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Am J Physiol Lung Cell Mol Physiol 291: L232-L243, 2006. First published February 10, 2006; doi:10.1152/ajplung.00530.2005
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Neutrophil elastase-initiated EGFR/MEK/ERK signaling counteracts stabilizing effect of autocrine TGF-beta on tropoelastin mRNA in lung fibroblasts

Sandra J. DiCamillo,1,* Shenghong Yang,1,* Maria V. Panchenko,2 Paul A. Toselli,1 Estee F. Naggar,1 Celeste B. Rich,1 Phillip J. Stone,1 Matthew A. Nugent,1 and Mikhail P. Panchenko1

Departments of 1Biochemistry and 2Medicine, Boston University School of Medicine, Boston, Massachusetts

Submitted 19 December 2005 ; accepted in final form 8 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neutrophil elastase (NE) plays an important role in emphysema, a pulmonary disease associated with excessive elastolysis and ineffective repair of interstitial elastin. Besides its direct elastolytic activity, NE releases soluble epidermal growth factor receptor (EGFR) ligands and initiates EGFR/MEK/ERK signaling to downregulate tropoelastin mRNA in neonatal rat lung fibroblasts (DiCamillo SJ, Carreras I, Panchenko MV, Stone PJ, Nugent MA, Foster JA, and Panchenko MP. J Biol Chem 277: 18938–18946, 2002). We now report that NE downregulates tropoelastin mRNA in the rat fetal lung fibroblast line RFL-6. The tropoelastin mRNA downregulation is preceded by release of EGF-like and TGF-{alpha}-like polypeptides and requires EGFR/MEK/ERK signaling, because it is prevented by the EGFR inhibitor AG1478 and the MEK/ERK uncoupler U0126. Tropoelastin expression in RFL-6 fibroblasts is governed by autocrine TGF-beta signaling, because TGF-beta type I receptor kinase inhibitor or TGF-beta neutralizing antibody dramatically decreases tropoelastin mRNA and protein levels. Half-life of tropoelastin mRNA in RFL-6 cells is >24 h, but it is decreased to ~8 h by addition of TGF-beta neutralizing antibody, EGF, TGF-{alpha}, or NE. Tropoelastin mRNA destabilization by NE, EGF, or TGF-{alpha} is abolished by AG1478 or U0126. EGF-dependent tropoelastin mRNA downregulation is reversed upon ligand withdrawal, whereas chronic EGF treatment leads to persistent downregulation of tropoelastin mRNA and protein levels and decreases insoluble elastin deposition. We conclude that NE-initiated EGFR/MEK/ERK signaling cascade overrides the autocrine TGF-beta signaling on tropoelastin mRNA stability and, therefore, decreases the elastogenic response in RFL-6 fibroblasts. We hypothesize that persistent EGFR/MEK/ERK signaling could impede the TGF-beta-induced elastogenesis/elastin repair in the chronically inflamed, elastase/anti-elastase imbalanced lung in emphysema.

epidermal growth factor receptor; mitogen-activated protein kinase kinase; extracellular signal-regulated kinases 1 and 2; transforming growth factor-beta; elastin

ELASTIN, A MAJOR EXTRACELLULAR MATRIX constituent of the mature lung, maintains the resilience and structural integrity of the airways and blood vessels (3, 23). Elastin production in the lung is developmentally regulated by reaching its maximum during the early postnatal period and is critical for the coordinated phases of alveolarization and vascular growth (26, 39). Elastin is an exceptionally durable polymer, assembled from cross-linked tropoelastin monomers, and once deposited as an intrinsic component of elastic fibers, it essentially does not turn over in the normal lung (6, 33). Smoking-induced chronic pulmonary inflammation and inherited deficiency of {alpha}1-protease inhibitor are some of the known risk factors associated with the excessive activity of elastases, primarily of neutrophil and macrophage origin, which predispose the lung to the degradation of interstitial elastin. Persistent elastolysis and accompanying elastin repair insufficiency in the chronically elastase/anti-elastase-imbalanced lung are thought to result in irreversible loss of alveoli and lead to emphysema in humans and experimental animals (9, 28, 35, 38). The molecular-cellular mechanisms underlying the insufficiency of elastin repair in the elastase/anti-elastase-imbalanced lung are still under investigation.

Elastogenesis is a finely orchestrated biological response of fibrogenic tissue and is normally found in cells of mesenchymal origin such as smooth muscle cells and fibroblasts. In the lung parenchyma, elastin deposition is thought to be a function of the septal interstitial and alveolar wall fibroblasts (26). The elastase/anti-elastase imbalance in the lung is associated with the release/induction of growth factors and cytokines (2, 29), which are known to positively [transforming growth factor-beta (TGF-beta) (22, 41)] and negatively [interleukin-1beta (IL-1beta) (1), tumor necrosis factor-{alpha} (TNF-{alpha}) (12), or basic fibroblast growth factor (bFGF) (31)] modulate elastin gene expression in lung fibroblasts. Previous studies in our laboratory demonstrated that exposure of neonatal rat lung fibroblasts to neutrophil elastase (NE) and/or pancreatic elastase results in the release of biologically active epidermal growth factor receptor (EGFR) ligand(s), such as polypeptides immunochemically related to epidermal growth factor (EGF) (5) and heparin-binding EGF-like growth factor (18), leading to EGFR activation and initiation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinases 1 and 2 (ERK) signaling to tropoelastin mRNA downregulation (5). Interestingly, in airway epithelial cells, NE was shown to release TGF-{alpha}-like polypeptides followed by EGFR transactivation and signaling to increase expression of a major respiratory mucin, Muc5ac (14). Recently, in two different experimental models of chronic obstructive pulmonary disease/emphysema in mice, it was demonstrated that NE actually colocalizes with TGF-{alpha} and TGF-beta in the alveolar septum (20), suggesting that these and probably other elastase/anti-elastase imbalance-induced growth factors/cytokines, possessing counterposing effects on elastin gene expression, may signal to the aberrant elastogenesis and obstructive remodeling evident in the chronically inflamed pulmonary interstitium.

