Introduction

Top Abstract Introduction Materials and methods Results Discussion References

Cell proliferation in the fetal lung is regulated by a complex integration of various inhibitory and stimulatory factors present in the cellular microenvironment. Locally produced peptide growth factors, such as the insulin-like growth factors (IGFs), play a major role in this process by providing cell-specific signals that regulate mitogenesis. IGF-I and IGF-II are small secreted peptides that promote cell proliferation in many tissues through autocrine and/or paracrine interaction with a cell-surface receptor, the type 1 IGF receptor (IGF-1R) (1). Stimulation of the IGF-1R leads to activation of various intracellular signaling pathways that promote cell cycle progression. There are multiple mechanisms that modulate IGF activity, including regulation of IGF transcription, localization, and clearance. Much of the post-translational regulation of IGF activity is controlled by IGF binding proteins (IGFBPs), a group of at least six related proteins that bind IGFs with high affinity (1). Evidence that the IGF system has a role in modulating fetal lung growth includes the findings that IGFs, IGFBPs, and IGF-1R are expressed in lung from early in gestation and that IGF-I is mitogenic for a variety of lung cells (2, 3). Production of IGF-I and expression of IGF messenger RNAs (mRNAs) are relatively stable during the latter portion of fetal lung development, whereas IGFBP expression in lung changes in a cell-specific manner throughout this period (4), suggesting that IGF bioactivity in fetal lung is regulated, in part, by IGFBPs.

There is considerable evidence that IGFBPs regulate cell proliferation. The most well-defined role for IGFBPs is to bind IGFs and regulate IGF bioavailability and subsequent IGF-1R activation (1, 7). In addition, IGFBPs may directly regulate cell proliferation independent of IGF binding (8). An emerging theme of recent studies is that many hormones, trophic factors, and mitogenic peptides influence cell proliferation by regulating IGFBPs. For example, retinoic acid and transforming growth factor- (TGF-) each decrease DNA synthesis through downregulation of IGFBP-3 production (8). Conversely, estradiol increases MCF-7 breast carcinoma cell DNA synthesis, in part, by suppressing IGFBP-5 production (9). Growth factors and hormones control the biologic effects of IGFBPs by regulating IGFBP production (10) and clearance (11, 12), and IGF affinity (13, 14). By utilizing the many mechanisms involved in controlling IGFBPs, these agents are able to influence mitogenesis within a tissue in a cell-specific manner.

At least five IGFBPs are produced by fetal lung cells in culture, and IGFBP production by fetal lung cells is regulated by agents that alter fetal lung growth or differentiation, such as IGF-I, glucocorticoids, and retinoic acid (15). Previous investigations have demonstrated that other growth factors involved in lung development, such as members of the fibroblast growth factor (FGF), TGF, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) families, regulate IGFBPs in many cell types (10, 16). The effect of these growth factors on fetal lung cell production of IGFBPs is unknown. The goals of this study were to determine whether IGFBP production by lung fibroblasts is regulated by growth factors that are important for lung organogenesis.

	Materials and Methods

Top Abstract Introduction Materials and methods Results Discussion References

Materials

The following reagents and materials were purchased: bovine calf serum (BCS) from Hyclone (Logan, UT); minimum essential medium (MEM), EGF, TGF-, keratinocyte growth factor (KGF), basic FGF (bFGF), and PDGF-BB from GIBCO BRL (Gaithersburg, MD); trypsin and deoxyribonuclease (DNase) I from Sigma Chemical Company (St. Louis, MO); and tissue culture flasks and multiwell plates from Costar (Cambridge, MA). The IGFBP-2 and IGFBP-4 antisera were gifts from Dr. David Clemmons (University of North Carolina at Chapel Hill, Chapel Hill, NC), and the IGFBP-6 antiserum was a gift from Dr. Nicholas Ling (Neurocrine Biosciences, San Diego, CA). IGF-free bovine serum albumin (BSA) (IGF-I < 10 pg/25 mg BSA) was a gift from Dr. Louis Underwood (University of North Carolina at Chapel Hill).

