EDITORIAL FOCUS
Functional components of basic fibroblast growth factor signaling that inhibit lung elastin gene expression

Isabel Carreras1, Celeste B. Rich1, Julie A. Jaworski1, Sandra J. Dicamillo1, Mikhail P. Panchenko1, Ronald Goldstein2, and Judith Ann Foster1

1 Department of Biochemistry and 2 The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we have demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in confluent rat lung fibroblasts via the binding of a Fra-1-c-Jun heterodimer to an activator protein-1-cAMP response element in the distal region of the elastin promoter. In the present study, we show that bFGF activates the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, resulting in the translocation of phosphorylated extracellular signal-regulated kinase 1/2 into the nucleus followed by increased binding of Elk-1 to the serum response element of the c-Fos promoter, transient induction of c-Fos mRNA, and sustained induction of Fra-1 mRNA. The addition of PD-98059, an inhibitor of mitogen-activated protein kinase kinase, abrogates the bFGF-dependent repression of elastin mRNA expression. Comparative analyses of confluent and subconfluent fibroblast cultures reveal significant differences in elastin mRNA levels and activator protein-1 protein factors involved in the regulation of elastin transcription. These findings suggest that bFGF modulates specific cellular events that are dependent on the state of the cell and provide a rationale for the differential responses that can be expected in development and injury or repair situations.

pulmonary fibroblasts; extracellular signal-related kinase; Fra-1; injury


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROTEIN ELASTIN is an essential extracellular component of the pulmonary airways and vasculature because of its ability to impart the elasticity necessary to sustain respiratory dynamics (17). Our laboratory group (13, 28) has been examining pulmonary fibroblast cell cultures as an in vitro model of pulmonary injury. These studies showed that elastase treatment of fibroblast cultures resulted in the release of basic fibroblast growth factor (bFGF) (28) and cell surface heparan sulfate proteoglycans (5). Interestingly, the addition of elastase-released products to untreated pulmonary fibroblasts led to repression of elastin gene expression (13), and this effect was mimicked by exogenous bFGF (28). bFGF is able to decrease elastin transcription through the binding of a Fra-1-c-Jun heterodimer to a hybrid activator protein (AP)-1-cAMP response element located in the distal region of the elastin promoter (26). Transient transfections of fibroblast cultures with an elastin promoter-reporter construct, mutated within the AP-1-cAMP response element, resulted in increased promoter activity, suggesting that this sequence serves to repress elastin transcription in pulmonary fibroblast cultures. Further data to support this hypothesis were the finding that the addition of bFGF blocking antibody to pulmonary fibroblast cultures resulted in an increase in elastin mRNA (28). In composite, data thus far obtained suggest that bFGF acts as a negative regulator of elastin transcription in contact-inhibited fibroblast cultures where elastin transcriptional levels are normally high (6).

