Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
Submitted 4 June 2003 ; accepted in final form 21 July 2003
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ABSTRACT |
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elastin; heparin-binding epidermal growth factor-like growth factor; epidermal growth factor receptor; fibroblast growth factor-2; heparan sulfate proteoglycan; transcription; extracellular signal-regulated kinase 1/2; Fra-1
Elastin is synthesized as a soluble precursor, tropoelastin, that is secreted and cross-linked to form insoluble elastic fibers. In rodents, lung elastin gene expression is high during late fetal and early postnatal development, when the majority of elastin mRNA is localized in the pulmonary vasculature and later in the forming alveolar septa and crests (32). Elastin gene expression decreases afterwards, and there is very little elastin synthesis in adults. Temporal and spatial association of elastin synthesis with specific developmental events suggests that elastin plays an active role in lung morphogenesis. Indeed, elastin deposition within the tips of the secondary septa is thought to be a driving force for alveolarization in rodent postnatal lung development (3, 8, 27). In addition, elastin-deficient mice exhibit retarded lung branching morphology at birth, suggesting an earlier contribution of elastin in terminal airway branching (50).
A number of animal models of emphysema, based on administration of elastolytic proteases, have been used to analyze the biochemical changes accompanying the loss of elastin from lung parenchyma. In these animal models, the rapid decrease of lung insoluble elastin levels is followed by a reactivation of elastin gene expression (45). The observed elastin resynthesis following the proteolytic damage is thought to represent an attempt to repair the proteolytic injury. However, human emphysema is characterized as a chronic disease resulting from repeated exposure to pathological insults that are thought to eventually compromise an effective repair. Mechanisms underlying the regulation of elastin gene expression in development and injury/repair situations are not well understood, although they may share some common mediators. The complexity of lung architecture makes it difficult to perform biochemical analyses in vivo. The injury/repair process is further complicated by the involvement of accompanying inflammatory responses that include an influx of cells and their release of cytokines. Thus the use of in vitro cell culture models allows a simple system for dissection of the effects of various factors and their mechanism of action.
Primary cultures of rat pulmonary fibroblasts synthesize an insoluble elastic matrix similar to that found in alveolar walls (5). Brief treatment of these cultured fibroblasts with pancreatic elastase solubilizes elastin (15). The resultant elastase digest exhibits an inhibitory activity on elastin gene expression when added exogenously to untreated fibroblasts (15). Previously, we have identified fibroblast growth factor-2 (FGF-2) as a major elastogenic inhibitory factor within the elastase digest (44). FGF-2 is a heparan sulfate proteoglycan (HSPG)-binding growth factor that is initially sequestered in the matrix of pulmonary fibroblasts. Elastase cleavage of the core proteins depletes cell-associated HSPG and mobilizes FGF-2 (4). In addition, elastase treatment of the cells decreases the equilibrium binding of FGF-2 to both HSPG and receptor sites, suggesting a modified FGF-2-dependent response in elastase-treated cells (4).
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is another HSPG-binding growth factor that has been implicated in lung pathogenesis induced by various factors (23, 38, 48, 53). HB-EGF is a potent mitogen for lung fibroblasts and epithelial cells (29, 53) and has been shown to stimulate cell migration in other cell systems (31, 47). HB-EGF is a member of the EGF family that consists of a number of other structurally related growth factors such as EGF, transforming growth factor (TGF)-, and amphiregulin (40). Unlike EGF or TGF-
, HB-EGF possesses a heparin-binding domain (46) that allows its interaction with cell surface and matrix HSPG and subsequent modulation of its biological activity (18). EGF family members bind and activate the tyrosine kinase receptor epidermal growth factor receptor (EGFR) to regulate diverse processes in various cell types (39). Importantly, EGF has been shown to decrease elastin gene expression in chick aortic smooth muscle cells (22) and lung fibroblasts (10). The effect of HB-EGF on elastin gene expression has not been examined. Because its activity is regulated by HSPG (18), and HSPG is a target of elastase treatment, HB-EGF may be an important signaling ligand in elastase-induced injury/repair.