The current study was performed to investigate the mechanism of NE-initiated EGFR/MEK/ERK cascade-mediated tropoelastin mRNA downregulation and to determine its overall impact on elastogenesis in lung fibroblasts. In this study we demonstrate that EGFR/MEK/ERK signaling inhibits insoluble elastin deposition via destabilization of tropoelastin mRNA stabilized by autocrine signaling of TGF-beta in RFL-6 lung fibroblasts.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Human NE was used as described previously (5). Murine epidermal growth factor (catalog no. 53003-018) was obtained from Invitrogen (Carlsbad, CA). Rabbit polyclonal anti-mouse EGF (neutralizing; catalog no. 06-102), rabbit polyclonal anti-ERK (COOH-terminal domain; catalog no. 06-182), and rabbit polyclonal anti-Smad2/3 (catalog no. 07-408) antibodies were received from Upstate Biotechnology. Rabbit polyclonal anti-phospho-ERK antibodies (catalog no. 9101) and U0126 were obtained from Cell Signaling Technology. Recombinant human TGF-{alpha} (catalog no. 239-A), TGF-beta1 (catalog no. 240-B), and mouse monoclonal anti-TGF-beta1–3 antibody (neutralizing; catalog no. MAB1835) were obtained from R&D Systems. Goat polyclonal anti-rat lung {alpha}-elastin antiserum (catalog no. RA75) was obtained from Elastin Products. Mouse anti-TGF-{alpha} monoclonal antibodies (catalog no. GF06), DMSO, AG1478, GM6001, TGF-beta type I receptor kinase inhibitor, anisomycin, and emetine were obtained from Calbiochem. Actinomycin D, cycloheximide, diisopropyl fluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF), 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB), sodium orthovanadate, Triton X-100, Tween 20, Nonidet P-40, mouse monoclonal anti-beta-actin antibody (catalog no. A5441), peroxidase-conjugated goat anti-rabbit antibodies (catalog no. A6154) were purchased from Sigma. Peroxidase-conjugated goat anti-mouse antibodies (catalog no. sc-2055) and peroxidase-conjugated donkey anti-goat antibodies (catalog no. sc-2020) were purchased from Santa Cruz. Recombinant human bFGF was received from Scios-Nova. Tissue culture reagents were obtained from Invitrogen and Sigma unless otherwise specified.

Cell culture and treatment. The rat fetal lung (gestation day 18) fibroblast cell line RFL-6 fibroblasts were obtained from the American Type Culture Collection (ATCC 192-CCL; Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (JRBioscience) containing 100 U/ml penicillin and 100 µg/ml streptomycin and supplemented with 5% fetal bovine serum (FBS; Atlas Biologicals), 1 mM sodium pyruvate, and 100 µM nonessential amino acids at 37°C in a humidified 5% CO2 atmosphere. For experiments, cells were seeded into 12-well cluster plates, 6-well cluster plates, 60-mm dishes, or 100-mm dishes at a seeding density of 25,000, 60,000, 120,000, or 350,000 cells per well/dish, respectively, and were cultured for 6 days. Once in a postconfluent ("hills and valleys") state, cells were placed into medium supplemented with 0.5% serum for 3 days and then were starved for a total of 14–16 h with three consecutive washes in serum- and antibiotic-free medium. Serum-free cell cultures were challenged with EGF and inhibitors as specified. Control cell cultures always received an equal amount of the solvent vehicle (i.e., PBS, DMSO, or ethanol) used with experimental cultures. The final concentration of DMSO or ethanol in the conditioned medium did not exceed 0.1% (vol/vol).

Northern blot analysis. Total RNA was extracted from cell cultures by using TRIzol reagent (Invitrogen). Samples of total RNA (7 µg/lane) were electrophoresed through 1.0% agarose-formaldehyde gels, capillary transferred to nylon membranes (Osmonics, Minnetonka, MN), and cross-linked to filters by ultraviolet irradiation (Stratalinker; Stratagene). To compare integrity and correct RNA loading, blots were stained with 0.04% methylene blue in 0.5 M sodium acetate. Membranes were prehybridized at 42°C for 2 h in a solution containing 50% formamide, 5x sodium chloride and sodium citrate (SSC: 1x SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5x Denhardt's reagent, 0.5% sodium dodecyl sulfate (SDS), and 100 µg/ml denatured salmon sperm DNA and were hybridized overnight in a solution containing 50% formamide, 5x SSC, 10% dextran sulfate, 0.5% SDS, and 32P-labeled fragments of 1.1 kb of rat tropoelastin cDNA (30), 0.85 kb of rat {alpha},beta,{gamma}-actin cDNA (30), 0.8 kb of rat lysyl oxidase cDNA [provided by Dr. Herbert Kagan, Boston University School of Medicine (BUSM)], 2.1 kb of mouse fibrillin 1 cDNA (provided by Dr. Robert Lafyatis, BUSM), 1.0 kb of rat fibulin 5 cDNA (provided by Dr. Martin Joyce-Brady, BUSM), 0.6 kb of rat {alpha}1(I) collagen cDNA, or 0.9 kb rat {alpha}2(I) collagen cDNA (provided by Dr. Barbara Smith, BUSM) generated using a nick translation labeling kit (Invitrogen). After hybridization, membranes were washed (2 times in 1x SSC and 0.1% SDS at 55°C for 1 h) and exposed at –80°C in a cassette with double intensifying screens to X-ray film (Abgene, Rochester, NY) at several different times to ensure that the bands could be quantified by densitometry within the linear range. Hybridization signals were quantitated with the use of a Molecular Dynamics laser scanning densitometer.

Western blot analysis. After treatment, cell cultures in 12-well cluster plates, 6-well cluster plates, 60- mm culture dishes, or 100-mm culture dishes were quickly rinsed twice with room temperature PBS and lysed with gentle rocking at 4°C for 10 min in 0.2, 0.5, 1.0, or 3.0 ml, respectively, of ice-cold RIPA lysis buffer containing 50 mM Tris·HCl (pH 8.0), 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 mM DFP, and 0.2 mM sodium orthovanadate. The lysates were centrifuged at 4°C for 15 min at 15,000 g, and clear supernatants were kept at –80°C. Forty-microliter aliquots of supernatants (40 µg of total protein) were mixed with 20 µl of 3x SDS-PAGE sample buffer with 2-mercaptoethanol and heated for 10 min at 100°C, and 40-µl aliquots (~25 µg of total protein) were loaded on 4% stacking and 9 or 12% separating SDS-PAGE minigel. Electrophoresis was performed at constant current (20 mA/0.75-mm-thick gel with 10–15 lanes). After electrophoresis, the proteins were electroblotted (16 h, 4°C, 65 V) onto a 0.45-µm pore size nitrocellulose membrane (Schleicher and Schuell). Subsequent steps were performed at room temperature, unless specifically indicated. Transferred proteins were stained briefly with 0.1% (wt/vol) Ponceau S in 5% acetic acid (Sigma) to check for even loading and transfer. Membranes were blocked in 5% (wt/vol) nonfat milk powder in TBST (10 mM Tris·HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 1 h, washed three times for 5 min with TBST, incubated for 1 h with 1:1,000-diluted (in TBST) primary antibodies, and then washed three times for 10 min with TBST and incubated for 1 h with 1:1,000-diluted (in TBST) peroxidase-conjugated secondary IgG. The membranes were washed two times for 10 min with TBST and once for 5 min with TBS, and immunodetection of proteins was performed with enhanced chemiluminescence by using a LumiGlo chemiluminescence detection kit (Kirkegaard and Perry Laboratories). After being blotted dry, membranes were placed in a sheet protector and exposed to X-ray film (Abgene). For densitometric analysis, films were scanned and signals were quantitated using a Molecular Dynamics laser scanning densitometer. Routinely, blots were stripped of bound antibodies in 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris·HCl, pH 6.8 at 70°C for 30 min, washed with TBST, and reprobed with 1:1,000-diluted primary antibodies and 1:1,000-diluted peroxidase-conjugated secondary IgG.