Many studies (24, 31-33) have focused on the ability of bFGF to influence cell cycle progression, cell growth, and cell differentiation. The majority of these actions appear to be communicated through activation of extracellular signal-regulated kinase (ERK1/2), with subsequent induction and/or modification of different AP-1 family members. The fact that ERK1/2 activation results in such a plurality of cell responses suggested to us that identification of the ERK1/2 pathway components affected by bFGF could provide insight into molecular mechanisms underlying the differential elastogenic responses Foster et al. (13) have reported in pulmonary fibroblast cultures. Consequently, the primary goals of the present study were to determine the signal pathway components by which bFGF decreases elastin expression in confluent pulmonary fibroblast cultures and to compare these findings to subconfluent, proliferating fibroblast cell cultures. The latter situation presents a potential model for examining the events expected after elastase treatment of cell cultures where cell-matrix contacts are disrupted, cell proliferation is initiated, and cell surface receptor signaling is altered (5, 13). We found that bFGF signals a decrease in elastin gene expression through activation of the ERK1/2 pathway that results in the induction of Fra-1 mRNA and protein, with an eventual decrease in elastin transcription. Furthermore, we show that subconfluent and confluent fibroblast cell cultures differ in their endogenous levels of elastin mRNA and AP-1 family members as well as in their response to bFGF stimulation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Human recombinant bFGF (18 kDa) was obtained from Scios-Nova (Mountain View, CA). Fra-1, c-Fos, phospho-Elk-1, AP-2, mitogen-activated protein (MAP) kinase (MAPK) phosphatase (MKP)-1, and c-Jun rabbit polyclonal antibodies and horseradish peroxidase-conjugated anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies to phospho-ERK1/2 were purchased from New England Biolabs (Beverly, MA). Indocarbocyanine (Cy3) goat anti-rabbit IgG was purchased from Jackson ImmunoResearch (West Grove, PA). Complementary, single-strand oligodeoxynucleotides representing the AP-1 consensus sequence (5'-CGCTTGATGACTCAGCCGGAA-3') (23), the elastin promoter sequences from the -573- to -546-bp (5'-GGCAGAACCTGTCTCTAGCCAGACCTG-3') and the -238- to -212-bp (5'-TGCGTGTGTTGTGTCAAGAAAAAAGCTC-3') regions, and the c-Fos serum response element (SRE) sequence (5'-ACAGGATGTCCATATTAGGACATC-3') (38) were synthesized by the DNA Protein Core Facility at Boston University Medical Center (Boston, MA). Duplex oligomers were prepared as previously described (18).

Isolation and treatment of cell cultures. Neonatal rat pulmonary fibroblast cells were isolated from the lungs of 3-day-old Sprague-Dawley rats and seeded in the first or second passage as previously described (28). Cells for subconfluent cell cultures were plated at 2 × 104/cm2 in 75-cm2 flasks in 5% fetal bovine serum (FBS)-DMEM and maintained overnight. The medium was removed, and fresh 5% FBS-DMEM was added for 4 h. This medium was replaced with 0.5% FBS-DMEM overnight (~20 h). Confluent cell cultures were plated at 2 × 104/cm2 in 75-cm2 flasks and maintained for 2 wk in 5% FBS-DMEM. The medium was changed twice weekly. After 2 wk, the medium was replaced with 0.5% FBS-DMEM and incubated overnight. Ten nanograms per milliliter of bFGF were then added, and the cell cultures were incubated for various times. The inhibitor PD-98059 (50 µM) or an equal amount of the solvent dimethyl sulfoxide (DMSO) was added to the cells 30 min before the treatment with bFGF (10 ng/ml). Cycloheximide (2 µg/ml) was added at the same time as bFGF (10 ng/ml).

Isolation and analysis of RNA. Total RNA was isolated and analyzed by Northern blotting as described previously by Wolfe et al. (41). Rat cDNA for glyceraldehyde-3-phosphate dehydrogenase was purchased from American Type Culture Collection (Manassas, VA). Mouse c-Fos cDNA was generously provided by Dr. Michael Birrer (National Cancer Institute, Bethesda, MD). Rat tropoelastin cDNA was described by Rich and Foster (27). Mouse histone H3.2 plasmid was provided by Dr. W. F. Marzluff (University of North Carolina, Chapel Hill, NC). Human Fra-1 cDNA was generously provided by Dr. P. R. Dobner (University of Massachusetts Medical School, Worcester, MA). For poly(A)+ RNA isolation, 500 µg of total RNA were passed over a mini-oligo(dT) cellulose spin column following manufacturer's protocol (5 Prime right-arrow 3 Prime, Boulder, CO).

Preparation of cell and nuclear extracts. The nuclear extracts were prepared as previously reported by Conn et al. (10). Total cell lysates were prepared from cells washed two times with ice-cold PBS and then extracted for 10 min at 4°C with ice-cold cell lysis buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA (pH 7.5), 1 mM EGTA (pH 9.0), 0.5% Nonidet P-40, 0.4 mM phenylmethylsulfonyl fluoride, and 0.2 mM sodium vanadate]. The cells were scraped and pelleted at 18,000 g at 4°C for 10 min, and the supernatant was stored at -80°C. Total protein for each sample was determined by the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL).