The goal of this study is to investigate the potential role of HB-EGF in regulating elastin gene expression in pulmonary fibroblast cultures. We first established that elastase treatment of matrix-laden pulmonary fibroblast cultures resulted in a detectable amount of soluble HB-EGF within the elastase-released products. Addition of soluble HB-EGF to undigested fibroblast cultures decreased the steady-state level of elastin mRNA through a mechanism similar to that reported for FGF-2 (6, 42). Furthermore, we show that HB-EGF treatment of pulmonary fibroblasts leads to an increase in endogenous FGF-2 mRNA and protein levels.
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MATERIALS AND METHODS |
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Cell cultures and treatment. Neonatal rat pulmonary fibroblast cells were isolated from the lungs of 3-day-old Sprague-Dawley rats as described (15). For most experiments, second-passage fibroblasts were plated in 75-cm2 flasks at 2 x 104 cells/cm2. In some experiments, cells were seeded in 35-mm plates at 10 x 104 cells/dish. Cells used for 125I-HB-EGF binding assays were plated at 5 x 104/well into 24-well plates (2 cm2/well; Costar, Cambridge, MA). After being plated, cells were maintained in DMEM containing 5% FBS for 2 wk. Before elastase treatment, the confluent, matrix-laden cultures were rinsed twice with Puck's saline (137 mM NaCl, 5.37 mM KCl, 1.10 mM KH2PO4, 1.08 mM Na2HPO4, 6.10 mM glucose, pH 7.4) and once with the digestion buffer (44 mM sodium bicarbonate, pH 7.4). Pancreatic elastase in sodium bicarbonate solution (5 µg/ml) was added to the cells, and the flasks were incubated at 37°C for 5 min. The control cultures were treated similarly except that they were incubated with sodium bicarbonate solution without the supplement of elastase. At the end of the 5-min period, digestion buffers covering the control and treated cells were collected, and diisopropyl fluorophosphate (DFP) was added at a final concentration of 1 µM to inhibit further elastase activity. Elastase digests were kept frozen in -80°C if not used immediately.
In experiments studying the effects of exogenously added HB-EGF and/or FGF-2, 2-wk-old fibroblasts were fed with fresh medium containing 0.5% FBS and were cultured for 24 h before ligand addition. Unless otherwise specified, 10 ng/ml of growth factors were added. In some experiments, cells were preincubated with either the inhibitors (AG-1478, 10 µM; PD-98059, 15-75 µM; U-0126, 25-75 µM) or an equal amount of solvent (DMSO or methanol) for 1 h before the addition of growth factors.
Analysis of elastase digest. The original elastase digests were concentrated to 1:20 of the original volume using the SPD1010 SpeedVac System (Thermo Savant, Holbrook, NY) and subjected to an SDS-PAGE clean up kit (Amersham Biosciences, San Francisco, CA). The resultant material was separated on 16% SDS-PAGE and analyzed by Western blot analysis.
125I-HB-EGF binding assays. 125I-HB-EGF was prepared using a modification of the Bolton-Hunter method (33). Labeled HB-EGF was equally active as unlabeled HB-EGF in an assay measuring its stimulation of DNA synthesis in BALB/c 3T3 cells. Equilibrium binding of 125I-HB-EGF was conducted on pulmonary fibroblast cultures treated with elastase for different times and at different concentrations. Immediately after elastase treatment, DMEM containing 5% FBS was added back to the cells to inhibit any residual elastolytic activity. The medium was replaced once again before the start of subsequent binding assay. Cultures were rinsed three times with ice-cold DMEM containing 25 mM HEPES and 0.05% gelatin and were incubated on ice for 10 min. 125I-HB-EGF was added to each cell well at a final concentration of 5 ng/ml, and the cells were incubated on ice at 4°C for 2 h. HSPG- and receptor-bound 125I-HB-EGF were determined separately using a combination of high-salt and low-pH extraction buffers (13). HSPG-bound 125I-HB-EGF was recovered by briefly washing the cells with buffer containing 20 mM HEPES (pH 7.4) and 2 M sodium chloride, followed by a second wash with PBS. Receptor-bound 125IHB-EGF was recovered by washing the cells twice with buffer containing 25 mM sodium acetate (pH 4.0) and 2 M sodium chloride. Nonspecific binding was determined for each extraction method with control cells by competing with increasing concentrations of unlabeled HB-EGF until no further reduction in label binding was observed (100 µg/ml). Less than 1% of the added 125I-HB-EGF was bound nonspecifically within the HSPG and receptor fractions, and these values were subtracted from all data.