Isolation and analysis of insoluble elastin. Elastin was purified from the RIPA buffer-insoluble portion of the whole cell lysates by hot alkali treatment in 0.1 NaOH at 95°C for 45 min. The hot alkali residue was then hydrolyzed in 6 N HCl for 24 h at 110°C, dried under a stream of nitrogen, and subjected to amino acid analysis (Beckman model 6300 with System Gold software; Palo Alto, CA) using an 80-min cycle (30). Total protein was calculated as the sum of the elastin protein, the protein in the hot alkali supernatant, and the protein present in RIPA buffer lysate. Insoluble elastin was normalized by calculating the ratio of the elastin amount (in µg) to the total protein amount (in mg) recovered from the same plate. Each normalized value is represented as the mean of four independent plate determinations (±SD).

Cell culture and electron microscopy. RFL-6 fibroblasts were seeded into p60 plates with a density of 120,000 cells/plate and cultured for 3 days in complete growth medium to reach visual confluency and then for 11 consecutive days in complete growth medium with or without 10 ng/ml EGF added. On day 14, cell cultures were rinsed briefly with PBS, fixed with 4.3% glutaraldehyde (Ted Pella) in 0.03 M sodium barbital/acetate buffer (pH 7.4) containing 0.07 M KCl, and then post-fixed in 1% osmium tetroxide (Ted Pella) prepared in the same buffer solution. The samples were dehydrated, embedded in a 1:1 Araldite 502 dodecenyl succinic anhydride epoxy mixture (Ciba), sectioned with an LKB ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Philips 300 electron microscope.

Elastin promoter-reporter plasmid construction. The BglII (blunted)-HindIII (–641 to +1 bp) fragment of rat elastin promoter (generously provided by Dr. Charles Boyd, University of Hawaii) was subcloned into pGL3-basic vector (Promega) using the SmaI and the HindIII sites.

Culture and transient transfection of cells. RFL-6 fibroblasts were seeded in complete growth medium supplemented with 10% FBS into 12-well cluster plates (175,000 cells/well) 24 h before transfection. Cells at ~90% confluence were starved at 37°C for 1 h in serum-depleted, antibiotic-free medium that was replaced with 1 ml of the same medium, containing preformed DNA/Lipofectamine2000 (Invitrogen) complex. The total amounts of DNA (purified with a plasmid midiprep kit from Qiagen) and Lipofectamine2000 were, respectively, 0.5 µg and 2 µl per well. After 5 h, transfection medium was aspirated and 2 ml of complete growth medium supplemented with 10% FBS were added per well, and the cells were maintained overnight. After 24 h, medium was replaced with 2 ml of the same culture medium containing vehicle (0.1% DMSO), AG1478 (10 µM), or U0126 (25 µM), and 1 h later cells were challenged with or without 10 ng/ml EGF. Twenty-four hours later, cells were quickly rinsed with PBS and harvested in 100 µl of reporter lysis buffer (Promega). Cell lysates were centrifuged at 4°C for 15 min at 15,000 g. A 5-µl supernatant aliquot was used to measure luciferase activity by using a luciferase assay system (Promega) and TD-20/20 luminometer (Turner Designs). The efficiency of transfection was consistently 50–60%, as determined by transfection of the cells with green fluorescent protein expression vector (Clontech).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Previously, investigators in our group have demonstrated that NE releases biologically active EGFR ligands, such as EGF-like polypeptides, and initiates EGFR-dependent MEK/ERK activation resulting in downregulation of tropoelastin mRNA levels in neonatal rat lung fibroblasts (5). In the present study we aimed to dissect the mechanism of EGFR/MEK/ERK-mediated tropoelastin mRNA downregulation, as well as to determine its impact on elastogenesis in lung fibroblasts. For this purpose, we performed experiments with the elastogenic lung fibroblast cell line RFL-6, because these cells not only express tropoelastin mRNA but also synthesize tropoelastin protein and deposit insoluble elastin.

We first examined whether RFL-6 fibroblasts respond to NE treatment by downregulating their tropoelastin mRNA levels as previously shown with neonatal rat lung fibroblasts (5). Indeed, NE at concentrations of 0.1–0.5 µg/ml caused a prominent decline in tropoelastin mRNA (Fig. 1A). Importantly, the NE-initiated tropoelastin mRNA downregulation was prevented in the presence of AG1478, an inhibitor of EGFR, or U0126, a MEK/ERK uncoupler, confirming the role of EGFR transactivation and MEK/ERK signaling in NE's effect on tropoelastin mRNA in RFL-6 cells as well (Fig. 1A).


Figure 1
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  		Fig. 1. Neutrophil elastase (NE) and epidermal growth factor (EGF) downregulate tropoelastin mRNA levels in RFL-6 fibroblasts via EGF receptor (EGFR)/MEK/ERK pathway. Postconfluent serum-starved cells (in 6-well cluster plates with 4 ml of medium per well) were incubated with or without 10 ng/ml EGF for the indicated times (B), in the absence or presence of 10 µM AG1478 or 25 µM U0126 for 1 h (A and C), and then for 18 h with or without 0.1 or 0.5 µg/ml NE (A), 10 ng/ml EGF, or 20 ng/ml basic fibroblast growth factor (bFGF; C), and total cellular RNA were isolated. Seven micrograms of each RNA sample were analyzed using Northern blotting for tropoelastin mRNA (A–C) and beta,{gamma}-actin mRNA (B). Methylene blue staining of 28S and 18S ribosomal RNAs was provided to ensure even RNA loading and integrity in different samples. The autoradiograms shown are representative of experiments that were repeated at least 3 times. TE, tropoelastin.