Gel mobility shift assay. The duplex oligomers described in Reagents were labeled with T4 polynucleotide kinase and separated from free [32P]ATP with a Sephadex G-50 column procedure (30). The labeled DNA oligomers and nuclear extracts were prepared and run as previously described by Rich et al. (26). For supershift experiments, 20 µg of nuclear extract proteins were combined with 4 µg of antibody for 30 min at room temperature before the addition of labeled oligomer. The reactions were incubated for an additional 30 min at room temperature before being run on the gel.

Western blot analysis. Cell and nuclear extracts were fractionated by SDS-PAGE as specified and electrophoretically transferred to nitrocellulose as Rich et al. (28) have described. After transfer, the nitrocellulose membrane was stained briefly with Ponceau S solution (Sigma, St. Louis, MO) and rinsed with distilled water to check for even loading and transfer. The membranes were probed with primary antibody at room temperature for 2-3 h. Appropriate secondary antibody was added for 30 min at room temperature. Immunodetection of proteins was visualized by the chemiluminescence method according to manufacturer's instructions (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

In-gel kinase assays. An in-gel myelin basic protein kinase assay was performed as previously described (34) with minor modifications. Briefly, 2-wk-old confluent cultures of fibroblasts in 35-mm dishes were serum starved for 24 h. After challenge with the ligand, the cell cultures were quickly rinsed twice with room temperature PBS and lysed with gentle rocking for 10 min at 4°C in 0.25 ml of ice-cold buffer containing 10 mM Tris · HCl (pH 7.5), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.4 mM phenylmethylsulfonyl fluoride, and 0.2 mM sodium vanadate. The lysates were centrifuged at 4°C for 30 min at 15,000 g. The clear supernatants were mixed with SDS-PAGE sample buffer containing 2-mercaptoethanol and heated for 10 min at 100°C. Then, 40-µg aliquots were loaded on 4% stacking and 12% separating SDS-PAGE gels that had been polymerized with 0.4 mg/ml of myelin basic protein. After electrophoresis, the gel was washed with 20% isopropanol in 100 mM Tris · HCl, pH 8.0, followed by a wash in 100 mM Tris · HCl, pH 8.0, containing 5 mM 2-mercaptoethanol. Then the gel was denatured in 6 M guanidinium hydrochloride followed by renaturation in 0.04% Tween 40. The gel was incubated at room temperature in kinase buffer containing 20 mM HEPES, pH 7.2, 10 mM MgCl2, and 2 mM 2-mercaptoethanol for 30 min followed by another incubation in kinase buffer containing 50 µM ATP and 50 µCi of [gamma -32P]ATP (NEN, Boston, MA) for 60 min at room temperature. The gel was washed with 1% sodium pyrophosphate in 5% trichloroacetic acid, stained with Coomassie blue R-250, and dried. Autoradiography was performed for 6-24 h at -80°C with an intensifying screen.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The MAPK pathway signals bFGF induction of Fra-1 and subsequent repression of elastin mRNA levels. The previous study by Rich et al. (26) demonstrated that the addition of bFGF to confluent pulmonary fibroblast cultures resulted in repression of elastin gene transcription. This repression was conveyed via induction of Fra-1 and the subsequent binding of this transcription factor to a distal promoter element as a heterodimer with c-Jun. Because bFGF has been shown to transmit its signal through the MAPK pathway in other cell systems (24, 31-33), the potential involvement of this signaling pathway in pulmonary fibroblasts was investigated. After bFGF was added to pulmonary fibroblasts, the kinase activities of p44/p42 ERK1/2 were determined by an in-gel assay at various times after exposure (Fig. 1). The data demonstrate that the addition of bFGF results in activation of ERK1/2 within 5 min, with the highest activity at 30 min, followed by a lower level of activity that persists for 8-12 h.