Isolation and analysis of RNA. Total RNA from cells cultured in 75-cm2 flasks was isolated with 4 M guanidinium thiocyanate as previously described (52). A single-step procedure using TRIzol reagent (GIBCO Life Sciences, Gaithersburg, MD) was used to isolate total RNA from cells cultured in 35-mm dishes. Equal amounts (10 µg unless otherwise specified) of RNA were fractionated in a 1% agarose-1.8% formaldehyde gel and transferred onto a nylon membrane (Schleicher and Schuell, Keene, NH) by capillary force. RNA was cross-linked to the membrane by ultraviolet irradiation. The membrane was hybridized with 32P-labeled cDNAs. Rat cDNA encoding tropoelastin was described earlier (43). Rat cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from American Type Culture Collection (Manassas, VA). Mouse histone 3/2b plasmid was provided by Dr. W. F. Marzluff (Univ. of North Carolina, Chapel Hill, NC). Bovine FGF-2 c-DNA plasmid was previously described (1).
Nuclear run-on assay. The assay was performed as described previously (52). Briefly, nuclei from control or HBEGF-treated cells were isolated and used to initiate in vitro transcription reaction where the newly synthesized mRNAs were labeled with [32P]UTP. The reaction was stopped 20 min after initiation. Samples containing equal amounts of radioactive counts were hybridized to nitrocellulose membranes (Schleicher and Schuell) on which empty and chimeric pBluescript plasmids containing specific cDNAs were immobilized. Rat actin cDNA was provided by Dr. B. Nadal-Ginard (Harvard Medical School, Boston, MA). After being washed, the membranes were subjected to autoradiography, and signal was quantitated by using a Molecular Dynamics laser scanning densitometer.
Preparation of nuclear extracts and cell lysates. Nuclear extracts were prepared as described (7). Briefly, cell layers were rinsed twice with prechilled Puck's saline and scraped with cold lysis buffer (containing 10 mM Tris·HCl, pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). The cell suspension was centrifuged at 315 g for 10 min, and the supernatant was discarded. The cell pellet was resuspended in the lysis buffer, and the suspension was centrifuged at 560 g for 10 min. After removing the supernatant, the remaining pellet (crude nuclei) was resuspended with the nuclear protein extraction buffer [20 mM HEPES, pH 7.9, 0.35 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, 25% glycerol, 0.5 mM dithiothreitol, 0.5 µg/ml (1 µM) leupeptin, 2.0 µg/ml (0.3 µM) aprotinin, 0.7 µg/ml (1 µM) pepstatin, 0.2 mM sodium vanadate, 100 µM sodium fluoride, 1 µM DFP] at 0.5-1 volume of the pellet size. The extract was incubated on ice for at least 15 min and then centrifuged for 20 min at 9,500 g. The supernatant was transferred into another tube and stored at -80°C. Total cell lysates were prepared with ice-cold lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris·HCl, pH 7.5, 1 mM EDTA, pH 7.5, 1 mM EGTA, pH 9.0, 0.5% Nonidet P-40, 0.4 mM PMSF, 0.2 mM sodium vanadate) as described (6). Protein concentrations were determined by the bicinchoninic acid assay (Pierce, Rockford, IL).