Cell Culture

Primary cultures of fetal rat lung fibroblasts were prepared using differential adherence as described previously (15), with some modification. Rat pups were delivered aseptically from 19-d-gestation timed pregnant Sprague- Dawley rats (Hilltop Laboratory Animals, Scottdale, PA). The lungs were removed and minced, and the cells were dispersed by incubating with trypsin (0.05%) and DNase (10 mg/ml) for 20 min at 37°C. Trypsin activity was stopped by adding MEM with penicillin (100 U/ml), streptomycin (100 U/ml), and 10% charcoal-stripped BCS (MEM/BCS-). BCS- (20) was prepared by incubating BCS with activated charcoal for 1 h at 55°C, followed by centrifugation and filtration through a 0.45-µm filter. The cell suspension was then passed through a 50-mm Nitex mesh filter and collected by centrifugation, then resuspended in a red blood cell lysis solution (15 mM Tris and 150 mM NH₄Cl, pH 7.2) for 10 min at 37°C. Cell clumps trapped by the filter were subjected to a second 20-min period of digestion (21). After centrifugation, the cells were resuspended in MEM/BCS- and incubated in 150-cm² tissue culture flasks for 45 min. The nonadherent cells were then removed and fresh MEM/BCS- was added to the flasks. The adherent cells were predominantly fibroblasts and are termed primary fibroblast cultures in this study. Previous studies have demonstrated that this method yields primary cultures of fibroblasts having approximately 85 to 90% purity as judged by morphology and vimentin staining (21). Cells were grown to appropriate confluence in 5% CO₂/95% air at 37°C and were used within 2 d of initial harvest.

Preparation of Conditioned Medium (CM) and Cell Lysate

Cells were detached by trypsinization and plated at a concentration of 3.5 × 10⁴ cells/cm² in 24-well plates (2-cm²-area wells). After 24 h in culture, the cells were washed twice with serum-free MEM (SFM), and incubated with 0.05% IGF-free BSA in SFM, with or without growth factors, for the indicated times. At the end of the experimental period, media were collected and frozen at 20°C. For assessment of cellular DNA content, 0.1% sodium dodecyl sulfate (SDS) was added to the cell layer in each well and collected after incubation for 1 h at 42°C. The DNA content for each well was determined using a DNA fluorimeter (Hoeffer Scientific, San Francisco, CA) as described by Rannels and colleagues (22), except that 0.1% SDS was used to facilitate fibroblast lysis. The DNA assay was linear from 340 to 3,500 ng DNA (25,000 to 250,000 cells, r = 0.996) with an intra-assay variation coefficient of 2.4%. For each treatment condition tested, DNA content varied by less than 15% from untreated control cells.

Ligand Blot Analysis of CM

Ligand blot analysis was performed as previously described (15). CM samples were lyophilized and resuspended in water. Laemmli buffer (4×) was then added and the samples were immediately heated to 95°C for 3 min and centrifuged, and the supernatant was subjected to electrophoresis through a 12.5% SDS-polyacrylamide gel under nonreducing conditions. The proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Inc., Keene, NJ) and incubated with 100,000 cpm/ml [¹²⁵I]IGF-I. After being washed, the membranes were exposed to Kodak Biomax MS autoradiography film (Eastman Kodak, Rochester, NY) at 70°C. Autoradiograms were analyzed by densitometry using the Image-Pro system (Media Cybernetics, Silver Spring, MD) to determine the relative abundance of each IGFBP species.

Immunoblot Analysis of CM

After ligand blotting, nitrocellulose membranes were washed in 50 mM Tris, 0.2 M NaCl, and 5% nonfat dry milk, and then incubated with a rabbit antiserum against bovine IGFBP-2 (1:2,000) (23), human IGFBP-4 (1:2,000) (23), or rat IGFBP-6 (1:500) (24). After being washed, blots were incubated with goat antirabbit immunoglobulin G biotin-surfactant protein conjugate (1:5,000; Jackson Laboratories, West Grove, PA). Blots were again washed and incubated with a streptavidin-peroxidase conjugate (1:5,000; Jackson Laboratories). Antibody complexes were visualized with Amersham ECL detection reagents (Amersham, Arlington Heights, IL) and Kodak Biomax MS autoradiographic film. Fetal rat serum was used as a positive control for IGFBP-2 and IGFBP-4. Negative controls (no primary antisera) recognized no proteins of relative molecular mass (M_r) less than 50,000. There was no significant cross-reactivity between IGFBPs for the antisera used (23, 24).