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Fig. 1.   Basic fibroblast growth factor (bFGF) induced extracellular signal-regulated kinase (ERK) 1/2 activity. Serum-starved pulmonary fibroblasts were treated with bFGF (10 ng/ml) for the indicated times, and total cell lysates were obtained. Forty micrograms of protein were analyzed by in-gel myelin basic protein kinase assay as described in MATERIALS AND METHODS. Dried gels were stained with Coomassie blue.

The data from this experiment allowed us to pursue the involvement of the MAPK pathway in affecting downstream targets, i.e., Fra-1 and elastin. Because these targets are essential to our focus, the effects of PD-98059, an inhibitor of MAPK kinase, on the bFGF-dependent induction of Fra-1 and repression of elastin mRNA were examined by Western and Northern blot analyses, respectively. Figure 2A presents a Western blot using an antibody to Fra-1 to examine nuclear extracts isolated at 0 and 22 h after treatment with either bFGF alone or bFGF with PD-98059. Results show that Fra-1 expression is induced by the addition of bFGF but that this induction is abolished in the presence of PD-98059. Furthermore, Northern blot analysis was performed with total RNA isolated 22 h after treatment of the cells under the conditions described in Isolation and treatment of cell cultures. The results shown in Fig. 2B demonstrate that the downregulation of elastin mRNA by bFGF is inhibited in the presence of PD-98059. These results link the bFGF activation of ERK1/2 to Fra-1 induction and elastin mRNA repression.


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Fig. 2.   PD-98059 inhibits bFGF-dependent upregulation of Fra-1 protein and downregulation of elastin mRNA. A: Fra-1 expression was analyzed in pulmonary fibroblasts after 22 h of treatment with bFGF in the presence and absence of 50 µM PD-98059. Fifty micrograms of nuclear proteins extracted from bFGF-treated cells were electrophoresed on a 12% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and probed with a Fra-1 antibody and a horseradish peroxidase-conjugated secondary antibody. Fra-1 protein was visualized by chemiluminescence. B: total RNA (10 µg) was extracted from pulmonary fibroblasts that were untreated or treated with bFGF in the presence and absence of 50 µM PD-98059, and Northern blot analysis was performed. Top: autoradiography of the Northern blot hybridization with 32P-labeled elastin cDNA probe. Bottom: Northern blot stained with methylene blue for detection of rRNA.

bFGF stimulates the binding of phosphorylated Elk-1 to the SRE of the c-Fos promoter, transient induction of c-Fos mRNA, and a sustained induction of Fra-1 mRNA. Because Elk-1 has been shown to be a common nuclear substrate for activated ERK1/2 (20), the effect of bFGF on this transcription factor was pursued. Western blot analysis using several commercial antibodies recognizing the phosphorylated form of Elk-1 did not reveal any detectable phosphorylated Elk-1 as a result of bFGF addition. This situation has been noted by other investigators (20) and may reflect the low endogenous levels of this protein. Therefore, to detect the possible phosphorylation of Elk-1 in response to bFGF treatment, gel shift analysis was performed with the SRE element of the c-Fos promoter (14). This approach has been used successfully to determine the phosphorylation of Elk-1 by examining its ability to bind the SRE as a component of the ternary complex (14). Figure 3A provides a gel shift analysis in which nuclear extracts isolated from bFGF-treated pulmonary fibroblast cultures were incubated with radiolabeled SRE probe. Several major complexes form, including two slow migrating complexes (complexes A and B) and several faster moving complexes (complex C). Within 5 min after bFGF administration, complexes A through C increase and return to basal levels by 4 h. The migration of complexes A and B is similar to those reported for the binding of serum response factor and ternary complex factor (20). Because phosphorylated Elk-1 is known to associate with SRE to form a ternary complex (14, 20), an antibody against phosphorylated Elk-1 was added to the nuclear extract obtained 5 min after bFGF treatment. AP-2 antibody was added to the nuclear extract as a negative control to test the specificity of the reaction. The addition of phosphorylated Elk-1 antibody abrogates the binding of complex B, demonstrating that phosphorylated Elk-1 is a component of complex B and, more importantly, showing that bFGF treatment results in the phosphorylation of Elk-1 (Fig. 3B).