Western blot analysis. Unless otherwise specified, 40 µg of nuclear extracts or cell lysates were separated on 10% SDS-PAGE before being electrotransferred overnight to nitrocellulose membranes (Schleicher and Schuell). The membranes were stained in Ponceau S dye (Sigma, St. Louis, MO) briefly to examine equal protein loading and transfer. The membranes were then blocked with 5% milk in TBST (10 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) before being probed with the primary antibody at room temperature for 2 h or at 4°C overnight. After being washed with TBST, the membranes were incubated with the secondary antibody for 45 min. Specific proteins were visualized by the chemiluminescence method according to the manufacturer's instructions (Kirkegaard & Perry Laboratories, Gaithersburg, MD). To probe the blot with a second antibody, the membrane was incubated in 68°C stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris·HCl, pH 6.8) for 30 min to remove any residual primary and secondary antibodies.
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RESULTS |
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Elastase-treatment decreases the binding of HB-EGF at cell surface receptors. We next examined whether elastase treatment affected HB-EGF binding to pulmonary fibroblasts. Because HB-EGF binds to both HSPG and receptor sites, we investigated the time- and dose-dependent effects of elastase treatment on HB-EGF binding at both sites (Fig. 2). Elastase addition resulted in a time-dependent decrease in HB-EGF binding at both HSPG (Fig. 2A) and receptor sites (Fig. 2B). Treatment of fibroblast cultures with 0.5 µg/ml of elastase reduced binding by nearly 50% at HSPG sites and by 35% at receptor sites within 10 min of enzyme addition, whereas binding to both HSPG and receptor sites was attenuated by >85% within 30 min of elastase administration. Treatment of fibroblast cultures with increasing elastase concentrations resulted in a dose-dependent decrease in HB-EGF binding to both HSPG (Fig. 2C) and receptor sites (Fig. 2D). Administration of 10 µg/ml of elastase for 15 min resulted in an 85% decrease in binding at both HSPG and receptor sites. Note that the extraction method used in this series of experiments does not allow differentiation between HB-EGF bound to matrix vs. cell surface HSPG. However, in an earlier study, Buczek-Thomas and Nugent (4) have suggested that elastase cleaves cell surface HSPG based on the evidence that FGF-2 binding to its cell surface receptor is decreased in elastase-treated cells, whereas no changes on the receptor expression is observed. Collectively, our results indicate that HB-EGF binding at both HSPG and receptor sites is diminished after elastase exposure that is consistent with the elastase-mediated depletion of cell-associated HSPG sites (4). The loss of HB-EGF binding to elastase-treated cultures suggests that the released HB-EGF is likely to have only a minimal effect on the injured cells immediately after enzyme treatment.
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HB-EGF downregulates elastin mRNA via EGFR activation. To determine how HB-EGF might individually affect elastin expression in pulmonary fibroblasts, we used matrix-laden cultures that were not treated with elastase in experiments similar to those in which we have studied FGF-2 effects (44). Cells were treated with HB-EGF for various times (Fig. 3A) or with different concentrations (Fig. 3B). Total RNA was isolated from control and treated cultures, and Northern blot analyses were performed to measure elastin mRNA levels in these cultures. HB-EGF downregulated elastin mRNA in a time- and dose-dependent manner. As shown in Fig. 3A, elastin mRNA downregulation became visible 8 h after ligand treatment, and the maximal inhibitory effect was seen at 48 h. As a control, GAPDH and histone 3/2b mRNA levels were not affected within the 48-h period. The unresponsiveness of an S-phase marker such as histone 3/2b mRNA to HB-EGF treatment is consistent with our previous observations that the primary pulmonary fibroblasts are "contact inhibited" (42). The effect of HB-EGF on elastin was saturated at a concentration of 10 ng/ml (Fig. 3B).
