Elastase release of basic fibroblast growth factor in pulmonary fibroblast cultures results in down-regulation of elastin gene transcription. A role for basic fibroblast growth factor in regulating lung repair.

We have reported previously that a factor released by elastase treatment of pulmonary fibroblast cultures is capable of down-regulating elastin gene expression. In the present study we have pursued the identification of the factor released by elastase treatment and the characterization of the level of elastin gene expression at which this factor exerts its effect. We have found by immunologic and biochemical procedures that elastase treatment results in the release of basic fibroblast growth factor (bFGF) that is bound within the matrix. Both purified bFGF and bFGF released by elastase from cell matrices decrease the transcriptional level of the elastin gene by 70-80% within 24 h. Transient transfections of pulmonary fibroblasts with a series of elastin promoter deletion constructs show that the region of the elastin gene responsive to bFGF is located within sequences spanning -900 to -200 base pairs. The biological implications of these findings coupled with our previous report are significant, since they demonstrate that elastase digestion of pulmonary fibroblast matrices not only results in the proteolysis of elastin but also results in the release of a potent regulator of elastin gene transcription whose activity can influence repair mechanisms.

Elastin is an extracellular protein whose intrinsic ability to passively expand and contract under gas and liquid pressure gradients renders it an important functional element in maintaining proper pulmonary function. The formation of insoluble elastin in the developing alveolar wall is an essential step in imparting the ability of the developing mammalian lung to meet the demands of gas exchange in respiratory dynamics. In pulmonary obstructive diseases such as emphysema, the continual loss of elastin from alveolar walls with concomitant enlargement of air spaces is a significant factor in the pathological process. This loss of elastin is thought to result from elastase activities arising from enzymes secreted by an influx of macrophages and neutrophils into the airways after prolonged physical or chemical insult (Albin et al., 1987;Sandhaus, 1987;Snider et al., 1991). Although metabolic labeling studies have shown that elastin can be resynthesized to pre-insult levels, ultrastructural evaluation suggests that the repair is disorganized and dysfunctional (Kuhn et al., 1976).
Previously we have reported on an in vitro model, which mimics conditions proposed to trigger the development and perpetuation of the emphysematous condition, i.e. elastase digestion of the extracellular matrix (Foster et al., 1990). In this model primary cultures of neonatal rat pulmonary fibroblasts, which synthesize a matrix containing insoluble elastin as well as other essential matrix components, are briefly exposed to elastase and the cellular response examined. Using this system we have found that the products released by elastase exposure are capable of significantly down-regulating steady-state levels of elastin mRNA in the control pulmonary fibroblast cultures. Since the amino acid analysis of the elastase-solubilized fraction exhibited a composition indicative of elastin, we hypothesized that elastin peptides were capable of autoregulating elastin gene expression in a negative manner (Foster et al., 1990).
The overall goals of the present study were to determine the level at which elastase solubilized products of pulmonary fibroblast cultures down-regulate elastin mRNA and to identify the elastin peptide responsible for this activity. We have found that the down-regulation of elastin expression occurs at the transcriptional level, and, contrary to our initial hypothesis, the factor responsible is not an elastin peptide but rather bFGF, 1 which is released from the matrix upon elastase treatment.

MATERIALS AND METHODS
Reagents-Human recombinant bFGF (18 kDa) was from Scios-Nova (Mountain View, CA). Mouse monoclonal anti-bovine bFGF, Type II IgG (Upstate Biotechnology Inc., Lake Placid, NY), was used for immunocytochemistry and Western blot analyses. Mouse monoclonal anti-bovine bFGF, Type I IgG (Upstate Biotechnology Inc.), was used to neutralize bFGF activity.
Establishment and Treatment of Cell Cultures-Neonatal rat pulmonary fibroblast cells were isolated from lungs of 2-3-day-old Sprague-Dawley rats and seeded in first passage in 75-cm 2 flasks at a density of 2 ϫ 10 4 cells/cm 2 in 20 ml of Dulbecco's modified Eagle's medium (DMEM) and 5% fetal bovine serum (FBS) as described previously (Foster et al., 1990). Medium was changed twice weekly. After 18 days, the cell cultures were treated with either elastase-generated digest (5-15 g/ml) or purified bFGF (2-50 ng/ml) for 24 h.