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Fig. 3.   bFGF rapidly induces Elk-1 binding to the serum response element (SRE) of the c-Fos promoter. A: 20 µg of nuclear proteins isolated from untreated and bFGF-treated pulmonary fibroblasts and 10 µg of poly(dI-dC) were incubated with 1 ng of 32P-labeled c-Fos SRE oligomer for the indicated times. The resulting complexes were resolved by native gel electrophoresis and visualized by autoradiography. Individual bands of the SRE binding complexes are labeled A, B, and C according to the nomenclature published by Liao et al. (20). B: 20 µg of nuclear proteins from pulmonary fibroblasts treated with bFGF for 5 min were incubated with 4 µg of phospho-Elk-1 (pElk-1) antibody or 4 µg of activator protein (AP)-2 antibody for 30 min at room temperature before the addition of radiolabeled DNA. These analyses were repeated 3 times with different cell extracts, different preparations of pElk-1 antibody, and different negative controls.

bFGF stimulation of ERK1/2 has been shown by others to induce c-Fos mRNA (7). Because the gel shift analysis shown in Fig. 3 suggests that bFGF might stimulate c-Fos gene transcription via phosphorylation and binding of Elk-1 to the SRE of the c-Fos promoter, we investigated the potential involvement of c-Fos in the bFGF-dependent signal pathway. Total RNA was isolated from pulmonary fibroblasts at early times after bFGF treatment, and Northern blot analysis was performed (Fig. 4A). Because the level of c-Fos mRNA has been shown to be controlled via mRNA stability as well as transient transcriptional regulation (9, 29), the addition of cycloheximide was included in these analyses to compare the effect of bFGF versus a general inhibitor of protein synthesis. The results show that bFGF induced the steady-state level of c-Fos mRNA within 15 min, with the highest level at 30 min, followed by a decline within 60 min. It is interesting that the addition of cycloheximide by itself resulted in a sustained increase in c-Fos mRNA for the 60-min period examined, suggesting that the c-Fos transcript is normally destabilized in these cell cultures (9, 29). The combination of bFGF and cycloheximide resulted in the highest and most sustained increase in c-Fos mRNA levels within the 60 min investigated but also revealed significant differences in the mechanism of induction. The fact that the addition of bFGF and cycloheximide together resulted in the highest level of c-Fos mRNA suggests that these factors are exerting their effects at different levels of c-Fos mRNA expression, namely transcriptional and posttranscriptional levels. Furthermore, the results agree with the hypothesis that the transient upregulation of c-Fos mRNA by bFGF is initiated through the phosphorylation and subsequent binding of Elk-1 to the SRE of the c-Fos promoter.


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Fig. 4.   Effect of bFGF on c-Fos and Fra-1 mRNAs. A: pulmonary fibroblasts were treated with bFGF (10 ng/ml), cycloheximide (chx; 2 µg/ml), or bFGF and cycloheximide together for the indicated times. Total RNA was extracted, and Northern blot analysis was performed with 32P-labeled cDNA probes for c-Fos, elastin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: total RNA (50 µg) from pulmonary fibroblasts that were untreated or treated for 4 h with bFGF or bFGF and cycloheximide were analyzed by Northern blot with Fra-1, c-Fos, and elastin cDNA probes. C: 10 µg of mRNA isolated from pulmonary fibroblasts treated with bFGF for the indicated times were analyzed by Northern blot for Fra-1 expression. This film was exposed for 1 wk at -80°C.

