Peroxisome proliferators alter lipid acquisition and elastin gene expression in neonatal rat lung fibroblasts

Stephen E. McGowan, Sheila K. Jackson, Melissa M. Doro, and Paula J. Olson

Department of Veterans Affairs Research Service and The University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-Delta -12,14-prostaglandin J2 (15-dPGJ2), decrease elastin gene transcription and the steady-state levels of tropoelastin (TE) and alpha -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, alpha -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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -smooth muscle actin (alpha -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 alpha -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-beta ), 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-gamma (PPAR-gamma ), 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-alpha and PPAR-gamma . 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-alpha and PPAR-gamma . More specific ligands for PPAR-gamma have been identified and include its apparent natural ligand 15-deoxy-Delta -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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-gamma 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-beta GAL expression plasmid that contains beta -galactosidase cDNA under the control of the thymidine kinase promoter. The CAT enzymatic activity was normalized for the beta -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 beta -aminoproprionitrile, and pulse labeled for 6 h with L-[3,4-3H]N-proline (DuPont NEN, Boston, MA) with or without 15-dPGJ2. beta -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 alpha -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 alpha -SMA (20). The quantities of protein loaded were 50 µg for the analyses of vimentin and alpha -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-alpha -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, alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 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 alpha 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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Fig. 1.   Concentration-dependent effects of peroxisome proliferators on tropoelastin (TE; A) and alpha 1(I)-procollagen mRNA (B). Exposures for 24 or 18 h to 5,8,11,14-eicosatetraynoic acid (ETYA) and 15-deoxy-Delta -12,14-prostaglandin J2 (15-dPGJ2), respectively, decreased steady-state levels of TE mRNA and procollagen mRNA. Northern analysis was performed, and nylon filters were probed sequentially with rat TE cDNA, ribosomal phosphoprotein P-0 (RP-0) cDNA, and rat alpha 1(I)-procollagen cDNA. RP-0 served as a control to normalize for minor inequalities in amounts of RNA loaded in the various lanes. Autoradiograms were subjected to densitometry, and density of bands representing TE or procollagen mRNA was normalized to density of the corresponding band for RP-0 mRNA for each sample. Ratios of density of TE mRNA or procollagen mRNA to RP-0 mRNA for samples from flasks that had been exposed to either ETYA or 15-dPGJ2 are expressed as a percentage of ratio for control flasks that received only diluent (DMSO). For ETYA and 15-dPGJ2, concentrations are means ± SE from 3 independent experiments. * P < 0.05 compared with control (no ETYA or 15-dPGJ2) by 2-way analysis of variance (ANOVA).

The time course of the decrease in TE and alpha 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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Fig. 2.   Time course of effects of peroxisome proliferators on TE and alpha 1(I)-procollagen mRNA. Lung fibroblast cultures contained either 75 µM ETYA (A), 15 µM 15-dPGJ2 (B), or vehicle (DMSO) for various periods of time. Northern analyses were performed, and data were analyzed as described in Fig. 1. RP-0 mRNA was used to normalize for minor differences in quantities of RNA loaded. At each time, abundance of TE mRNA in a peroxisome proliferator-treated culture is expressed relative to that in a corresponding control flask that was cultured for the same duration, in parallel, in absence of ETYA or 15-dPGJ2. For TE and alpha 1(I)-procollagen, points represent means ± SE from 3 independent experiments. * P < 0.05 compared with control (no ETYA or 15-dPGJ2) by 2-way ANOVA.

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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Fig. 3.   15-dPGJ2 decreases elastin gene transcription. A representative in vitro transcription with nuclei that were isolated from lung fibroblasts that had been exposed to 15 µM 15-dPGJ2 for 12 h or that remained unexposed (control) is shown. 32P-labeled RNA was hybridized to cDNAs for rat TE, rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or rat RP-0. There was no detectable hybridization to control cDNAs that contained only the parent plasmids pBluescript SK(-) or pBR322 (data not shown).

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-gamma 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-gamma 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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Fig. 4.   Cycloheximide prevents 15-dPGJ2 from decreasing TE mRNA. A representative Northern analysis showing effect of cycloheximide on ability of 15-dPGJ2 to lower steady-state level of TE mRNA is shown. Cultures were exposed to cycloheximide and/or 15-dPGJ2 for 12 h, as indicated, and total RNA was isolated and subjected to Northern analysis. These results are representative of 3 other experiments that were performed. +, Presence; -, absence.