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We then examined whether EGFR activation is involved in HB-EGF-induced downregulation of elastin mRNA by treating cells with AG-1478, a selective tyrphostin-type EGFR tyrosine kinase inhibitor (35). AG-1478 treatment abolished elastin mRNA downregulation induced by HB-EGF, but not by FGF-2, which signals via a different receptor tyrosine kinase (Fig. 3C). These results indicate that activation of EGFR tyrosine kinase is an integral part of the HB-EGF-induced signaling pathway leading to downregulation of elastin mRNA.
HB-EGF inhibits elastin transcription. We next examined whether HB-EGF-induced downregulation of elastin mRNA is caused by transcriptional suppression. Elastin gene transcription levels in control and HB-EGF-treated cells were measured by nuclear run-on analyses. As shown in Fig. 4A, HB-EGF treatment led to a significant decrease (74.2 ± 6.4%) in the level of elastin transcription in 48 h, whereas the transcription level of actin was not affected. Quantitation of the data shows that after 48 h of HB-EGF treatment, elastin gene transcription was inhibited to an extent comparable with the decrease of elastin mRNA (Fig. 4B). A time course of elastin transcription levels in response to HB-EGF is given in Fig. 4C. The effect on elastin transcription was apparent as early as 4 h (20% inhibition), suggesting that this is a primary effect of HB-EGF and not a secondary downstream event. We conclude from these data that transcriptional inhibition accounts for HB-EGF-dependent downregulation of elastin mRNA.
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HB-EGF induces ERK1/2 activation and subsequent Fra-1 nuclear accumulation. EGFR activation can induce a number of signal transduction pathways (39). Among these, the best-characterized pathway is the ERK1/2 pathway. In this pathway, EGFR tyrosine phosphorylation initiates a signaling cascade that involves sequential activation of Ras, c-Raf, MEK1/2, and ERK1/2, which in turn transduces the signal into the nucleus. We first examined whether ERK1/2 is activated by HB-EGF signaling in these cells. Cell lysates were prepared after different periods of HB-EGF treatment and subjected to Western blot analysis. Figure 5A shows that HB-EGF induced a time-dependent phosphorylation of ERK1/2, with the levels of phosphorylated ERK1/2 (pERK1/2) peaking at 15-30 min and returning close to basal levels at 10 h. Levels of total ERK1/2 did not change in response to HB-EGF treatment.
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Activator protein-1 transcription factors are among the primary targets of ERK1/2 activation (51). We have previously shown, in the same cells, that FGF-2-induced ERK1/2 signaling leads to phosphorylation of Elk-1 and induction of Fra-1, which results in increased c-Jun/Fra-1 heterodimer complex binding to a distal negative element in the elastin promoter (6, 42). We examined whether similar nuclear signaling is induced by HB-EGF. Cells were treated with HB-EGF for different times, and the levels of nuclear pERK1/2 and Fra-1 were evaluated by Western blot analysis (Fig. 5B). HB-EGF treatment resulted in a time-dependent increase in nuclear pERK1/2 levels, whereas no apparent changes in the levels of total ERK1/2 were observed. The level of Fra-1 was significantly elevated after 6 h of HB-EGF treatment, and it was still clearly above basal level at 24 h. The levels of c-Jun, a binding partner of Fra-1, were not affected. Figure 5C presents the Ponceau S staining of two representative lanes loaded with either nuclear extract or total cell lysate, indicating the uniqueness of protein distribution in the nuclear compartments.
MEK1/2 inhibitor PD-98059 (12) was used to test the functional linkage between ERK1/2 activation and Fra-1 induction. PD-98059 inhibited HB-EGF-induced ERK1/2 activation in a dose-dependent manner (Fig. 5D). We then examined the effect of PD-98059 on HB-EGF-dependent Fra-1 induction. As shown in Fig. 5E, the majority of HB-EGF-initiated Fra-1 induction was blocked by PD-98059, indicating that the nuclear accumulation of Fra-1 is downstream of ERK1/2 activation. The residual Fra-1 induction is likely due to PD-98059's incomplete inhibition of ERK1/2 activation, as evidenced in Fig. 5D.