Our previous study (26) showed that bFGF treatment resulted in an induction of Fra-1 protein within 1-4 h after treatment. Because the present study is focused on the signaling pathway leading to elastin gene downregulation, the effect of bFGF on Fra-1 mRNA was examined in two ways. Figure 4B shows a Northern blot of RNA isolated 4 h after the addition of bFGF and bFGF plus cycloheximide. The steady-state level of Fra-1 mRNA was induced by bFGF, and this induction was unaffected when bFGF and cycloheximide were added together. Although not shown, the addition of cycloheximide by itself did not increase Fra-1 mRNA levels. The mRNA levels of elastin and c-Fos are included for comparison. Figure 4C provides a Northern blot analysis showing a bFGF-dependent increase in the steady-state levels of Fra-1 mRNA that is evident after 1 h and sustained within the 18-h treatment time with bFGF.

Subconfluent and confluent fibroblast cell cultures differ in elastin expression, levels of AP-1 family members, and response to bFGF. Our next series of experiments dealt with a comparison of bFGF signaling in cells actively proliferating versus those that are confluent and contact inhibited. The former situation is pertinent to situations in which elastase activity results in the loss of matrix and subsequent proliferation of cells (35, 39). For these comparative studies, pulmonary fibroblast cells were plated at different densities and left in culture for specified times after bFGF addition. Figure 5 provides a Northern blot showing that elastin mRNA is not detectable in subconfluent, proliferating fibroblasts, and, consequently, there was no observable effect of bFGF on elastin expression. Furthermore, the addition of bFGF to subconfluent cultures appears to decrease the level of histone mRNA with time, suggesting that bFGF by itself is not acting as mitogen.


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Fig. 5.   bFGF differentially affects levels of elastin and histone mRNAs in subconfluent and confluent pulmonary fibroblasts. Neonatal rat pulmonary fibroblast cells were plated at 2 × 104/cm2 in 75-cm2 flasks in 5% fetal bovine serum (FBS)-DMEM. The day after seeding, medium was removed and fresh 5% FBS-DMEM was added. For subconfluent cells, cultures were maintained in 5% FBS-DMEM for 4 h. For confluent cells, cultures were maintained in 5% FBS-DMEM for 2 wk. After ~20 h in 0.5% FBS-DMEM, subconfluent and confluent cultures were treated with bFGF (10 ng/ml) for 4 or 18 h, and total cellular RNA was extracted. Ten micrograms of RNA were analyzed by Northern blot with elastin and histone cDNA probes.

To compare the components of the bFGF signaling pathway in the two different cell culture conditions, our initial approach involved a comparison of ERK1/2 activation in the context of MKP-1 levels. Figure 6 provides a composite of three separate experiments in which the protein levels of phosphorylated ERK1/2 (p44 and p42) and MKP-1 were determined in both total cell lysates and nuclear extracts. This examination of ERK1/2 activation in separate cell extracts was predicated on our finding that cell lysates prepared for the in-gel kinase assay reported in Fig. 1 did not adequately reflect the contribution or actual level of proteins within the nucleus. Because these latter considerations are important to the ability of phosphorylated ERK1/2 to impact on transcriptional events, total cell lysates and nuclear extracts were obtained. A Ponceau S solution stain of each extract is provided (Fig. 6, insets) to illustrate the uniqueness of the compartments examined. The results obtained are interesting not only in the comparison of confluent and subconfluent fibroblast cultures but also in the understanding of the activation of ERK1/2 in confluent cultures. The data provided in Fig. 1 show that the addition of bFGF results in activation of ERK1/2 within 5 min, with highest activity at 30 min, followed by a lower level of activity that persists for 8-12 h. This conclusion was verified in the cell lysate measurement of phosphorylated ERK1/2 as shown in Fig. 6. On the other hand, the nuclear extract shows that phosphorylated ERK1/2 levels are high and sustained, with little fluctuation over the 8-h period after bFGF addition. Therefore, it appears that the initial transient levels of ERK1/2 activation seen in the cytoplasm translate into a high sustained increase within the nucleus. In contrast to this situation, subconfluent fibroblast cultures display a high but brief activation of ERK1/2 in the cytoplasm, with little to no sustained activation in the nucleus. A major reason for the differences of the duration of ERK1/2 activation between confluent and subconfluent cultures may be due to the endogenous levels of MKP-1. As seen in Fig. 6, the cytoplasmic and nuclear levels of MKP-1 are relatively high in subconfluent cultures compared with those in confluent cultures. The addition of bFGF to either culture does not appear to alter the intrinsic levels of MKP-1 found in the nucleus within the 8-h period examined, suggesting that MKP-1 levels are specified by the cell state and are not bFGF dependent.