 
As expected, exogenously added EGF mimicked the effect of NE on tropoelastin mRNA in a time-dependent manner. Thus the inhibitory effect of EGF on tropoelastin mRNA reached its steady-state maximum after 18 h, whereas actin mRNA levels did not change (Fig. 1B). The EGF-induced tropoelastin mRNA downregulation was prevented by AG1478 or U0126 (Fig. 1C). Of note, only U0126 but not AG1478 prevented the bFGF-induced tropoelastin mRNA downregulation (Fig. 1C), consistent with the efficiency and selectivity of these pharmacological tools in the RFL-6 cell culture model.

Treatment of RFL-6 cells with increasing concentrations of NE led to accumulation in the cell conditioned medium of a number of proteins, including 24- and 22-kDa EGF-like polypeptides (Fig. 2A), as well as 29-, 27-, 15-, and 14-kDa TGF-{alpha}-like polypeptides (Fig. 2B), and coincided with prominent ERK activation in the cells (Fig. 2C). After collection of condition medium samples, NE activity was inactivated by addition of DFP. Addition of the conditioned medium samples containing NE-released EGF-like and TGF-{alpha}-like polypeptides to control RFL-6 cells resulted in ERK activation, which was similar to that induced by NE itself or by exogenously added EGFR ligands such as EGF or TGF-{alpha} (Fig. 2D). Importantly, activation of ERK in all cases was prevented in the presence of AG1478 or U0126 (Fig. 2D), consistent with the EGFR-dependent and MEK-mediated mode of ERK activation.


Figure 2
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  		Fig. 2. NE initiates EGFR/MEK-dependent ERK activation by releasing EGF-like and TGF-{alpha}-like polypeptides in RFL-6 fibroblasts. A–C: postconfluent serum-starved cells (in 100-mm plates with 8 ml of medium per plate) were treated with or without indicated concentrations of NE for 30 min, and then conditioned medium samples and total cell lysates were harvested in the presence of 1 mM diisopropyl fluorophosphate (DFP). Conditioned medium samples were chilled on ice and centrifuged at 15,000 g for 10 min at 4°C, and supernatants were concentrated 25 times to the final volume of 300 µl in Amicon Ultra-4 10,000 centrifugal filter devices and dialyzed against PBS. Fifty microliters of each conditioned medium sample were separated by 12% SDS-PAGE (in nonreducing conditions) and analyzed by Western blotting with anti-EGF antibody (A); 10 µl of each conditioned medium sample was separated by 15% SDS-PAGE (in reducing conditions) and analyzed by Western blotting with anti-TGF-{alpha} antibody (B), and ERK activation status in total cell lysates was determined by Western blotting with phospho-ERK antibodies (C). In A–C, Ponceau S-stained patterns of conditioned medium or whole cell lysate proteins are presented, and the electrophoretic mobility of prestained protein standards (Bio-Rad) is shown at right. D: postconfluent, serum-starved cells (in 12-well cluster plates) were incubated for 30 min in 500 µl of serum-free medium in the absence or presence of 10 µM AG1478 or 50 µM U0126, and then cells were challenged for 30 min with 75 µl of the concentrated and dialyzed conditioned medium samples isolated from the control and NE-treated cells (A and B) or with 1 µg/ml NE, 10 ng/ml EGF, or 10 ng/ml TGF-{alpha} as indicated. Status of ERK activation and total ERK were determined in 25 µg of total protein from each cell lysate by Western blotting with phospho-ERK and total ERK antibodies, respectively. The autoradiograms shown are representative of experiments that were repeated at least 3 times. p-ERK, phospho-ERK; WB, Western blot.

 
TGF-beta is known as a potent proelastogenic factor in lung fibroblasts (22). RFL-6 cells were shown to synthesize biologically active TGF-beta and express TGF-beta type II and type I receptors (41). We examined whether tropoelastin mRNA expression levels in RFL-6 cells are maintained by autocrine signaling of TGF-beta. Indeed, treatment of RFL-6 cells with TGF-beta type I receptor kinase inhibitor led to a 10-fold concentration-dependent decrease of tropoelastin mRNA levels, whereas actin mRNA levels were almost unaffected by this inhibitor (Fig. 3A). Exogenously added TGF-beta1 induced a moderate, less than two-fold stimulatory effect on tropoelastin mRNA levels, which was prevented in the presence of TGF-beta type I receptor kinase inhibitor as well (Fig. 3B). It is important to note that NE and EGF failed to further downregulate the remaining ~10% of tropoelastin mRNA levels, which were independent of endogenous TGF-beta/TGF-beta type I receptor kinase signaling (Fig. 3B, right). To further confirm the proelastogenic activity of endogenous TGF-beta, we treated RFL-6 cells with increasing concentrations of TGF-beta neutralizing antibodies and assessed tropoelastin protein expression levels. This treatment resulted in a dramatic downregulation of tropoelastin protein (Fig. 3C), demonstrating that tropoelastin expression in RFL-6 cells is dependent on the autocrine production and signaling of TGF-beta. The data suggest that EGFR/MEK/ERK signaling downregulates tropoelastin mRNA by overriding the stimulatory effect of endogenous TGF-beta signaling in lung fibroblasts.


Figure 3
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  		Fig. 3. Tropoelastin mRNA and protein expression in RFL-6 fibroblasts require autocrine TGF-beta signaling. Postconfluent, serum-starved cells (in 6-well cluster plates with 4 ml of medium per well) were treated with or without indicated concentrations of TGF-beta type I receptor kinase inhibitor for 18 h (A), with or without 2 µM TGF-beta type I receptor kinase inhibitor for 1 h and then with or without 0.1 or 0.5 µg/ml NE, 10 ng/ml EGF, or 2 ng/ml TGF-beta1 for 18 h (B), or with or without indicated concentrations of TGF-beta neutralizing antibody for 24 h (C). Total cellular RNA were isolated, and 3 (A) or 7 µg (B) of each sample was analyzed by Northern blotting for tropoelastin mRNA and beta,{gamma}-actin mRNA expression. Methylene blue staining of 28S and 18S ribosomal RNAs was provided to ensure even RNA loading and integrity. Total cell protein lysates were isolated, and 25 µg of each sample were analyzed by Western blotting for tropoelastin and beta-actin (C). The autoradiograms shown are representative of experiments that were repeated at least 3 times.

