmin, and -SMA (20). The quantities
of protein loaded were 50 µg for the analyses of vimentin and
-SMA, 100 µg for desmin, and 125 µg for smooth muscle myosin.
The proteins were transferred to nitrocellulose overnight at 35 V and
4°C. The following antibodies (all obtained from Sigma Chemical and
diluted 1:500) were used to probe the immunoblots: mouse monoclonal
anti-vimentin, rabbit polyclonal anti-desmin, mouse monoclonal
anti-
-SMA, and mouse monoclonal anti-smooth muscle myosin heavy
chain. After transfer, the nitrocellulose filters were blocked for 1 h
at room temperature in a solution containing PBS, 0.1% Tween 20, and
5% nonfat dry milk (solution A).
Incubations with the first antibodies were performed for 2 h at room
temperature. Washes were in solution A
except for desmin when the solution was 0.5 M NaCl rather than 0.145 M
NaCl (26). The second antibody was an affinity-purified, F(ab')2 rabbit anti-mouse
immunoglobulin G-peroxidase conjugate (Sigma Chemical) for vimentin,
-SMA, and myosin and an affinity-purified goat anti-rabbit
immunoglobulin G-peroxidase conjugate (Sigma Chemical) for desmin.
After a final wash, the peroxidase reaction was developed using the
enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
The filters were exposed to Kodak X-OMAT AR-5 film in the dark for
varying lengths of time (see Figs. 6 and 7). These
analyses were conducted at least three times with cellular extracts
from different cell cultures or animals.
Histochemical staining of intracellular lipid droplets. LFs were cultured on glass chamber slides (Nunc, Naperville, IL) that had been coated for 2 h at room temperature with a solution of 8 µg/ml of human fibronectin in PBS. Postconfluent cells were exposed to 75 µM ETYA or 10 µM 15-dPGJ2 for 24 h, and the cell layers were washed with PBS and then fixed for 30 s with 70% ethanol. The slides were then incubated for 1.5 min with a saturated solution of oil red O in 70% ethanol, rinsed for 3 s in 50% ethanol, and washed two times with deionized water (11). The slides were then counterstained for 2 min with Gill hematoxylin and mounted in aqueous mounting medium.
Statistical analyses. The data are expressed as means ± SE. Student's t-test was used when two experimental groups were compared. Multigroup analyses were done with a two-way analysis of variance, followed by a post hoc test with the multivariate general linear hypothesis module of Systat (Systat, Evanston, IL). Differences were considered significant when P was <0.05.
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RESULTS |
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Peroxisome proliferators decrease TE mRNA in
postconfluent LFs. LFs that had been maintained in
2.5% bovine calf serum were exposed to various concentrations of ETYA
for 24 h or 15-dPGJ2 for 18 h, and
RNA was isolated and subjected to Northern analysis. The results are
shown in Fig. 1 and demonstrate that both
agents produced a concentration-dependent decrease in TE (Fig.
1A) and 1(I)-procollagen mRNA (Fig.
1B). Higher concentrations of ETYA were required to decrease TE because 50 µM ETYA produced no
detectable decease, whereas 75 µM ETYA produced a mean decrease in TE
mRNA of ~50%. ETYA also decreased
1(I)-procollagen mRNA in a
concentration-dependent manner when concentrations exceeded 50 µM.
The prostanoid 15-dPGJ2 decreased
both TE and procollagen mRNA in a continuous fashion over the
concentration range from 5 to 20 µM. The highest concentration of
15-dPGJ2 that was tested, 30 µM,
did not produce a further reduction in TE or procollagen mRNA beyond
that observed at 20 µM. Concentrations of ETYA at or above 100 µM
were cytotoxic and lowered the DNA content of the cultures during
exposures lasting longer than 24 h.
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The time course of the decrease in TE and
1(I)-procollagen
mRNA was also examined with 75 µM ETYA (Fig.
2A) or
15 µM 15-dPGJ2 (Fig.
2B). Significant reductions in TE
mRNA were observed after 12 h of exposure to either agent, with an
additional decrement evident after 24 h. Lengthening the exposure to 48 h did not further reduce TE mRNA after exposure to either ETYA or
15-dPGJ2. The maximal reduction in
procollagen mRNA was observed after 12-24 h, and neither agent
produced an additional decrement when longer exposures were used.
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ETYA can reduce the production of PGs by the cyclooxygenase and lipoxygenase pathways. Therefore, it could decrease TE mRNA through a mechanism that involves the inhibition of PG synthesis rather than through PPARs. To address this issue, we examined the effects of indomethacin and esculatin on TE mRNA. Neither agent significantly reduced the steady-state levels of TE mRNA, which were 100 ± 5 and 89 ± 14% (SE; n = 3 cultures) of the control levels, respectively.