Statistical Analysis

Each experiment was performed using at least two separate primary cell-culture preparations. Statistical differences between groups within an experiment were determined by analysis of variance and Dunnett's test for multiple comparisons using SigmaStat (version 1.0; Jandel Corp., San Rafael, CA). Results were considered significant for P values less than 0.05. For skewed data sets, results were log-transformed before statistical analysis.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

IGFBP Accumulation in Response to Various Growth Factors

Ligand blot analysis of primary fetal rat lung fibroblast CM demonstrated IGFBPs of 39,000 to 43,000, 32,000, and 24,000 M_r. The 39,000- to 43,000- and 24,000-M_r IGFBPs have been identified previously as IGFBP-3 and IGFBP-4, respectively, and the 30,000- to 32,000-M_r band primarily as IGFBP-2 (15). Although previous experiments using specific IGFBP-5 antisera and more recent experiments using a more sensitive immunoblot technique have failed to identify intact (30,000-M_r) IGFBP-5 in lung cell CM (unpublished observations), IGFBP-5 is produced by these cells (15) and may be present in CM in amounts below the detection limit of the antisera used. IGFBP-6 is also produced by fetal rat lung fibroblasts. Immunoblot analysis of CM using a specific antisera against IGFBP-6 was undertaken and demonstrated the presence of a faint band at 25,000 to 26,000 M_r (data not shown). This IGFBP-6 immunoreactive band does not correspond to any visible IGFBP band using [¹²⁵I]IGF-I ligand blot techniques, likely because of the low affinity of IGFBP-6 for IGF-I (25).

To determine the effect of peptide growth factors on the production of IGFBPs by fetal lung fibroblasts, near-confluent cells were exposed to bFGF (5, 25, and 50 ng/ ml), TGF-1 (0.2, 1, and 10 ng/ml), KGF (5, 25, and 50 ng/ ml), EGF (5, 25, and 50 ng/ml), or PDGF-BB (2.5, 25, and 50 ng/ml) for 24 h and CM was harvested for analysis by ligand blot. As shown in Figures 1 and 2, 24 h exposure of cells to bFGF at doses greater than 5 ng/ml increased CM IGFBP-4 up to 2.1-fold compared with cells exposed to SFM only, but had no effect on the 30- to 32-kD band or IGFBP-3. Exposure of cells to TGF-1 for 24 h had no effect on accumulation of IGFBPs in CM. KGF at doses above 5 ng/ml increased the accumulation of IGFBP-3 up to 2-fold and decreased the intensity of the 30- to 32-kD band, but had no effect on IGFBP-4 in CM. EGF at all doses decreased the 30- to 32-kD IGFBP, but did not affect the accumulation of IGFBP-3 or IGFBP-4 in CM. PDGF-BB exposure (doses greater than 2.5 ng/ml) resulted in a small decrease in IGFBP-3 and up to a 4.2-fold increase in the accumulation of IGFBP-4 at 24 h.

View larger version (30K): [in this window] [in a new window]   Figure 1.   Autoradiographs of representative ligand blots from experiments showing the abundance of IGFBPs in CM following growth-factor treatment of fetal lung fibroblasts for 24 h. Fibroblasts were exposed to SFM alone (lanes 2, 4, 6, and 9), bFGF (25 ng/ml, lane 3), TGF- (10 ng/ml, lane 5), KGF (25 ng/ml, lane 7), PDGF-BB (25 ng/ml, lane 8), or EGF (25 ng/ml, lane 10) for 24 h, and CM was collected and analyzed by ligand blot as described in MATERIALS AND METHODS. Mr estimates (shown on the left of lanes 1 and 9) are based on the analysis of fetal rat serum run in a parallel lane (lanes 1 and 11).