The generation of elastase matrix products was performed essentially as we have previously detailed (Foster et al., 1990) but with several differences. After collection of the medium from elastase digestion and treatment to inhibit elastase, the mixture was either used directly or was lyophilized, dissolved in 0.1 original volume of 44 mM NaHCO 3 and then dialyzed against 44 mM NaHCO 3 using a membrane * This work was supported by National Institutes of Health Grant HL 46100 and HL 46902 and a Whitaker Foundation biomedical engineering research grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. with a molecular weight exclusion of 6,000. The protein concentrations of the original and concentrated digest were determined by the BCA protein assay (Pierce).
The Type I neutralizing monoclonal antibody to bFGF (Upstate Biotechnology Inc.) was used to inhibit the activity of bFGF in the cell cultures. Specified amounts of antibody were added to the cell cultures at the same time as 10 ng/ml bFGF or 5 g/ml elastase-generated digest and the cells incubated for 24 h.
Immunocytochemistry and Confocal Analysis-Pulmonary fibroblasts were cultured on dual chamber glass slides (Nunc) for 10 or 14 days. Cell cultures were treated with elastase (0.5 g/ml) or with buffer alone for 10 min at 37°C and then washed once with PBS and fixed with 3.7% formaldehyde for 30 min at room temperature. Fixed cultures were washed twice with PBS and incubated in blocking buffer (PBS, 3% BSA) for 10 min at room temperature. They were then incubated overnight at 4°C in PBS, 1% BSA, with one chamber on each slide receiving the primary antibody (anti-bFGF Type II, 5 g/ml). After the primary antibody incubation, both chambers on each slide were incubated with fluorescein-conjugated secondary antibody (horse antimouse IgG) at a final dilution of 1:300 in PBS, 1% BSA for 1 h at room temperature. Cultures were then examined using a Leica confocal laser scanning microscope (CLSM) in conjunction with Voxel View Imaging software run on a Silicon Graphics workstation (Trinkaus-Randall et al., 1993). Control cells, not incubated with anti-bFGF, were used to establish CLSM voltage settings such that no fluorescence is visible within the control cultures and these same settings were used to analyze the primary antibody-containing cultures. In 2 regions of each sample treated with anti-bFGF, 6 separate 2-m z-series optical sections were taken from the apical to the basal surface of the cells. The relative fluorescent intensity in each optical section was quantitated in the x,y plane.
Western Blot Analysis-Digests recovered from elastase-and mocktreated pulmonary fibroblast were made 1 M in diisopropyl fluorophosphate to inhibit elastase activity and then directly analyzed by 16% SDS-PAGE. Recombinant bFGF and a set of protein molecular weight standards were included on each gel analysis. The samples were electrophoretically transferred to nitrocellulose by the method of Towbin et al. (1979). The blot was treated as we have described previously (Jensen et al., 1995) using the Type II monoclonal antibody to bFGF (1 g/ml) and goat anti-mouse IgG (Bio-Rad) conjugated to horseradish peroxidase and then treated with Amersham ECL reagents and exposed to x-ray film for specified times.

125
I-bFGF Binding and Release-Equilibrium binding of 125 I-bFGF was conducted with confluent pulmonary fibroblasts (Nugent and Edelman, 1992). 125 I-bFGF was prepared using a modification of the Bolton-Hunter method that we have described previously (Nugent and Edelman, 1992). Second passage cells (5 ϫ 10 4 /well) were plated into 24-well plates (2 cm 2 /well, Costar, Cambridge, MA) in 1 ml of DMEM, 10% FBS and incubated at 37°C for 3 days. Prior to initiating the binding assay, the culture medium was removed and the cells were washed once with binding buffer (DMEM, 25 mM HEPES, 0.05% gelatin) at 4°C. Fresh binding buffer was added (0.5 ml/well), and the cells were incubated at 4°C for 10 min. 125 I-bFGF (20 ng/ml) was added, and the cells were incubated at 4°C for 2.5 h. At the end of the binding incubation, non-bound 125 I-bFGF was removed by washing the cells four times: three times with cold binding buffer followed by one wash with PBS at room temperature. The cells were then incubated with 44 mM NaHCO 3 , pH 7.4, with and without elastase (0.5 g/ml) for various times (0 -30 min) at room temperature. At each time point the enzyme solution was removed, the cells were washed once with PBS and extracted with 1 N NaOH, and the amount of cell-bound 125 I-bFGF was quantitated with a ␥ counter. The amount of 125 I-bFGF bound to the cells at each time point was subtracted from the amount bound at time zero (1.06 Ϯ 0.06 ng/well) to determine the amount released. Observed first order rates were determined by non-linear least squares curve fitting (Kaleida-Graph version 3.0.4, Synergy Software, Reading, PA).