 
We next examined whether the effect of EGFR/MEK/ERK signaling on tropoelastin mRNA is reversible and whether continuous activation of this pathway may signal to downregulate TGF-beta production. For this purpose, cells were treated with or without EGF for 18 h, and then conditioned medium was replaced with fresh medium with or without EGF, and cells were cultured for an additional 24 h. Conditioned medium and cell layers harvested after the first and second cell treatments were processed for the assessment of TGF-beta protein (Fig. 4A) and tropoelastin mRNA (Fig. 4B). As expected, stimulation with EGF resulted in sustained tropoelastin mRNA downregulation (Fig. 4B). Importantly, EGF withdrawal resulted in a full recovery of tropoelastin mRNA expression levels, demonstrating the reversibility of the observed phenomena (Fig. 4B). However, levels of the endogenous biologically active 25-kDa form of TGF-beta secreted into the conditioned medium remained unchanged in all samples and did not correlate with the EGF-induced changes in tropoelastin mRNA levels (Fig. 4A), suggesting that EGFR/MEK/ERK cascade signaling does not inhibit TGF-beta production but, rather, interferes with the downstream components of the TGF-beta signaling pathway in RFL-6 cells.


Figure 4
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  		Fig. 4. EGFR/MEK/ERK signaling to tropoelastin mRNA downregulation is reversible and does not interfere with autocrine TGF-beta production or TGF-beta-induced Smad2 cytoplasm-nucleus translocation in RFL-6 fibroblasts. A and B: postconfluent cells (in 60-mm plates with 5 ml of 0.5% serum-containing medium per plate) were treated with or without 10 ng/ml EGF for 18 h and then washed for 1 h with two sequential medium changes and thereafter challenged again with or without 10 ng/ml EGF for 23 h. Conditioned medium samples and cells were harvested after the first and second EGF treatments as indicated. Conditioned medium samples were chilled on ice and centrifuged at 15,000 g for 10 min at 4°C. Supernatants were concentrated 10 times to the final volume of 500 µl in Amicon Ultra-4 10,000 centrifugal filter devices and dialyzed against PBS, and 25 µl of each sample were separated by 12% SDS-PAGE (in nonreducing conditions) and analyzed by Western blotting for TGF-beta (A). Ponceau S-stained patterns of proteins present in the conditioned medium samples are shown at bottom. Total cellular RNAs were isolated, and 7 µg of each total RNA sample was analyzed by Northern blotting for tropoelastin mRNA (B). Methylene blue staining of 28S and 18S ribosomal RNAs was provided to ensure even RNA loading and integrity. C: postconfluent serum-starved cells (in 12-well cluster plates with 2 ml of medium per well) were treated with or without 10 ng/ml EGF, 1 µg/ml NE, 2 µM TGF-beta type I receptor kinase inhibitor, or 100 µg/ml TGF-beta neutralizing antibody for 30 min and then with or without 2 ng/ml TGF-beta1 for 30 min. Cells were briefly rinsed with cold PBS, put on ice, and gently extracted for 5 min with 150 µl of hypotonic lysis buffer containing 20 mM HEPES-Na, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 10 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM PMSF, and 0.5 mM DFP. Cytosolic extracts were carefully removed from the wells, and the cytoskeleton/nuclei fraction was extracted with 150 µl of ice-cold RIPA buffer containing 500 mM NaCl for 30 min. Fifty microliters of each cytosolic and nuclear extract fraction were separated by 9% SDS-PAGE and analyzed by Western blotting with Smad2 antibody. Ponceau S-stained patterns of proteins present in cytosolic and nuclear extracts are shown at bottom. The autoradiograms shown are representative of experiments that were repeated at least 3 times.

 
TGF-beta is known to signal via activation and translocation of cytosolic Smad proteins into the nucleus. In addition, it has been suggested that EGFR/MEK/ERK signaling may interfere with the TGF-beta-induced nuclear translocation of Smads (36). We therefore examined whether NE or EGF might interfere with the TGF-beta-induced Smad2 translocation. For this purpose, RFL-6 cells were placed into fresh serum-free medium (to minimize the effect of endogenous TGF-beta) and treated with or without exogenous TGF-beta1 in the absence or presence of EGF, NE, TGF-beta type I receptor kinase inhibitor, or TGF-beta neutralizing antibody. Cells were then fractionated into nuclear and cytosolic fractions and assessed for the levels of Smad2 protein. Treatment of RFL-6 cells with TGF-beta1 led to increased Smad2 protein levels in the nuclear fraction (Fig. 4C, left) and a proportional decrease in the cytosolic fraction (Fig. 4C, right). As expected, the TGF-beta1-induced cytoplasm/nucleus translocation of Smad2 protein was prevented by TGF-beta type I receptor kinase inhibitor and also in the presence of TGF-beta neutralizing antibody (Fig. 4C). Nevertheless, stimulation of RFL-6 cells with EGF or NE did not prevent the TGF-beta1-induced Smad2 protein cytosplasm/nucleus redistribution (Fig. 4C), suggesting that EGFR/MEK/ERK cascade signaling inhibits TGF-beta-stimulated tropoelastin mRNA levels downstream of TGF-beta-dependent activation and nuclear translocation of Smad protein complex, most likely at the level of Smad protein interactions with transcriptional coactivators and/or corepressors (36).

It is well documented that in lung fibroblasts, TGF-beta exerts its prominent proelastogenic effect via an increase in tropoelastin mRNA stability (15, 21). We found that the tropoelastin mRNA is very stable with an estimated half-life of >24 h (Fig. 5, A, left, and B). Addition of the TGF-beta neutralizing antibody prominently decreased tropoelastin mRNA half-life to ~8 h (Fig. 5,A, middle, B), demonstrating that autocrine TGF-beta signaling is responsible for the high stability of tropoelastin mRNA. To dissect the cause of EGFR/MEK/ERK-mediated tropoelastin mRNA downregulation in RFL-6 cells, we examined whether the inhibitory effect might be due to tropoelastin mRNA destabilization. Consistent with the reduction in steady-state tropoelastin mRNA, treatment of cells with EGF (Fig. 5, A, right, and B), TGF-{alpha} (Fig. 6, A, middle, and B), or NE (Fig. 6, A, right, and B) led to a decrease of tropoelastin mRNA half-life to ~8h. The ability of TGF-beta neutralizing antibody, EGF, TGF-{alpha}, or NE to destabilize tropoelastin mRNA was not reflective of a general effect on mRNA stability, because actin mRNA stability did not change (Figs. 5A and 6A). We also determined whether an EGF-, TGF-{alpha}-, or NE-dependent decrease in tropoelastin mRNA stability could be prevented by AG1478 or U0126. Indeed, both inhibitors prevented tropoelastin mRNA destabilization by EGF (Fig. 5, C and D), TGF-{alpha} (Fig. 6, C and D), or NE (Fig. 6, C and D), indicating that EGFR/MEK/ERK signaling downregulates tropoelastin mRNA via decrease of its half-life.