View larger version (59K): [in this window] [in a new window]   Figure 2.   Densitometric analysis of ligand blots showing the abundance of IGFBPs in CM following growth-factor treatment for 24 h. Fetal lung fibroblasts were treated with SFM alone, bFGF (a), TGF-1 (b), KGF (c), EGF (d), or PDGF-BB (PB; e) for 24 h; CM was then collected and analyzed by ligand blot. Densitometric analysis of ligand blot bands is shown as the mean ± SEM of at least three wells expressed as a percent of IGFBP abundance in CM from untreated cells. Similar results were obtained using at least two separate primary cell culture preparations. *P < 0.05 compared with SFM controls.

As noted previously (15), the pattern of IGFBP accumulation in CM of fetal rat lung fibroblasts changes between 24 and 48 h. CM IGFBP-3 and IGFBP-2 in CM exposed to SFM without additional growth factors or serum increases between 24 and 48 h, whereas IGFBP-4 does not significantly increase in abundance after 24 h (15). To determine the IGFBPs secreted into CM by 48 h of growth-factor exposure, fetal lung fibroblasts were plated as described above and then exposed to bFGF (25 and 50 ng/ ml), TGF-1 (1 and 5 ng/ml), KGF (25 and 50 ng/ml), EGF (25 and 50 ng/ml), or PDGF-BB (25 and 50 ng/ml) for 48 h, and CM was harvested for analysis by ligand blot. Basic FGF did not alter IGFBP-3 accumulation in fetal lung fibroblast CM over 48 h (Figures 3 and 4), but IGFBP-4 was increased up to 2.7-fold by bFGF (maximum response at 25 ng/ml). A 48-h exposure of cells to 1 ng/ml TGF-1 decreased IGFBP-4 accumulation but did not alter IGFBP-3 or the abundance of the 30,000- to 32,000-M_r IGFBP in CM. IGFBP-3 accumulation was increased by both KGF and EGF, with the greatest response observed at 25 ng/ml for KGF and 50 ng/ml for EGF. The 30,000 to 32,000 ligand blot band was decreased more than 50% in CM of 25 ng/ml EGF-treated cells. The increase in CM IGFBP-4 was much greater after 48 h of exposure to either EGF or PDGF-BB compared with 24 h of exposure (maximum 6-fold increase and 14.9-fold increase in IGFBP-4 at 48 h for EGF and PDGF-BB, respectively).

View larger version (39K): [in this window] [in a new window]   Figure 3.   Autoradiographs of representative ligand blots from experiments showing the abundance of IGFBPs in CM following growth-factor treatment of fetal lung fibroblasts for 48 h. Fibroblasts were exposed to SFM alone (lanes 2, 4, and 8), bFGF (25 ng/ml, lane 3), TGF-1 (5 ng/ml, lane 5), KGF (25 ng/ml, lane 6), EGF (25 ng/ml, lane 7), or PDGF-BB (25 ng/ml, lane 9) for 48 h, and CM was collected and analyzed by ligand blot. Mr estimates (shown on the left of lanes 1, 4, and 8) are based on the analysis of fetal rat serum run in a parallel lane (lane 1).

View larger version (45K): [in this window] [in a new window]   Figure 4.   Densitometric analysis of ligand blots showing the abundance of IGFBPs in CM following growth-factor treatment for 48 h. Fetal lung fibroblasts were treated with SFM alone, bFGF (a), TGF-1 (b), KGF (c), EGF (d), or PDGF-BB (PB; e) for 48 h; CM was then collected and analyzed by ligand blot. Densitometric analysis of ligand blot bands is shown as the mean ± SEM of at least three wells expressed as a percent of IGFBP abundance in CM from untreated cells. Similar results were obtained using at least two separate primary cell culture preparations. *P < 0.05 compared with SFM controls.