Isolation and Analysis of RNA-Total RNA was isolated and analyzed by Northern blotting as described previously (Wolfe et al., 1993). Duplicate 10-g samples of total RNA were fractionated on a 1.1% agarose, 6% formaldehyde gel and electrophoretically transferred to a Nytran filter (Foster et al., 1988). Prior to electrophoretic transfer, the gel was cut in half so that one of each duplicate sample could be stained with acridine orange to monitor sample loading and RNA integrity and to establish electrophoretic size markers. After electrophoretic transfer of the gel to Nytran, hybridization was performed with 32 P-labeled rat tropoelastin (Rich and Foster, 1989) and rat actin (gift of Dr. Nadal-Ginard) cDNAs as detailed previously (Wu et al., 1992). The filter was first exposed to x-ray film at Ϫ80°C using an intensifying screen and then analyzed on a phosphoimager cassette for quantitation using a Molecular Dynamics PhosphorImager.
Transcription Nuclear Run-on Analysis-Nuclei from control, elastase-generated digest (5 g/ml), and bFGF-treated (10 ng/ml) pulmonary fibroblast cultures were isolated and subjected to nuclear run-on analyses as detailed elsewhere (Wolfe et al., 1993). Two separate sets of primary cell cultures were used for these analyses with each set run in duplicate. Ten micrograms of pBluescript DNA and chimeric pBluescript DNA containing cDNAs for tropoelastin, lysyl oxidase (gift of Dr. Philip Trackman), actin, and histone 2B were applied to nitrocellulose using a slot blot apparatus. The prehybridized filters were incubated with recovered run-on solutions (4 ϫ 10 6 cpm/ml) at 65°C for 72 h, then washed and treated with RNase (Wolfe et al., 1993). The nitrocellulose filters were first exposed to x-ray film at Ϫ80°C for 5 days and then analyzed on a phosphoimager cassette (24 -48 h) for quantitation using a Molecular Dynamics PhosphorImager.
Transient DNA Transfections and CAT Assays-Pulmonary fibroblasts were transfected with plasmid DNA (30 g) purified by two successive CsCl density gradient centrifugations using the calcium-phosphate co-precipitation method as we have previously reported (Wolfe et al., 1993). A series of deletion constructs containing fragments of the human elastin gene promoter driving a CAT reporter were used for transfection assays (generously supplied by Drs. Bashir and Rosenbloom and detailed in Wolfe et al., 1993). Deletion constructs comprised sequences Ϫ2200 to ϩ2 bp, Ϫ900 to ϩ2 bp, Ϫ500 to ϩ2 bp, and Ϫ195 to ϩ2 bp of the elastin promoter (Wolfe et al., 1993). Transfection efficiencies were assessed by co-transfection with 5 g of pCMV ␤-gal (Clontech). Second passage cells were plated in 100-mm tissue culture plates at a density of 3 ϫ 10 4 cells/cm 2 in DMEM/5% FBS medium and incubated overnight. The medium was changed, the calcium phosphate/ DNA precipitate added, and the cell cultures incubated for 20-22 h. After removal of the medium, the cell layer was washed twice with DMEM and the cells then treated with 3 ml of 15% glycerol in 0.15 M NaCl, 1.4 mM Na 2 HPO 4 , 25 mM Hepes, pH 7.12, for 30 s. Subsequently, the cells were washed twice with DMEM and then incubated in DMEM/0.5% FBS for 24 h. bFGF or a vehicle control in 0.5% FBS were then added for 24 h. Four separate sets of pulmonary fibroblasts and two different preparations of plasmid DNAs were used for these analyses. Determination of CAT and ␤-galactosidase activities were performed as described previously (Wolfe et al., 1993), with the one exception that quantitation of acetylated and nonacetylated forms of [ 14 C]chloroamphenicol was analyzed by PhosphorImager analysis of the intact thin layer plate.