Because PD-98059 did not completely inhibit HBEGF-induced ERK1/2 activation, we tested the effect of another MEK1/2 inhibitor, U-0126 (14). U-0126 inhibited HB-EGF-induced ERK1/2 activation more effectively than PD-98059 (Fig. 6A vs. Fig. 5D), which is consistent with other reports (14, 34). Total ERK1/2 levels were not altered by U-0126 treatment. Cells were then preincubated with 25 µM U-0126 and treated for 24 h with HB-EGF, and elastin mRNA levels in these cells were measured. As shown in Fig. 6B, U-0126 abrogated HB-EGF-dependent elastin downregulation, indicating that the ERK1/2 pathway mediates HB-EGF-induced downregulation of elastin gene expression.
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HB-EGF and FGF-2 have additive inhibitory effects on elastin gene expression. Because FGF-2 and HBEGF are released by elastase, and both are potent elastin repressors, we examined the effect of the addition of the two ligands together on elastin mRNA levels. Each ligand was used at a concentration of 10 ng/ml, which represented their maximal inhibitory effects on elastin mRNA (Fig. 3B and data not shown). Cells were treated for 48 h with HB-EGF, FGF-2, or the two ligands in combination. Interestingly, coaddition of these two ligands resulted in a greater inhibitory effect on elastin mRNA than observed with either ligand alone (Fig. 7). GAPDH levels were not affected by any of the treatments. These results show that the elastin-suppressing effects of these two elastase-released growth factors are additive.
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HB-EGF induces FGF-2 mRNA and protein levels. HB-EGF is thought to indirectly regulate some cellular processes by inducing the levels of other growth factors (2, 36, 53). Because of the major contribution of FGF-2 as an elastin downregulator in pulmonary fibroblasts (44), we examined whether HB-EGF treatment induces FGF-2, which may potentially contribute to HBEGF-dependent inhibition of elastin transcription. Cells were treated with HB-EGF for different times. Total cellular RNA was prepared and subjected to Northern analysis to measure the levels of FGF-2 mRNA. As shown in Fig. 8A, HB-EGF treatment led to a time-dependent induction of FGF-2 mRNA, as the level of FGF-2 mRNA was increased gradually to its peak in 8-12 h. Next, cellular FGF-2 protein levels in HB-EGF-treated cells were examined by Western analysis. In pulmonary fibroblast cell lysates, three forms of FGF-2 were detected, with apparent molecular masses of 18, 22, and 23.5 kDa (Fig. 8B), very similar to those described by others as different products of translation initiated from alternative start codons (37). HB-EGF treatment increased the levels of all three forms of FGF-2 protein. The induction was visible at 8 h and maximal at 24 h. Together, these results demonstrate the potential of the two elastase-released growth factors to work together in regulating elastin gene expression.
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DISCUSSION |
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Addition of HB-EGF to undigested pulmonary fibroblasts resulted in a significant decrease of elastin gene transcription through ERK activation. Furthermore, HB-EGF treatment led to a time-dependent induction of Fra-1, although not affecting the levels of its binding partner, c-Jun. These signaling events are similar to those described for FGF-2 (6, 42), implying that a common nuclear signaling pathway is used by both ligands to inhibit elastin transcription. These results suggest that elastase-released HB-EGF, together with FGF-2 (44), may act in a paracrine manner to repress elastin gene expression.
Like other EGF family members, soluble HB-EGF is processed from its transmembrane precursor, i.e., pro-HB-EGF (40). Interestingly, pro-HB-EGF by itself is biologically active and can function as a juxtacrine factor (19). The membrane-anchored proform signals only in adjacent cells, whereas its cleaved product can function in a paracrine mode and impact nonadjacent cells. Besides the obvious physical difference between their modes of action, it has been demonstrated that the biological effects of the membrane-bound form vs. the soluble, released form of HB-EGF can be different (24). Together, these observations led to the hypothesis that shedding or proteolytic release of soluble HB-EGF from the cell surface can act as a switch to regulate certain biological processes. Several metalloproteinases from the ADAM (a disintegrin and a metalloproteinase) family are thought to be responsible for HB-EGF ectodomain shedding in different cell types (17). By using a hydroxamate-based inhibitor of EGFR ligand shedding and the neutralizing antibody against HBEGF, others have suggested the involvement of HBEGF shedding in submandibular gland epithelial morphogenesis (49) and cutaneous wound healing (47). In the latter study, which used a model based on mechanical disruption of the cell culture layer, HB-EGF processing was found to be associated with keratinocyte proliferation and migration during the repair process (47).