Because of the possibility that the 30,000- to 32,000-M_r band may include both intact IGFBP-5 and IGFBP-2, immunoblot analyses were performed to determine the identity of the IGFBP altered by EGF and KGF. The intensity of the immunoreactive IGFBP-2 band changed in parallel to the 30,000- to 32,000-M_r ligand blot band for 24-h CM from EGF- and KGF-treated cells (not shown) and for 48-h CM from EGF-treated cells (Figure 5). To confirm that the changes in the 24,000-M_r ligand blot band represented changes in IGFBP-4, immunoblot analyses were performed on CM from cells exposed to bFGF, EGF, and PDGF-BB. The major immunoreactive band recognized by the IGFBP-4 antisera migrated at 24,000 M_r, as expected (Figure 6). A smaller band at approximately 14,000 M_r likely identifies a proteolytic fragment of IGFBP-4. In addition, faint bands at 28,000 (likely representing glycosylated IGFBP-4) and 18,000 M_r (another proteolytic fragment of IGFBP-4) were visualized in some samples. The 24,000 IGFBP-4 band increased in parallel with the 24,000-M_r ligand blot band identified in CM from cells treated with bFGF and PDGF-BB for 24 and 48 h and EGF for 48 h. In addition, the IGFBP-4 fragment was less prominent in 48-h CM samples from cells treated with EGF compared with cells exposed to SFM alone (14,000- M_r band intensity decreased 55% to 75% compared with CM from cells incubated with SFM).

View larger version (29K): [in this window] [in a new window]   Figure 5.   Immunoblot analysis of CM from fetal rat lung fibroblasts following growth-factor treatment for 24 h. Fibroblasts were exposed to SFM alone (lane 1), EGF (25 ng/ml, lane 2), or KGF (25 ng/ml, lane 3) for 48 h. Following analysis by ligand blot, CM samples were incubated with IGFBP-2 antiserum and sites of antibody binding were visualized using chemiluminescence as described in MATERIALS AND METHODS.

View larger version (42K): [in this window] [in a new window]   Figure 6.   Immunoblot analysis of CM from fetal rat lung fibroblasts following growth-factor treatment for 24 and 48 h. Fibroblasts were exposed for 24 h to SFM alone (lane 1), bFGF (25 ng/ ml, lane 2), or PDGF-BB (25 ng/ml, lane 3); or for 48 h to SFM alone (lanes 4 and 7), EGF (25 ng/ml, lane 5), PDGF-BB (25 ng/ ml, lane 6), or bFGF (25 ng/ml, lane 8). Following analysis by ligand blot, CM samples were incubated with IGFBP-4 antiserum and sites of antibody binding were visualized using chemiluminescence as described in MATERIALS AND METHODS. Mr estimates (shown on the left of lane 1) are based on comparison with the corresponding ligand blot.

Growth Factor Cooperativity in Regulating IGFBP Accumulation

To determine whether there were cooperative effects of growth factors on IGFBP accumulation in CM, cells were exposed to PDGF-BB (25 ng/ml), EGF (25 ng/ml), or bFGF (25 ng/ml) alone or in combination for 24 (Figure 7) or 48 h (Figure 8). As described above, EGF increased IGFBP-3 in 48-h CM. Addition of neither PDGF-BB nor bFGF altered the accumulation of IGFBP-3 in response to EGF (data not shown). The inhibition of IGFBP-2 accumulation following 48 h incubation with EGF was not significantly altered by coincubation of EGF and PDGF-BB or coincubation of EGF and bFGF. For IGFBP-4, the combination of EGF and bFGF resulted in greater accumulation of IGFBP-4 than with EGF or bFGF alone (IGFBP-4 increased 1.7-fold by EGF and 1.6-fold by bFGF compared with a 3.7-fold increase following incubation with EGF plus bFGF). The increase in accumulation of CM IGFBP-4 following 24-h coincubation with EGF and PDGF-BB or bFGF and PDGF-BB was not significantly greater than the increase noted with PDGF-BB alone. More pronounced effects were noted following 48 h incubations with combinations of growth factors. CM IGFBP-4 accumulations were twice that expected from a summation of the effects of either growth factor alone for the combination of PDGF-BB and EGF, PDGF-BB and bFGF, and EGF and bFGF (IGFBP-4 increased 9.8-fold by PDGF-BB, 4.0-fold by EGF, and 1.8-fold by bFGF, compared with a 27.2-fold increase for PDGF-BB plus EGF, a 37.3-fold increase for PDGF-BB plus bFGF, and a 13.0-fold increase for EGF plus bFGF).