Peroxisome proliferators also decrease TE protein. Cultures that were exposed to 15 µM 15-dPGJ2 for 18 h and unexposed control cultures contained 177.1 ± 3.9 (n = 3) and 322.0 ± 21.9 ng of soluble elastin/µg DNA, respectively. When the results from three separate experiments were combined, 15-dPGJ2 reduced the soluble elastin content in the culture medium to 50.2 ± 6.4% of the control level, which is similar to the reduction in TE mRNA after 18 h (Fig. 2B). Exposure to 87.5 µM ETYA for 24 h also reduced the quantity of soluble elastin to 85.4 ± 26.0 ng/µg DNA (n = 3 cultures) compared with 280.6 ± 45.7 ng/µg DNA for the DMSO-exposed control culture. This reduction by ETYA is similar in magnitude to that of the decrement in TE mRNA that was observed at 24 h (Fig. 2A). A decrease in TE was also observed when LFs were metabolically labeled with [3H]proline and immunoreactive TE was recovered from the medium by immunoprecipitation and subjected to SDS-PAGE. Densitometric analysis of the resultant fluorogram showed 2,075 ± 107 density units of 75-kDa TE for control cultures and 683 ± 94 density units for cultures that were exposed to 15 µM 15-dPGJ2 for 18 h (P < 0.01; n = 3). The incorporation of [3H]amino acid into total protein was not decreased in the presence of 75 µM ETYA or 15 µM 15-dPGJ2, which were 93.2 ± 11.6 and 132.1 ± 11.1% of control levels, respectively (n = 5 cultures for both ETYA and 15-dPGJ2). However, 100 µM ETYA did significantly decrease incorporation to 41.3 ± 11.5% of the control level.
Peroxisome proliferators decrease elastin gene transcription. Postconfluent LF cultures were exposed to 75 µM ETYA or 15 µM 15-dPGJ2 for 12 h, and nuclei were isolated from the exposed cultures as well as from control cultures that were exposed only to the vehicle DMSO. Elastin transcripts were reduced to 5 ± 3% of the corresponding control level in cultures exposed to 15-dPGJ2 (P < 0.01; n = 4 cultures). A representative autoradiogram is shown in Fig. 3. The 95% reduction in elastin transcripts after 12 h is greater than the ~55% reduction in the steady-state level that was observed at this time. Therefore, a decrease in transcription can completely account for the steady-state level of TE mRNA, and a concomitant decrease in the steady-state level of TE mRNA half-life is unlikely. Exposure to ETYA also decreased elastin gene transcription (data not shown).
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Mechanisms of action of 15-dPGJ2 on elastin gene
expression. Cycloheximide, a general inhibitor of protein
synthesis, was used to evaluate whether the
15-dPGJ2-induced decrease in TE
mRNA required new protein synthesis. A representative (one of four experiments) Northern analysis is shown in Fig.
4 and demonstrates that the
15-dPGJ2-mediated decrease in TE
mRNA was observed in the absence of cycloheximide
(lane 3) but not in its presence (lane 4). Cycloheximide by itself
did not significantly alter TE mRNA (lane
2) compared with the control culture. The ratios of
the density of the bands containing TE mRNA to the density of their
respective bands containing ribosomal phosphoprotein P-0 mRNA for Fig.
4 were 0.226 for the control culture, 0.215 for cycloheximide (95% of
the control value), 0.122 for
15-dPGJ2 (54%), and 0.283 for
cycloheximide plus 15-dPGJ2
(125%). Therefore, new protein synthesis is required for
15-dPGJ2 to decrease
TE mRNA, consistent with an
indirect effect of peroxisome proliferators on elastin gene expression
rather than with a direct effect of a previously synthesized PPAR and
its ligand binding directly to an element in the transcriptional
regulatory region of the elastin gene. To further evaluate whether
15-dPGJ2 decreases TE mRNA by the
direct binding of a PPAR to the elastin 5'-regulatory region, LFs
were transiently transfected with a reporter plasmid construct
containing 3.1 kb of 5'-flanking DNA immediately upstream from
the TE translational start site in the presence or absence of a
PPAR- expression plasmid. Some cultures were then exposed to
15-dPGJ2. The results shown in
Fig. 5 demonstrate that neither 15-dPGJ2 alone nor along with
cotransfection with an expression plasmid containing PPAR-
cDNA
decreased elastin promoter-driven transcription of the CAT reporter
construct. These findings further support the conclusion that the
peroxisome proliferators decrease TE mRNA by an indirect rather than by
a direct mechanism.