Fibroblast Growth Factor Mimics the Action of Matrix Degradation Products on Elastin mRNA Levels-We have reported
previously that the addition of elastase-generated, matrix degradation products to pulmonary fibroblasts cultures resulted in the down-regulation of elastin protein and mRNA levels. In attempts to purify the active agent in the elastase solubilized products, we initially focused our efforts on the possibility that the active compound is an elastin fragment released by elastase treatment. This hypothesis was based on the amino acid composition of the protein/peptide fraction released by elastase treatment, which exhibited a composition very similar to that of rat insoluble elastin. We found that the more we fractionated the digest, to purify elastin peptides, the less activity we recovered. These observations, together with our inability to demonstrate either reproducible or significant activity with different purified rat insoluble elastin digests and rat tropoelastin preparations, led us to explore the possibility that a low abundant but potent growth factor or cytokine was released from the matrix by elastase treatment. Two such factors, i.e. bFGF and TGF-␤, are known to be associated with matrix molecules and both are capable of modulating elastin gene expression (Liu and Davidson, 1988;Kahari et al., 1992;Brettell and McGowan, 1994). Although TGF-␤ did not alter elastin mRNA levels (data not shown), bFGF had a significant effect. The Northern blot given in Fig. 1 compares the addition of elastasegenerated peptides and purified bFGF on the steady-state levels of elastin and actin mRNAs. The addition of elastase digest to the pulmonary fibroblasts resulted in a 78 Ϯ 10% reduction in elastin mRNA consistent with our previous findings (Foster et al., 1990). Interestingly, the addition of 10 ng/ml FGF also decreased elastin mRNA levels to comparable levels in the three separate experiments, i.e. 80 Ϯ 10%. The levels of actin mRNAs were not changed by either treatment, showing that the decrease in elastin mRNA levels is not indicative of a generalized response.
Elastase-generated Peptides and Basic Fibroblast Growth Factor Decrease Levels of Elastin Transcription-Nuclear run-on assays were performed on nuclei isolated from pulmonary fibroblasts cultures treated with either elastase-generated digest or bFGF. Fig. 2A provides the nuclear run-on blots obtained from a typical analysis performed, and Fig. 2B provides a quantitative analysis of data obtained from three separate sets of pulmonary fibroblasts run in duplicate. The transcriptional level determined for each gene in the control cultures is given as 100%, and the levels obtained after addition of either elastase digest or bFGF to the cells are plotted as a percent of that control. These results demonstrate that both the elastase-generated digest and bFGF down-regulate elastin transcriptional levels by approximately 83-87%, which is in very close agreement with the reduction of the steady-state levels of elastin mRNA. Transcription levels of lysyl oxidase and actin do not change significantly with either treatment. The transcriptional level of histone 2B remained relatively constant among control, elastase digest, and bFGF-treated cells, suggesting that neither treatment results in a general increase in the number of cells entering S phase. However, we did not render the cells quiescent or attempt to synchronize them in G 0 so that we cannot exclude the possibility that either the digest or bFGF contributes to cell cycle progression within the limited 24-h period examined.
Basic Fibroblast Growth Factor Is Released after Treatment of the Pulmonary Fibroblasts Cultures with Elastase and Is the Active Factor in the Down-regulation of Elastin mRNA Levels--Since the effect of bFGF on elastin transcriptional and mRNA steady-state levels closely mirrored that of the elastase digest, we investigated the possibility that bFGF was both present in the matrix of the pulmonary fibroblast cultures and that it was released into the medium by elastase treatment. Several approaches were used to address these possibilities. Second passage fibroblast cells were plated onto dual chamber slides, allowed to grow for 10 -14 days, and then processed for immunocytochemistry analysis. Photomicrographs of CLSM images of control and elastase-treated pulmonary fibroblasts stained with a monoclonal antibody to bFGF are shown in Fig. 3. The images presented represent sections near the basal surface of the cells (6 m down from the apical surface). Intracellular staining was apparent in both control and elastase-treated cultures. However, significant extracellular staining was only observed in control cultures, suggesting that elastase treat-ment specifically released bFGF from the extracellular matrix. Both control and elastase-treated cells stained positively for bFGF. While both intracellular and extracellular staining was apparent, the most intense staining was within the cells in both conditions. However, the overall fluorescence intensity was significantly greater in the control cultures compared to the elastase-treated cultures. When the fluorescence intensity was quantitated in 12 separate paired images from each condition, a 78.7 Ϯ 5.0% decrease was observed in elastase-treated cultures compared to controls. The intracellular fluorescence intensity was also decreased but to a lesser extent by elastase (43.8 Ϯ 7.2%; n ϭ 12 cells per image). Thus the overall decrease in bFGF staining reflects the dramatic loss of bFGF from the matrix.