View larger version (42K): [in this window] [in a new window]   Figure 7.   Densitometric analysis of ligand blots showing the abundance of IGFBPs in CM following growth-factor treatment for 24 h. Fetal lung fibroblasts were treated with SFM alone, PDGF-BB (PB, 25 ng/ml), EGF (25 ng/ml), bFGF (25 ng/ml), or combinations of these peptides for 24 h, and CM was analyzed by ligand blot. Densitometric analysis of ligand blot bands is shown as the mean ± SEM of at least three wells expressed as a percent of IGFBP abundance in CM from untreated cells for IGFBP-2 (a) and IGFBP-4 (b). Similar results were obtained using at least two separate primary cell culture preparations. *P < 0.05 compared with SFM controls; +P < 0.05 compared with PDGF-BB; P < 0.05 compared with EGF; **P < 0.05 compared with bFGF.

View larger version (33K): [in this window] [in a new window]   Figure 8.   Densitometric analysis of ligand blots showing the abundance of IGFBPs in CM following growth-factor treatment for 48 h. Fetal lung fibroblasts were treated with SFM alone, PDGF-BB (PDGF-BB, 25 ng/ml), EGF (25 ng/ml), bFGF (25 ng/ml), or combinations of these peptides for 48 h, and CM was analyzed by ligand blot. Densitometric analysis of ligand blot bands is shown as the mean ± SEM of at least three wells expressed as a percent of IGFBP abundance in CM from untreated cells for IGFBP-2 (a) and IGFBP-4 (b). Similar results were obtained using at least two separate primary cell culture preparations. *P < 0.05 compared with SFM controls; +P < 0.05 compared with PDGF-BB; P < 0.05 compared with EGF; **P < 0.05 compared with bFGF.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

Perhaps because of its importance in regulating cell proliferation, there are multiple mechanisms that regulate IGF bioactivity. IGFBPs provide an important mechanism for post-translational control of IGF activity and are widely expressed in lung during fetal life. Previous investigations have demonstrated that IGFBPs produced by fetal lung cells are regulated by IGF-I, glucocorticoids, and retinoic acid (15). The present study extends these observations and establishes that IGFBPs are also regulated by peptide growth factors that influence lung organogenesis.

Exposure of fetal lung fibroblasts to bFGF, EGF, or PDGF-BB resulted in large increases in IGFBP-4 accumulation in CM. These growth factors increase IGFBP-4 in a variety of mesenchymal and epithelial cell types through both transcriptional and post-transcriptional mechanisms (10, 16). Other agents that increase IGFBP-4 in fetal lung fibroblasts include dibutyryl cyclic adenosine monophosphate (dcAMP) and retinoic acid (15). Increased intracellular cAMP increases IGFBP-4 in many cell types by increasing IGFBP-4 mRNA (26, 27). This response is likely mediated by the cAMP response element within the IGFBP-4 promoter (28). Thus, a PDGF-BB-stimulated increase in intracellular cAMP likely mediates the increase in IGFBP-4 noted following incubation of fetal lung fibroblasts with PDGF-BB. In support of this, PDGF-BB activates adenylate cyclase and increases intracellular cAMP through interaction with the  isoform of its cell-surface receptor (29). Fetal lung fibroblasts express the -PDGF receptor (-PDGFR) and increase tyrosine kinase activity in response to PDGF-BB (30). Interestingly, PDGFR- expression increases following serum withdrawal (31), a mechanism that may contribute to the much greater increase in IGFBP-4 in CM from lung fibroblasts treated with PDGF-BB for 48 compared with 24 h. Because EGF and bFGF also may increase intracellular cAMP (32, 33), a similar mechanism may explain the response of IGFBP-4 to these agents. However the synergistic effects of coincubation with combinations of PDGF-BB, bFGF, and EGF on IGFBP-4 abundance argue that these growth factors activate more than one mechanism to increase IGFBP-4. One potential explanation for synergistic activity between PDGF-BB and bFGF is suggested by the finding that PDGF-BB increases FGF receptor 1 (FGFR-1) expression (34). A progressive increase in FGF-R density following PDGF-BB exposure could explain the greater effect of coincubation of PDGF-BB and bFGF on IGFBP-4 noted at 24 compared with 48 h of exposure.