In the present study, we show that brief treatment of matrix-laden pulmonary fibroblasts with elastase results in the release of soluble HB-EGF that is similar in size to the shed form of HB-EGF reported by others (16). This implies that pancreatic elastase may cleave a proteolytic-susceptible site in pro-HB-EGF similar to that targeted by the HB-EGF "sheddase" or activate a metalloproteinase, which in turn processes pro-HBEGF. Interestingly, others have shown that elastases release the mature form of TGF-, another EGF family member, from the cell surface (28, 30). These observations, together with our findings, suggest that elastase treatment of cells can lead to processing of membrane-bound growth factor precursors, with the end products similar to those resulting from metalloprotease-mediated ectodomain shedding.
Elastase-released HB-EGF may function to induce cell proliferation and migration at the matrix-injured sites, similar to that reported by Tokumaru and colleagues (47) for an in vitro wound model. It is important to note that the equilibrium binding of HB-EGF to elastase-treated cells was decreased immediately after the injury due to the loss of cell-associated HSPG (Fig. 2). Studies of FGF-2 binding to elastase-treated cells show that the number of HSPG sites on the cell surface gradually recovers after the initial loss (4). Therefore, elastase-treated cells can be responsive to the signal of HB-EGF if given time for recovery. Previous studies from our group have proposed that the cellular responses, such as those induced by growth factors, are dependent on the proliferative potential of the cells (6). In this regard, it is significant to point out that we have recently reported that one of the major effects of elastase treatment is to release cells from contact inhibition, therefore reinitiating both cell proliferation and subsequent communication between the cells and the newly synthesized matrix components (41). Thus although in undigested matrix-laden fibroblasts the major effect of HB-EGF is to inhibit elastin gene expression, different cellular responses, such as proliferation or increased migration, can be induced in cells primed with brief elastase treatment. Whether soluble HBEGF plays a role in inducing cell proliferation and/or recruitment of cells to the site of injury is a question we are investigating.
Because FGF-2 and HB-EGF are both released by elastase, we investigated the effect of coaddition of the two growth factors on elastin gene expression. We found that FGF-2 and HB-EGF have additive inhibitory effects on elastin gene expression. This is an interesting observation considering the similarity between the two signaling pathways. It suggests that pulmonary fibroblasts have a sufficient intracellular signaling capacity to quantitatively respond to simultaneous activation of EGFR and FGF receptor. Furthermore, we demonstrate that HB-EGF induces the synthesis of FGF-2 in pulmonary fibroblast cultures, suggesting that the two factors participate in cross-regulatory mechanisms to mediate cellular responses in the pulmonary fibroblasts. Interestingly, cross-regulation between FGF-2 and HB-EGF has been shown in vascular smooth muscle cells (11, 36) and lung fibroblasts (53). The latter study shows that the induced FGF-2 was involved in the proliferation of lung fibroblasts stimulated by HBEGF. How FGF-2 and HB-EGF might function together to promote repair of elastase injury is an intriguing question and one we are pursuing.
In conclusion, our results demonstrate that HB-EGF is a potent regulator of elastin gene expression in "normal" undigested fibroblast cell cultures. The fact that HB-EGF downregulates elastin gene expression by a mechanism that appears similar to FGF-2, albeit via activating a different receptor, is interesting. A more complicated situation that we are currently addressing is how these released growth factors impact cells within areas that have sustained elastolytic injury.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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