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Analysis of cytoskeletal proteins in freshly isolated
and cultured LFs. Whole cellular extracts from freshly
isolated and postconfluent cultured LFs were subjected to SDS-PAGE and
immunoblotting (Fig. 6) with antibodies specific for
vimentin (lanes 1 and
2), desmin (lanes
3 and 4), and
-SMA (lanes 5 and
6). Lanes
1, 3, and
5 are from cultured LFs after one
subcultivation, whereas lanes 2,
4, and
6 are from freshly isolated LFs. Under
both conditions, LFs contain similar quantities of desmin as well as
vimentin intermediate filaments. However, culturing the cells was
associated with an increase in
-SMA (lane
5), which is characteristic of a more myofibroblastic
phenotype of the cultured cells. Figure 7 shows an
immunoblot probed with anti-smooth muscle myosin heavy chain antibody.
Lanes 1 and
2 show cellular extracts from cultured
LFs after the first subcultivation, and lanes
3 and 4 are from LFs after the second subcultivation. Lanes
5-7 are freshly isolated LFs, whereas
lane 8 is a rat aortic extract used as
a positive control for myosin heavy chain in smooth muscle cells.
Smooth muscle myosin heavy chain is expressed in cultured LFs but is not or only very weakly expressed in uncultured LFs. These findings also support the contention that LFs assume a more smooth muscle-like, myofibroblastic phenotype in culture that increases over two serial subculturings. They also demonstrate that the LF isolation procedure effectively excludes smooth muscle cells.
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15-dPGJ2 increases ALBP and lipid
droplets and decreases -SMA in LF cultures.
The preceding findings suggest that cultured LFs assume a more
myofibroblastic phenotype when they are cultured. Others have shown the
lipid-laden LFs lose their lipid droplets when they are cultured in
fetal bovine serum. Therefore, we cultured LFs in fetal bovine serum
and examined the effects of
15-dPGJ2 on
-SMA and ALBP mRNA,
markers of the myofibroblastic and adipocyte-like phenotypes,
respectively. The results of a representative Northern analysis are
shown in Fig. 8A and
indicate that 15-dPGJ2 decreases
-SMA mRNA and increases ALBP mRNA. These data suggest that
peroxisome proliferators can interrupt the transition to a
myofibroblastic phenotype and cause the cultured LFs to revert to a
more adipocyte-like phenotype. Figure
8B shows that, in contrast to cultured
LFs,
-SMA mRNA has a much lower abundance in freshly isolated LFs. These data corroborate the findings obtained with an antibody that
detects more
-SMA protein in cultured than in freshly isolated LFs.
To further confirm that the peroxisome proliferators induce an
adipocyte phenotype, cultured LFs were stained to identify lipid
droplets. Figure 9 shows that
postconfluent, control LFs have largely lost their lipid droplets (Fig.
9A) but that exposing them to ETYA
for 48 h restores the lipid droplets, which stain with oil red O (Fig.
9B). A similar change in phenotype
has been observed in LFs that have been exposed for 48 h to
15-dPGJ2 (data not
shown).
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DISCUSSION |
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The lipid-laden subpopulation of rat lung interstitial fibroblasts or
LFs share characteristics with both adipocytes and myofibroblasts. Others (1) have shown that morphologically the pulmonary LFs contain
cytoplasmic lipid droplets similar to those observed in adipocytes and
contractile microfilaments similar to those observed in myofibroblasts.
Our study demonstrates that when pulmonary LFs are cultured in fetal
bovine serum, they lose their lipid droplets and assume a more
myofibroblastic phenotype by increasing their expression of -SMA and
producing smooth muscle myosin. Maksvytis et al. (17) showed that when
these cells are cultured in neonatal rat serum, which is rich in
triglycerides and free fatty acids, they maintain or reacquire their
lipid droplets. We have shown that peroxisome proliferators increase
lipid droplet accumulation in LFs that have been cultured in fetal
bovine serum and have previously lost their lipid droplets (Fig. 9).
The accumulation of lipid is accompanied by an increase in the
expression of the ALBP gene, which is also expressed in differentiated
adipocytes. ETYA and 15-dPGJ2 also
decrease
-SMA mRNA, indicating that they downregulate the expression
of an mRNA that is characteristic of myofibroblasts. Therefore,
peroxisome proliferators reproduce the effects that Maksvytis et al.
observed with fatty acid-rich neonatal rat serum, suggesting that the
fatty acids, which have also been shown to be ligands for PPARs, may be
promoting lipid droplet accumulation through a pathway similar to that
utilized by the peroxisome proliferators (29). Others (14, 31) have shown that myoblast cell cultures can be converted to adipocyte-like cells in the presence of BRL-49653, a thiozolidinedione and specific ligand for PPAR-
. Similar to our observations with LFs, the
expression of the ALBP gene increased in myocytes and muscle-specific
-actin gene expression decreased (31). Approximately 24 h after the addition of BRL-49653, the myocytes began to express PPAR-
and C/EBP-
, which are critically involved in adipocyte differentiation (31). Others (10) have stimulated a conversion of myoblasts to
adipocytes by introducing retroviral constructs that express PPAR-
and C/EBP
.