In contrast to PDGF-BB and bFGF, there was a delayed response of IGFBP-4 to EGF exposure in the present study. These results are similar to previous investigations in rat granulosa cells that showed no increase in IGFBP-4 in response to EGF receptor (EGFR) activation until after 24 h exposure (18), and suggest that new protein synthesis is required for stimulation of IGFBP-4 by EGF. One mechanism by which growth factors and hormones regulate IGFBP-4 abundance is through release of specific IGFBP-4 proteases or protease inhibitors (12, 35). The finding of IGFBP-4 fragments in lung cell CM in the present study suggests that IGFBP-4 abundance in lung cell CM is regulated by proteolysis. Moreover, IGFBP-4 proteolytic activity has also been found in A549 lung cell CM (3). Although studies are needed to define further the role of proteolysis in regulating IGFBP-4 abundance, our results showing a decrease in CM IGFBP-4 proteolytic fragments following EGF exposure suggest that EGF stimulates the synthesis of an IGFBP-4 protease inhibitor that suppresses IGFBP-4 degradation.

As has been demonstrated previously for IGF-I (15), IGFBP-3 abundance in CM of primary lung fibroblasts is regulated by EGF, KGF, and PDGF-BB. The response of IGFBP-3 to PDGF-BB was variable and was dependent on both cell density and time in culture. In subconfluent cells, PDGF-BB decreased IGFBP-3 abundance at 24 h but tended to increase IGFBP-3 in confluent cell cultures or after 48 h of incubation for subconfluent cells (data not shown). The upregulation of IGFBP-3 by EGF is consistent with the pattern of regulation reported in other cell types and may involve upregulation of IGFBP-3 mRNA (10). Another mechanism by which EGF could increase IGFBP-3 accumulation is suggested by the findings that EGF stimulates IGF-I production (36) and that IGF-I increases fetal lung fibroblast CM IGFBP-3 (15). The lack of immediate effect on CM IGFBP-3 following EGF exposure suggests that the effect of EGF on IGFBP-3 requires time for the synthesis of an intermediate protein involved in IGFBP-3 regulation, such as IGF-I. Other mechanisms by which growth factors regulate IGFBP-3 post-transcriptionally include altering IGFBP-3 protease activity (11), IGFBP-3 internalization (13), or cell surface attachment (14). The role of these mechanisms in regulating IGFBP-3 for fetal lung fibroblasts is currently under investigation.

The regulation of IGFBPs by KGF appears to be a novel finding and emphasizes that, even within growth-factor families, there are significant differences in the regulation of IGFBPs. KGF and bFGF are both members of the FGF family of peptide growth factors. KGF increased fetal lung fibroblast CM IGFBP-3 at both time points and decreased 24-h CM IGFBP-2, whereas bFGF increased IGFBP-4. KGF interacts with a specific receptor, the FGFR-2 (IIIb) or KGF receptor (KGFR) (37). Although bFGF also interacts with the KGFR, it does so with a greatly reduced affinity (37). The divergence in IGFBP responses to KGF and bFGF implies that the effects of bFGF on IGFBP abundance are not mediated by the KGFR but by another FGFR. The downregulation of IGFBP-2 by KGF in the present study was attenuated as exposure times increased beyond 24 h. In primary cultures of lung fibroblasts, epithelial cells, and SV-40 transformed type II epithelial cells, CM IGFBP-2 and/or IGFBP-2 mRNA are increased in serum-deprived cells (15, 38). These results suggest that cell quiescence alters the intracellular pathways that control IGFBP-2 abundance and partially overrides the effects of KGF on IGFBP-2.