Figure 5
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  		Fig. 5. EGF and TGF-beta neutralizing antibodies decrease tropoelastin mRNA half-life in RFL-6 fibroblasts. A: postconfluent cells (in 60-mm plates with 5 ml of 0.5% serum-containing medium per plate) were incubated for 8 h with or without 10 ng/ml EGF or 20 µg/ml TGF-beta neutralizing antibody and then treated with 20 µg/ml 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB) for 0, 4, 6, 8, 10, and 12 h. Total cellular RNAs were isolated, and 7 µg of each RNA sample were analyzed by Northern blotting for tropoelastin mRNA and beta,{gamma}-actin mRNA. Methylene blue staining of the 28S and 18S ribosomal RNA was provided to ensure even loading and integrity of RNA in different samples. B: tropoelastin mRNA signals were quantitated using scanning laser densitometry, relative tropoelastin mRNA levels (% of zero time) remaining at 4, 6, 8, 10, and 12 h after addition of DRB were plotted vs. time on semilogarithmic coordinates, and tropoelastin mRNA half-lives (t1/2) were determined. Each line shows values for normalized tropoelastin mRNA signal intensities obtained in 3 separate experiments. Data are means ± SD. C and D: postconfluent cells (in 60-mm plates with 5 ml of 0.5% serum-containing medium) were treated with 10 µM AG1478 (C) or 25 µM U0126 (D) for 1 h and then with or without 10 ng/ml EGF for 8 h. Thereafter, 20 µg/ml DRB were added to the cells for 0, 4, 8, and 12 h. Total cellular RNAs were isolated, and 7 µg of each RNA sample were analyzed by Northern blotting for tropoelastin mRNA. Methylene blue staining of the 28S and 18S ribosomal RNA was provided to ensure even loading and integrity of RNA in different samples. The autoradiograms shown are representative of experiments that were repeated at least 3 times.

 

Figure 6
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  		Fig. 6. TGF-{alpha} and NE decrease tropoelastin mRNA half-life via EGFR/MEK/ERK signaling in RFL-6 fibroblasts. A: postconfluent, serum-starved cells (in 6-well cluster plates with 4 ml of medium per well) were incubated for 8 h with or without 10 ng/ml TGF-{alpha} or 0.25 µg/ml NE and then treated with 20 µg/ml DRB for 0, 4, 8, and 12 h. Total cellular RNAs were isolated, and 5 µg of each RNA sample were analyzed by Northern blotting for tropoelastin mRNA and beta,{gamma}-actin mRNA. Methylene blue staining of the 28S and 18S ribosomal RNA was provided to ensure even loading and integrity of RNA in different samples. B: tropoelastin mRNA signals were quantitated using scanning laser densitometry, relative tropoelastin mRNA levels (% of zero time) remaining at 4, 8, and 12 h after addition of DRB were plotted vs. time on semilogarithmic coordinates, and tropoelastin mRNA t1/2 were determined. Each line shows values for normalized tropoelastin mRNA signal intensities obtained in 3 separate experiments. Data are means ± SD. C and D: postconfluent, serum-starved cells (in 6-well cluster plates with 4 ml of medium per well) were treated with 10 µM AG1478 (C) or 25 µM U0126 (D) for 1 h and then with or without 10 ng/ml TGF-{alpha} or 0.25 µg/ml NE for 8 h. Thereafter, 20 µg/ml DRB was added to the cells for 0, 4, 8, and 12 h. Total cellular RNAs were isolated, and 5 µg of each RNA sample were analyzed by Northern blotting for tropoelastin mRNA. Methylene blue staining of the 28S and 18S ribosomal RNA was provided to ensure even loading and integrity of RNA in different samples. The autoradiograms shown are representative of experiments that were repeated at least 3 times.

 
Changes in elastin gene transcription have been shown to correlate with EGFR/MEK/ERK-dependent tropoelastin mRNA downregulation in neonatal rat lung fibroblasts (18). We addressed this possibility in RFL-6 cells by performing elastin promoter-reporter transcriptional assays. The cells were transiently transfected with the –641 to +1-bp rat elastin promoter-luciferase construct and treated with or without EGF for 24 h, and luciferase activity was measured. EGF caused only a moderate ~40% decline in elastin promoter activity, which was prevented by AG1478 but not by U0126 (Fig. 7A), suggesting that the inhibitory effect of EGF/EGFR signaling on the elastin promoter is MEK/ERK independent. Of note, EGF-dependent inhibition of endogenous tropoelastin protein levels assessed in these samples was equally sensitive to both inhibitors (Fig. 7B), suggesting that the moderate MEK/ERK-independent inhibition of elastin gene transcription contributes little to the net effect of EGF on tropoelastin mRNA downregulation in RFL-6 cells.


Figure 7
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  		Fig. 7. EGF-induced inhibition of elastin promoter activity requires EGFR tyrosine kinase activity but does not require MEK/ERK coupling in RFL-6 fibroblasts. Confluent, serum-starved cells (in 12-well cluster plates with 1 ml of medium per well) were transfected with the rat elastin –641 to +1-bp promoter-luciferase construct and recovered in culture medium containing 5% FBS for 24 h, and then replaced with the same medium (2 ml per well) with or without 10 µM AG1478 or 25 µM U0126. After 1 h, cells were challenged with or without 10 ng/ml EGF for 24 h. Cells were harvested in reporter lysis buffer and luciferase activity was measured. A: luciferase activity, normalized to total cell lysate protein, is represented as an average of 3 independent determinations (means ± SD; n = 5 experiments). B: 25 µg of total protein from individual cell lysates were analyzed by Western blotting for expression of tropoelastin and beta-actin proteins. The autoradiogram shown is representative of an experiment that was repeated 5 times.

 
To clarify whether the EGFR/MEK/ERK-dependent decline in tropoelastin mRNA stability might depend on de novo transcription and translation, we analyzed the effect of general transcription and translation inhibitors. Thus treatment of the cells with actinomycin D or DRB, as well as with cycloheximide, emetine, or anisomycin, prevented the EGF-dependent tropoelastin mRNA downregulation (Fig. 8), indicating that the effect of EGFR/MEK/ERK signaling on tropoelastin mRNA requires transcriptional activation and synthesis of a protein factor(s) involved in the regulation of tropoelastin mRNA stability.