Western blot analysis was performed on extracts obtained from elastase and vehicle-treated cultures to establish the presence of bFGF in the elastase-generated digest. Fig. 4 provides a blot comparing the immunoreactivity of proteins released by elastase digestion of the pulmonary fibroblast cell cultures with recombinant bFGF. The treatment of pulmonary fibroblasts with elastase results in the release of an immunoreactive polypeptide possessing an apparent molecular weight of 18,000, which exhibits an electrophoretic mobility very similar to recombinant bFGF. Significantly this immunoreactive polypeptide is not detectable in mock-treated cells. A minor, higher molecular weight band was visible in the control lane FIG. 1. Effect of elastase digest and bFGF on the steady-state levels of tropoelastin and actin mRNAs. Panel A, Northern blot analysis was performed on 10-g samples of total RNA using elastin and actin cDNA probes. Eighteen-day-old rat pulmonary fibroblast cell cultures were treated for 24 h as follows: lane 1, mock treatment with vehicle solution (500 l of 44 mM NaHCO 3 ); lane 2, treatment with elastase-generated digest (5 g/ml medium); lane 3, treatment with bFGF (2 ng/ml medium); lane 4, treatment with bFGF (10 ng/ml). Panel B, duplicate 10-g samples of total RNA were fractionated on the Northern gel and stained with acridine orange to monitor sample loading and RNA integrity. Each of the lanes are designated with a "d" to represent a duplication of the samples described in panel A.

FIG. 2. Effect of elastase digest and bFGF on the transcriptional activity of the elastin gene. Panel A,
18-day-old rat pulmonary fibroblast cell cultures were treated with either elastase-generated digest (5 g/ml) or bFGF (10 ng/ml) for 24 h. Nuclei from control and treated cultures were isolated and nuclear run-on assays performed in the presence of [ 32 P]UTP for 20 min. The nascent 32 P-labeled transcripts were hybridized to slots of filter-bound elastin, actin, lysyl oxidase, and histone 2B cDNAs, and pBluescript DNA. The nitrocellulose filters were exposed to x-ray film at Ϫ80°C for 5 days. Panel B, three separate sets of pulmonary fibroblast cultures were treated and subjected to nuclear run-on analysis as described above. Quantitation of individual slots was performed using a Molecular Dynamics PhosphorImager after a 24-h exposure. In order to combine three separate analyses, the transcriptional level of the various genes in the control cultures was set at 100% for each experiment and the levels obtained after addition of either elastase digest or bFGF were plotted as a percent of that control. Values represent the average Ϯ standard deviation. only. We do not know if this band is related to bFGF or simply interacts nonspecifically with the antibody used.
To further confirm that elastase can facilitate bFGF release from the pulmonary fibroblast matrix, the release of 125 I-bFGF from fibroblast matrices was measured in the presence and absence of elastase. 125 I-bFGF was incorporated into the matrices of pulmonary fibroblast by incubation of the cells with 20 ng/ml 125 I-bFGF at 4°C. Under these conditions cellular internalization is inhibited and bFGF is predominantly bound to heparin sulfate within the matrix (ϳ95% of all bFGF bound) (Nugent and Edelman, 1992). The 125 I-bFGF-labeled cultures were then incubated in buffer with or without elastase for various times. As shown in Fig. 5, the presence of elastase accelerated the rate of bFGF release from the matrix 22-fold (apparent first order rate constants, k obs ϭ 0.014 min Ϫ1 for control; k obs ϭ 0.314 min Ϫ1 for elastase-treated cells).
In order to test directly whether bFGF is the factor in the elastase digest that is responsible for the down-regulation of elastin mRNA, a neutralizing antibody to bFGF was added to fibroblast medium together with either the elastase-generated peptides or bFGF. Fig. 6A shows a Northern blot and Fig. 6B the quantitative analyses of three experiments performed with separate sets of primary fibroblasts. A preparation of nonimmune serum was used to establish nonspecific interactions. Although not shown, rRNA and actin mRNA were used to determine loading and specificity. Whereas the addition of 20 or 40 l of antibody to cells simultaneously exposed to bFGF (10 ng) partially blocked elastin mRNA down-regulation, the addition of 40 l of antibody together with the elastase digest not only blocked the down-regulation but also resulted in a small but reproducible increase in mRNA levels over control. Although not shown, the addition of 40 l of antibody to control cultures also results in a 10% increase in elastin mRNA. These data directly link the active agent in the elastase digest to bFGF and further suggest that elastin gene expression may be negatively regulated by endogenous bFGF in the control pulmonary fibroblasts.