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Fig. 5.   15-dPGJ2 and perioxisome proliferator-activated receptor (PPAR)-gamma do not decrease elastin gene transcription by altering binding of a PPAR to proximal elastin promoter regulatory region. Cultured lipofibroblasts (LFs) were transfected with a plasmid containing 3.1 kb of rat elastin DNA immediately upstream from translational start site ligated to chloramphenicol acetyltransferase (CAT) gene along with an expression construct containing cDNA for beta -galactosidase. Some cells were also cotransfected with an expression plasmid containing mouse PPAR-gamma cDNA. Some cultures were treated with 10 µM 15-dPGJ2 for 48 h after transfection, when cells were lysed and used to assay CAT and beta -galactosidase enzymatic activity. CAT activity has been normalized to beta -galactosidase activity for each culture (n = 3 for each condition) and is expressed relative to that in control cultures that received only vehicle (DMSO).

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 alpha -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 alpha -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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Fig. 6.   Western immunoblot comparing quantities of intermediate filament proteins and actin in lung LFs immediately after isolation and after culture. Equal quantities of protein from LFs after the 1st subcultivation (lanes 1, 3, and 5) or immediately after isolation (lanes 2, 4, and 6) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose filter as described in MATERIALS AND METHODS. Filter was cut and probed with antibodies for vimentin (lanes 1 and 2), desmin (lanes 3 and 4), and alpha -smooth muscle actin (lanes 5 and 6). Primary antibodies were bound to peroxidase-conjugated secondary antibodies, and peroxidase reaction was developed with enhanced chemiluminescence system. Filters were exposed to film for 2 min. Comigration of proteins of various known molecular sizes is shown at right.


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Fig. 7.   Western immunoblot comparing quantities of myosin heavy chain in lung LFs immediately after isolation and after culture. Equal quantities of protein from cultured LFs after 1 subcultivation (lanes 1 and 2), after 2 subcultivations (lanes 3 and 4), or immediately after isolation (lanes 5-7) were subjected to SDS-PAGE. After transfer, nitrocellulose filter was probed with a monoclonal antibody to smooth muscle myosin heavy chain. Immunoblot was developed as described in Fig. 6, and fluorogram was exposed for 30 min. Lane 8, protein from an extract of rat aorta.

15-dPGJ2 increases ALBP and lipid droplets and decreases alpha -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 alpha -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 alpha -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, alpha -SMA mRNA has a much lower abundance in freshly isolated LFs. These data corroborate the findings obtained with an antibody that detects more alpha -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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Fig. 8.   Actin and adipocyte lipid binding protein (ALBP) mRNA in cultured and freshly isolated LFs. A: LFs were cultured in absence (lane 1) or presence of 15 µM 15-dPGJ2 for 12 (lane 2) or 24 h (lane 3), and RNA was isolated and subjected to Northern analysis. Filter was probed with cDNAs for actin that hybridize with cytoplasmic beta -actin and alpha -smooth muscle actin (alpha -SM-actin), ALBP, and RP-0. Similar results were obtained in 2 other experiments. B: RNA that was purified from freshly isolated LFs was subjected to Northern analysis and probed with cDNAs for actin and RP-0. Unlike in cultured LFs, alpha -SM-actin mRNA was below level of detection in LFs that were isolated at postnatal day 8 from 2 separate litters of rats (lanes 1 and 2).


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Fig. 9.   Lipid droplets accumulate in cultured lung LFs that have been exposed to ETYA. Postconfluent LFs were exposed to vehicle (DMSO; A) or 100 µM ETYA (B) for 24 h. Cell layers were stained with oil red O to demonstrate lipid droplets and were then counterstained with Gill hematoxylin.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -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-gamma . Similar to our observations with LFs, the expression of the ALBP gene increased in myocytes and muscle-specific alpha -actin gene expression decreased (31). Approximately 24 h after the addition of BRL-49653, the myocytes began to express PPAR-gamma and C/EBP-alpha , 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-gamma and C/EBPalpha .

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, alpha 1(I)-procollagen, and alpha -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-alpha 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-alpha or RXR-alpha mRNA. The observation that procollagen and alpha -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 alpha -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 alpha -SMA in vivo. The pulmonary LF contains alpha -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 alpha -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 alpha -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.

    ACKNOWLEDGEMENTS

This research was supported by a Grant-in-Aid from the American Heart Association and by the Department of Veterans Affairs Research Service.

    FOOTNOTES

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.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(6):L1249-L1257
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