Figure 8
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  		Fig. 8. Transcription and translation inhibitors prevent EGF-induced tropoelastin mRNA downregulation in RFL-6 fibroblasts. Postconfluent cells (in 6-well cluster plates with 4 ml of 0.5% serum-containing medium per well) were first treated with or without 8 µg/ml cycloheximide (CHX), 1 µg/ml emetine (EMT), 2.5 µg/ml anisomycin (ANSM), 20 µg/ml DRB, or 10 µg/ml actinomycin D (ACTN D) for 1 h, and secondly with or without 25 ng/ml EGF for 12 h, and total cellular RNA was isolated. Seven micrograms of each RNA sample were analyzed by Northern blotting for tropoelastin mRNA expression. Equal loading and integrity of RNA samples were confirmed by methylene blue staining of 28S and 18S ribosomal RNAs. The autoradiogram shown is representative of experiment that was repeated 3 times.

 
To evaluate the impact of EGFR/MEK/ERK signaling on elastogenesis, we measured the insoluble elastin in control and chronic EGF-treated RFL-6 cell cultures. For this purpose, cells were grown to confluence, and upon reaching high steady-state levels of tropoelastin mRNA expression (data not shown), cells were treated with or without EGF for 5 consecutive days. Chronic exposure of cells to EGF constitutively downregulated tropoelastin mRNA (Fig. 9A) and protein (Fig. 9B) expression levels and resulted in a proportional decline in insoluble elastin by RFL-6 cells (Fig. 9C). Ultrastructural examination confirmed abundant amorphous elastin deposits in control cultures and very few elastin deposits in cultures chronically treated with EGF for 11 days (Fig. 9D). Importantly, the inhibitory effects of chronic EGF treatment on tropoelastin mRNA (Fig. 9A), protein (Fig. 9B), and insoluble elastin (Fig. 9C) were prevented in the presence of AG1478. We conclude that tropoelastin mRNA destabilization/downregulation induced by EGFR signaling results in a proportional inhibition of tropoelastin protein expression and a concomitant decrease of insoluble elastin in lung fibroblast cultures.


Figure 9
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  		Fig. 9. EGFR/MEK/ERK cascade signaling inhibits elastogenesis in RFL-6 fibroblasts. Cells were seeded into 60-mm plates, grown for 3 consecutive days to reach a visual confluent state, and then continuously treated in 5% serum-containing medium (6 ml per plate) in the absence or presence 10 ng/ml EGF with or without 10 µM AG1478, 10 µM U0126, or 10 µM GM6001. EGF and inhibitors were re-added to the cells with a 48-h cycle of medium change. After treatment for 5 days, cell cultures were harvested and total cellular RNA, protein, and insoluble elastin were isolated. A: 7 µg of total RNA from each sample were analyzed by Northern blotting for tropoelastin mRNA, lysyl oxidase (LOX-1) mRNA, fibrillin 1 mRNA, fibulin 5 mRNA, {alpha}1(I) collagen mRNA, and {alpha}2(I) collagen mRNA expression. Methylene blue staining of the 28S and 18S ribosomal RNA was provided to ensure even loading and integrity of RNA in different samples. B: 25 µg of total protein from each sample were analyzed by Western blotting for tropoelastin and beta-actin expression. The autoradiograms shown are representative of experiments that were repeated at least 3 times. C: elastin values were calculated as the ratio of the elastin amount (µg) to the total protein amount (mg) recovered from the same plate and are represented as an average of 4 independent determinations (means ± SD). The experiment was performed 3 times with similar results; data from a representative experiment are shown. D: after 11 days in culture with or without 10 ng/ml EGF, cells were fixed and electron microscopy was performed on horizontal sections of the cultures. Representative images of serial sections are presented. Note large amorphous deposits of extracellular elastin in the culture grown in the absence of EGF compared with little elastin accumulation in the EGF-treated culture. Arrowheads indicate elastin. N, nucleus.

 
It has been shown that EGFR signaling can stimulate expression of potentially elastolytic matrix metalloproteinases (MMPs) in some cell culture models (7, 13). Therefore, we examined the possible contribution of MMP-mediated elastolysis on EGF's effect on insoluble elastin levels by culturing cells in the presence of the broad-spectrum hydroxamic acid-based metalloprotease inhibitor GM6001. We found no protective effect of GM6001 on EGF-dependent inhibition of insoluble elastin levels (Fig. 9C), suggesting no contribution of the elastolytic metalloprotease(s) in EGF's effect on elastin in this experimental setting.

Insoluble elastin deposition depends on coordinate expression and function of other extracellular matrix components such as lysyl oxidase (LOX), fibrillins, and fibulins (11, 25, 32, 40). We determined whether the tropoelastin mRNA/protein downregulation and decreased insoluble elastin are associated with any change in expression of these proelastogenic genes in EGF-treated RFL-6 cells. Interestingly, EGF downregulated LOX mRNA levels, but in contrast to tropoelastin mRNA, LOX mRNA downregulation was not prevented in the presence of U0126 (Fig. 9A). This finding suggests that a different signaling pathway controls the EGFR-dependent downregulation of LOX mRNA in RFL-6 cells. Expression levels of fibrillin 1 and fibulin 5 mRNAs were not downregulated in EGF-treated cells (Fig. 9A), suggesting that these genes are not involved in the observed EGFR/MEK/ERK-dependent inhibition of insoluble elastin under these conditions.

It is interesting to note that {alpha}1(I) collagen and {alpha}2(II) collagen mRNA levels were upregulated by EGF in an AG1478- and U0126-sensitive manner (Fig. 9A), suggesting that the common EGFR/MEK/ERK pathway perhaps reciprocally modulates both type 1 collagen mRNAs and tropoelastin mRNA levels in RFL-6 fibroblasts.


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In the current study, we took advantage of the elastogenic lung fibroblast cell line RFL-6, which constitutively expresses high levels of tropoelastin mRNA and protein and readily deposits insoluble elastin, to dissect the inhibitory mechanism of NE-initiated EGFR/MEK/ERK-dependent tropoelastin mRNA downregulation, as well as to determine its overall impact on elastogenesis. Several observations were made. 1) We found that, similar to primary neonatal rat lung fibroblasts (5), NE signals to tropoelastin mRNA downregulation in RFL-6 cells via release of EGF-like ligands (found in both type of cells) and TGF-{alpha}-like polypeptides (found only in RFL-6 cells) and subsequently activates the EGFR/MEK/ERK cascade, suggesting that this might be a common response of the various elastogenic cells to elastolytic injury. 2) We demonstrated that the EGFR/MEK/ERK-dependent tropoelastin mRNA downregulation actually translates into major reduction of tropoelastin protein expression and insoluble elastin levels, signifying the overall inhibitory impact of EGFR/MEK/ERK cascade signaling on elastogenesis in lung fibroblasts. 3) We found that autocrine production and signaling of TGF-beta stabilizes tropoelastin mRNA in RFL-6 fibroblasts and is mainly responsible for the high steady-state tropoelastin mRNA levels in these cells. 4) Finally, we established that NE, as well as TGF-{alpha} and EGF, signals to downregulate tropoelastin mRNA by destabilizing tropoelastin mRNA stabilized by autocrine TGF-beta signaling. In summary, our findings are indicative of the counterposing interactions of the TGF-beta and EGFR/MEK/ERK pathways in the regulation of elastogenesis in lung fibroblasts.