The FGF Response Element Is Located in the Distal Region of the Human Elastin Gene Promoter-Pulmonary fibroblast cells were transiently transfected with a series of human elastin promoter deletion constructs in the absence and presence of 10 ng/ml bFGF. A representative CAT assay for these elastin gene constructs in control (Ϫ) and bFGF-treated (10 ng/ml medium) cells (ϩ) is provided in Fig. 7. The amount of extract from each transfection assayed for CAT activity was normalized to 100 g FIG. 3. Immunolocalization of bFGF in control and elastasetreated pulmonary fibroblast cultures. Pulmonary fibroblasts were treated with buffer alone (A) or with elastase (B) (0.5 g/ml; 10 min). Cells were stained with a monoclonal antibody to bFGF and a fluorescein-conjugated secondary antibody. Fluorescence was visualized by confocal laser scanning microscopy. Images are ϳ6 m below the apical surface of the cells.
FIG. 4. Elastase treatment of pulmonary fibroblast cultures results in the release of bFGF. Eighteen-day-old rat pulmonary fibroblast cell cultures were treated with elastase (0.5 g/ml) or with buffer alone for 10 min at 37°C. Media recovered from elastase-and mock-treated pulmonary fibroblast were made 1 M in diisopropyl fluorophosphate to inhibit elastase activity and 50 g directly analyzed by 16% SDS-PAGE. Recombinant bFGF and a set of protein molecular weight standards were included with each gel analysis. Western blot analysis was performed as detailed under "Materials and Methods." FIG. 5. Elastase stimulates bFGF release from pulmonary fibroblast matrix. 125I-bFGF was bound to cell matrix (1.06 Ϯ 0.06 ng/well) and the cells treated with (q) or without (⅜) elastase for the indicated times. The amount released from the cell layer (average Ϯ S.E. of triplicates) was determined at each time point. of protein and adjusted for transfection efficiency by co-transfection and measurement of ␤-gal activity. The results of four separate transfection experiments using two different preparations of plasmid DNAs are given in Fig. 8. In the control fibroblast cultures, the elastin gene deletion constructs dictate a basal pattern of activity reflecting several functional regulatory subregions similar to that reported in other cell types Kahari et al., 1990;Wolfe et al., 1993). With the addition of bFGF to cells, activity of the Ϫ195 to ϩ2 bp promoter fragment (set at 100% in the control) did not change significantly, whereas activities driven by both the Ϫ500 to ϩ2 and the Ϫ900 to ϩ2 bp fragments decreased by ϳ35% and ϳ65%, respectively. Although not shown, a Ϫ2200 to ϩ2 bp fragment exhibited ϳ45% decrease in cells exposed to bFGF. In composite, these results demonstrate that the down-regulation of elastin gene transcription measured directly by nuclear runons is reflected in the transient transfection assays with elastin gene promoter fragments driving CAT activity, although the effects measured by transfection assays are not as great as those found for endogenous gene activity. There are several possible explanations for this difference, which we will detail under "Discussion." The data are consistent with the proposal that the bFGF response element(s) is located between Ϫ900 and Ϫ195 bp of the elastin gene promoter. Since both the Ϫ900 and Ϫ500 bp fragments confer a responsiveness to bFGF, we cannot narrow the specific sequences targeted nor can we suggest that only one element may be involved.