Fibroblasts are not terminally differentiated cells and are usually a heterogeneous population, even in culture (28). Therefore, the alteration of fibroblast phenotype by PPARs may involve different mechanisms than those involved in the differentiation of the adipocyte or the reversal of the myoblast phenotype because the preadipocyte and the myoblast are, at least to some extent, "committed cell lines." The LF is a unique cell in that it undergoes phenotypic alterations during lung development that involves a decrease in proliferation, a loss of lipid droplets, and transient expression of the elastin gene (21). Therefore, studying the LF can provide insights into a developmental biological "program" that cannot be appreciated by studying the more classic preadipocyte and myoblast models.
ETYA and 15-dPGJ2 also have
downregulatory effects on elastin,
1(I)-procollagen, and
-SMA
gene expression. These occur in a concentration- and time-dependent
manner, and the effects on TE appear to occur at the level of
transcription. The decrease in TE mRNA occurs relatively rapidly and
before the appearance of lipid droplets, which are visible after 24 h
of exposure or longer. The fall in TE mRNA follows a similar time
course to the increase in ALBP mRNA, which also occurs by 12 h. A
decrease in elastin transcription appears to completely account for the
decrease in TE mRNA, and transcription is nearly completely abrogated
after a 12-h exposure to
15-dPGJ2. It appears
that the decrease in elastin gene transcription results from an
indirect effect rather than from a direct effect of a PPAR on an
element(s) in the portion of the 5'-flanking region of the
elastin gene that was analyzed. This does not preclude an interaction
with responsive elements further upstream or in the first intron.
Others (23) have recently shown that peroxisome proliferators that can
signal through PPAR-
decrease the expression of various genes in the
liver. These genes are not involved in lipid metabolism, and although
PPARs are required for the decrease in gene expression, this decrease
was not accompanied by alterations in the levels of PPAR-
or RXR-
mRNA. The observation that procollagen and
-SMA mRNAs also decrease
is consistent with the involvement of an intermediary factor(s) that
affects the expression of several genes and merits closer examination.
Our findings also illustrate that the lipid-laden subpopulation of
interstitial fibroblasts shares several characteristics with the
hepatic lipocyte or stellate cell, as others (25) have contended. In
vivo, both types of cells contain desmin as well as vimentin
intermediate filaments. When cultured, both the hepatic lipocyte and
the pulmonary LF contain -SMA and sarcomeric (smooth muscle) myosin,
and both lack sarcomeric myosin in vivo. However, the hepatic and
pulmonary lipid-storage cells differ in their expression of
-SMA in
vivo. The pulmonary LF contains
-SMA protein in the neonatal lung;
this protein is also expressed in some of the interstitial fibroblasts
in the adult lung, and it is particularly abundant in the cells located
at the septal tips (22). In contrast, the hepatic lipocyte lacks
-SMA in the normal liver, although it expresses this protein, as
well as sarcomeric myosin, in experimental cirrhosis in animals (30).
Culturing either the hepatic lipocyte or the pulmonary LF on plastic is
associated with changes in their synthesis of extracellular matrix
molecules. The cultured hepatic lipocyte synthesizes more collagen, and
type I collagen becomes more abundant than type III collagen, which is
the predominant fibrillar collagen in the liver (9). Retinoic acid
decreases collagen production by cultured hepatic lipocytes and by
cultured IMR-90 human fetal lung fibroblasts, which, like the LFs,
express
-SMA in vitro (7, 13). To our knowledge, studies of the effects of peroxisome proliferators on hepatic lipocytes have not been
reported.
The functions of the pulmonary LF during perinatal lung development currently remain incompletely defined. A study (33) with fetal LFs that were cocultured with alveolar type II cells showed that, in the presence of neonatal rat serum, LFs acquire triglycerides and transfer them to alveolar type II cells. Berk et al. (2) and Liu et al. (15) showed that freshly isolated LFs contain TE mRNA and that they produce both TE and insoluble elastin in vitro. Thus the LF appears to maintain both lipid-storage and elastin-producing capabilities in vivo, and their degree of commitment to either function in vitro is dependent on the culture conditions and duration in culture. The plastic phenotype of the perinatal LF makes it an interesting and informative cell for examining relationships between alveolar septal formation and surfactant production during pulmonary alveolar development.
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ACKNOWLEDGEMENTS |
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This research was supported by a Grant-in-Aid from the American Heart Association and by the Department of Veterans Affairs Research Service.
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FOOTNOTES |
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Address for reprint requests: S. McGowan, C33B G.H., Dept. of Internal Medicine, Univ. of Iowa Hospitals and Clinics, 200 Hawkins Dr., Iowa City, IA 52242.
Received 24 March 1997; accepted in final form 2 September 1997.
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