The regulation of tropoelastin mRNA expression in lung fibroblasts involves both transcriptional and posttranscriptional mechanisms. Whereas transcriptional mechanisms are responsible for tropoelastin mRNA upregulation during fetal development and early postnatal lung maturation, posttranscriptional mechanisms inhibit tropoelastin mRNA expression in the normal mature lung (27). Interestingly, our previous study on neonatal rat lung fibroblasts revealed a correlation between EGFR/MEK/ERK-mediated downregulation of tropoelastin mRNA and transcriptional inhibition of the elastin gene (18). We observed a modest effect of EGF on the elastin gene transcription rate in RFL-6 cells. Moreover, although tropoelastin mRNA downregulation and destabilization required EGFR/MEK/ERK signaling, the effect of EGF on elastin transcription was independent of MEK/ERK activation. In summary, our data suggest that the EGFR utilizes a signaling pathway different from MEK/ERK to downregulate elastin gene transcription in RFL-6 fibroblasts. It is plausible that the impact of elastin gene transcription on the net inhibitory effect of EGFR/MEK/ERK signaling on elastogenesis may be specific to the cell type.

The TGF-betas are potent positive autocrine modulators of elastogenesis in lung fibroblasts (15, 21, 22, 41) and are known to stabilize tropoelastin mRNA half-life, presumably via the canonical Smad-dependent pathway (16). In agreement with these data, we found that autocrine TGF-beta production and signaling are responsible for the high tropoelastin mRNA stability and tropoelastin protein expression in RFL-6 cells. At least two distinct functional elements have been described in the primary structure of tropoelastin mRNA involved in the regulation of its stability. One element is localized to a GA-rich sequence of the 3'-untranslated region (8), and another one is localized to exon 30 of tropoelastin mRNA (42). It has been suggested that TGF-beta signaling stabilizes tropoelastin mRNA in lung fibroblasts by interfering with the binding activity of an unidentified destabilizing factor to exon 30 (42). Our findings suggest that tropoelastin mRNA levels are regulated primarily at the posttranscriptional level via counterposing interactions between the TGF-beta and EGFR/MEK/ERK signaling pathways. Moreover, the inhibitory effect of EGFR/MEK/ERK signaling on tropoelastin mRNA requires active transcription and translation. It is tempting to speculate that EGFR/MEK/ERK cascade signaling initiates the transcription and synthesis of a factor(s) that might inhibit TGF-beta-dependent stabilization of tropoelastin mRNA in lung fibroblasts.

We established that the EGFR/MEK/ERK-dependent downregulation of tropoelastin mRNA/protein expression ultimately results in the proportional inhibition of insoluble elastin. Extracellular deposition of elastin is a multistep process not only depending on expression of tropoelastin protein but also requiring proper processing and assembly of the secreted tropoelastin monomers into elastic fibers upon their interaction with different extracellular matrix proteins, such as LOX (10, 11) and LOX-like (LOXL) proteins (19) and scaffolding microfibrils, consisting of fibrillins (32), as well as the specific elastin-associated integrin ligand fibulin 5 (25, 40). Interestingly, tropoelastin mRNA downregulation in chronically EGF-treated cell cultures was associated with a prominent downregulation of LOX mRNA levels. However, the inhibitory effect of EGF on insoluble elastin levels was similar to that found on tropoelastin mRNA and protein (~2.5-fold reduction from control levels), and no significant difference in the specific content of desmosine and isodesmosine cross-links was found in insoluble elastin isolated from control and EGF-treated cultures (DiCamillo SJ, Yang S, Stone PJ, and Panchenko MP, unpublished data). This finding suggests that LOX protein in chronically EGF-treated cell cultures may still be present at sites of elastogenesis in sufficient amounts. Alternatively, another possibility is that LOXL proteins might substitute for any deficiency of LOX protein (10, 19). In contrast, fibrillin 1 and fibulin 5 mRNA expression levels were not decreased by EGFR/MEK/ERK signaling, implying that alterations in the levels of these elastin-associated proteins are unlikely to contribute to the downregulation of elastogenesis in EGF-treated cells.

The elastase/anti-elastase imbalance is thought to play an important causative role in the pathogenesis of emphysema. The excessive activity of NE serves as a hallmark of the elastase/anti-elastase imbalance in the emphysematous lung (4, 34, 35, 37). We describe in this article how NE, by releasing EGF-like and TGF-{alpha}-like EGFR ligands, transactivates the EGFR/MEK/ERK cascade that signals to destabilize tropoelastin mRNA and thereby downregulates elastogenesis in a RFL-6 lung fibroblast cell culture model. In fact, the pathophysiological role of constitutive ERK activation in cigarette smoke-induced emphysema in animals, as well as in emphysema in humans, has been proposed (24). Moreover, the progression of cigarette smoke-induced experimental emphysema in mice reveals a significant amount of NE, TGF-{alpha} and TGF-beta in the alveolar interstitium (20). Other studies have demonstrated that early neonatal lung-targeted overexpression of TGF-{alpha} leads to fragmented and disorganized elastic fibers in the alveolar septae, disruption in alveoli formation, and distal airspace enlargement in mice (17). On the basis of these findings and our data, it is tempting to speculate that constitutive signaling of the EGFR/MEK/ERK cascade in the chronically inflamed, elastase/anti-elastase-imbalanced pulmonary interstitium may counteract the stabilizing effect of TGF-beta on tropoelastin mRNA and downregulate TGF-beta-dependent elastogenesis and, thus, contribute to the ineffective repair of elastin in the emphysematous lung.


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This work was supported by National Heart, Lung, and Blood Institute Grant 5 P01-HL-046902.


   ACKNOWLEDGMENTS
 
We thank Judith A. Foster for stimulating discussions and Barbara Smith, Herbert Kagan, Martin Joyce-Brady, Robert Lafyatis, and Charles Boyd for generosity in sharing reagents.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. P. Panchenko, Dept. of Biochemistry, Boston Univ. School of Medicine, 715 Albany St., Boston, MA 02118 (e-mail: panchenko{at}biochem.bumc.bu.edu)

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.

* S. J. DiCamillo and S. Yang contributed equally to this work. Back


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