DISCUSSION
In a previous publication from our laboratory, we reported on the development of an in vitro model for examining mechanisms operative in the development and propagation of pulmonary emphysema. Experimental data obtained from that model showed that elastase digestion of pulmonary fibroblast matrices results in the release of a factor(s) that could influence elastin gene expression in a positive or negative fashion dependent upon whether the cultures had been treated with elastase prior to the addition of the elastase digest (Foster et al., 1990). In the present study, we have focused on the ability of the elastase digest to down-regulate elastin gene expression in control cell cultures (no elastase treatment) since this is the simpler of the two situations. Within this system, we initially pursued the hypothesis that an elastin fragment was responsible for down-regulation by an autoregulatory mechanism. However, we switched our efforts after numerous attempts to purify putative elastin active fragments with no success. We therefore considered the possibility that elastase digestion might release a matrix sequestered factor present in low amounts relative to elastin peptides, but potent in terms of its ability to modulate elastin gene expression. Although there are several growth factors/cytokines that have been shown to down-regulate elastin gene expression (Berk et al., 1991;Kahari et al., 1992), the pulmonary fibroblast system utilized is devoid of any inflammatory cells that might secrete exogenous factors. Basic fibroblast growth factor was a likely candidate to target within pulmonary fibroblast cells, especially since Brettell and McGowan (1994) have already shown that bFGF decreases elastin production in cell cultures similar to those FIG. 6. Neutralizing antibody to bFGF inhibits the activity of the elastase digest in pulmonary fibroblasts cultures. Panel A, Northern blot analysis was performed on 10-g samples of total RNA using elastin and actin cDNA probes (latter mRNA is not shown). Eighteen-day-old rat pulmonary fibroblast cell cultures were treated with either elastase-generated digest (5 g/ml) or bFGF (10 ng/ml) for 24 h in the absence or presence of 20 and 40 l of Type I neutralizing monoclonal antibody (Ab) to bFGF.  figure) and 5 g of pCMV ␤-gal in the (ϩ) presence (10 ng/ml of medium) or (Ϫ) absence of bFGF (see "Materials and Methods" for details). Aliquots of cell lysates, containing equal amounts of ␤-galactosidase activity, were subjected to thin layer chromatography in order to separate 14 C-acetylated chloroamphenicol derivatives and the resultant thin layer plate exposed to x-ray film. A control lane (C) with CAT enzyme is also shown. used in our studies. These latter investigators demonstrated that bFGF was capable of decreasing elastin mRNA and protein levels and suggested that regulation could be exerted at the transcriptional level based on transient transfections of a CAT reporter gene driven by a 2.26-kilobase human elastin gene promoter fragment. Additionally, Davidson et al. (1993) have shown previously that bFGF inhibits the TGF-␤ stimulation of elastin production in smooth muscle cells. In the present study, we have directly measured transcription levels of the elastin gene by nuclear run-on experiments and have found that both elastase digest and bFGF decrease elastin gene transcription levels by comparable and significant amounts. More importantly, we have shown that bFGF is a factor released by elastase digestion of pulmonary fibroblast cell matrices thereby attributing its activity to the biologically relevant situation of injury/repair known to exist in the development and progression of pulmonary emphysema (Sandhaus, 1987;Snider et al., 1991).
Although we have shown that bFGF is a potent modulator of elastin gene transcription within an environment mimicking elastase-induced injury, we were unable to specifically localize the bFGF response region within the promoter except for excluding its existence within the proximal promoter region, i.e. Ϫ195 to ϩ2. The magnitude of the effect found by transient transfections is not as great as that measured for the endogenous gene response. The reasons for our inability to localize more precisely the region of bFGF responsiveness and the magnitude of that response may reside in the fact that we are using a heterologous system consisting of human elastin promoter sequences within rat cells or may reflect the use of an inappropriate reporter activity (CAT) to investigate down-regulation. We will address both of these issues in future studies by using rat elastin gene promoter constructs driving a luciferase reporter. We do not plan to switch to stable transfections since we have already found that creating stable transfectants by repeated passages of pulmonary fibroblasts results in a dramatic decrease in elastin synthesis as well as a complete loss of elastin gene regulation. 2 The results obtained in this study are important to developing hypotheses for mechanisms underlying normal and abnormal repair of pulmonary elastin. Our previous work suggested that the ability of the fibroblast cells to synthesize tropoelastin after elastase digestion was dependent on both its location relative to the site of injury and its competency to respond to factors released by elastase digestion of matrix components. Since bFGF has been identified as one of the components released by elastase treatment of fibroblast cultures, this suggests that bFGF may act as a repressor of elastin gene transcription in cells remote from an elastase injury, perhaps to prevent a general fibrotic response. Cells adjacent to the elastase-induced injury might not be immediately affected by bFGF release due to damage to FGF receptor or FGF-binding proteoglycan. Additionally, the cells adjacent to elastase activity might be selectively more responsive to potential up-regulators of elastin as the result of differential proteolytic damage.