The regulation of IGFBPs by peptide growth factors may be dependent on tissue-specific, developmental, or species-specific factors. Tissue-specific IGFBP regulation is illustrated in the present study by the increase in fetal lung fibroblast IGFBP-4 production following bFGF treatment, a result similar to that observed in fetal rat osteoblast-like cells (19), but different from the response of IGFBP-4 to bFGF for fetal rat glial cells (39). Also, the response of IGFBP-2 to EGF for fetal lung fibroblasts and rat intestinal epithelial cells (40) differs from that noted for newborn rat astroblasts (41). Other results suggest that differences in IGFBP regulation could be related to either species or developmental stage as well. Our results showing increased lung fibroblast IGFBP-4 following PDGF-BB or EGF exposure contrasts with the lack of response of adult human dermal fibroblast IGFBP-4 to PDGF-BB (23) or EGF (42). IGFBP-3 accumulation in CM is increased following EGF treatment of lung fibroblasts (present study) and cells derived from newborn rat brain (41), but is decreased following EGFR activation for mouse mammary cells (43). These variations in the regulation of IGFBPs by growth factors support the hypothesis that the multiple mechanisms involved in controlling IGFBP abundance provide a mechanism by which growth factors can control cell proliferation that is specific to the tissue, species, and/or developmental stage of the animal.

The significance of growth-factor regulation of IGFBP production in fetal lung cells is unknown. In other cell-culture systems, changes in IGFBP abundance are one mechanism by which hormones and trophic factors regulate DNA synthesis. IGFBPs can decrease cell proliferation through their effects on IGF-I bioavailability by inhibiting IGF-I binding to the IGF-1R (7) or by direct, IGF-independent mechanisms, triggered through a specific cell-surface receptor (8). Conversely, increased DNA synthesis following treatment of cells with IGFBP-3 may involve prevention of IGF-1R downregulation (44). Speculation on the physiologic significance of increased IGFBP-3 and decreased IGFBP-2 in response to KGF and EGF is derived from the observations that both KGF and EGF can act as epithelial cell mitogens (2, 45). Given that increased IGFBP-3 and decreased IGFBP-2 increase DNA synthesis (40, 46) and both IGFBP-3 and IGFBP-2 protein are primarily localized to epithelial cells in lung (5), we speculate that these changes in IGFBP abundance promote epithelial cell mitogenesis during alveolar genesis. In the present study, IGFBP-4 showed the most pronounced changes in abundance in response to exogenous growth factors. Several lines of evidence suggest that IGFBP-4 inhibits DNA synthesis. Cell proliferation is inhibited by increasing IGFBP-4 production (47) or adding exogenous IGFBP-4 (48), and increased DNA synthesis follows a decrease in IGFBP-4 production or activity (49). In fetal lung, IGFBP-4 is primarily expressed by mesenchymal cells (6). Because IGF-1R activation is necessary for fetal lung fibroblast proliferation (2), increased IGFBP-4 production is likely to decrease DNA synthesis in these cells. Thus, the ability of EGF and PDGF-BB to increase IGFBP-4 may be a mechanism by which the effects of IGF-I on fibroblast proliferation can be regulated. The lung fibroblasts used in this study are from the canalicular period of lung development, a period characterized by mesenchymal thinning and decreased fibroblast proliferation (50), and are associated with high PDGF-B and TGF- mRNA expression in lung (51, 52). These findings (52) suggest that elevated PDGF-B and TGF- expression during the canalicular period increases mesenchymal IGFBP-4 production and is a mechanism by which IGF-stimulated fibroblast proliferation is inhibited during acinar development.

Lung organogenesis requires complex cell communication mechanisms to translate myriad nutritional, developmental, and mechanical signals into cellular actions. At each step, autocrine and/or paracrine cell signals mediated by peptide growth factors are crucial in directing cell proliferation and differentiation. Increasing evidence suggests that interactions between growth factors determine the response of cells to these diverse stimuli. IGF-I is an important mitogen for lung cells, and IGFBPs are abundant, highly regulated proteins secreted by lung cells and capable of altering cell proliferation directly or by regulating IGF-I bioavailability. The demonstration that growth factors regulate IGFBPs in lung is consistent with other studies showing that IGFBP expression is regulated by hormones or factors important in the growth, differentiation, or function of that tissue (1, 16). This study suggests that IGFBPs play an important role in determining the proliferative response of cells to changes in growth-factor production during lung development.