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Fig. 6.   bFGF-dependent ERK1/2 activation and mitogen-activated protein kinase phosphatase (MKP)-1 expression differ in subconfluent and confluent pulmonary fibroblasts. Subconfluent and confluent pulmonary fibroblasts were untreated or treated with bFGF for the indicated times, and total cell lysates (A) and nuclear extracts (B) were obtained. Fifty micrograms of protein were analyzed by Western blot with antibodies to phospho-ERK1/2 (pERK1 and pERK2) and MKP-1. The total amounts of pERK1 and pERK2 detected on the nitrocellulose were quantitated by laser densitometry and plotted over the time of bFGF treatment. Values are means ± SD from 3 assays. Insets: protein pattern in cell lysates (A) and nuclear extracts (B) on a nitrocellulose lane stained with Ponceau S solution and nuclear extracts and cell lysates immunostained with antibodies to MKP-1.

To further compare the components of the bFGF signaling pathway in the two different cell culture conditions, Western blot analysis was used to examine the steady-state levels of various AP-1 family members in nuclear extracts (Fig. 7). The choice of specific AP-1 proteins was based on the previous study by Rich et al. (26) of bFGF effects in confluent fibroblasts. The major differences between the cell cultures are the components affected by bFGF and the intrinsic levels of AP-1 transcription factors. The only AP-1 family member affected by bFGF in confluent fibroblasts is Fra-1, whereas other differences between the cultures are endogenous and not altered by bFGF addition. Specifically, the steady-state levels of c-Fos and Fra-1 are higher in subconfluent cultures, whereas c-Jun levels are higher in the confluent cells. The higher levels of Fra-1 and c-Fos in subconfluent proliferating cells is in agreement with a study (15) in other cell types that has demonstrated that Fra-1 and c-Fos are maintained at high levels in proliferating cells.


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Fig. 7.   bFGF stimulation of AP-1 proteins in subconfluent and confluent bFGF-treated pulmonary fibroblasts. Subconfluent and confluent pulmonary fibroblasts were stimulated for the indicated times with bFGF, and nuclear extracts were obtained. Fifty micrograms of nuclear extract were subjected to Western blot analysis with antibodies specific for pERK1/2, c-Jun, c-Fos, and Fra-1.

Because our previous study (26) showed that a Fra-1-c-Jun heterodimer is responsible for the transcriptional downregulation of elastin by bFGF in confluent cells, gel shift analyses were performed to compare the ability of AP-1 proteins to bind the identified bFGF response element (Fig. 8). Nuclear extracts were prepared from confluent and subconfluent fibroblast cultures at specified times after bFGF addition. Comparative gel shift assays were performed with three probes containing 1) the elastin promoter bFGF response element (-564 to -558 bp) (26), 2) an elastin promoter AP-1 element (-229 to -223 bp) identified by Kahari et al. (19), and 3) the consensus AP-1 element found in the collagenase gene promoter (23). Results show that the three AP-1 sequences displayed very different binding patterns with the nuclear proteins isolated from subconfluent and confluent cells. The elastin bFGF response element does not exhibit any complex formation with nuclear proteins isolated from subconfluent fibroblasts even though the levels of Fra-1 are very high. These results suggest that the ability of Fra-1 to bind this element may depend on the level of its heterodimer partner, i.e., c-Jun and its competitor c-Fos or its phosphorylation state. It is interesting to note that Fra-1 appears as multiple bands in the subconfluent cultures, suggesting that it is phosphorylated (15). In contrast to extracts obtained from subconfluent cultures, nuclear extracts from confluent cultures form a major complex with the bFGF element in a situation where c-Jun levels are high, c-Fos levels are low, and Fra-1 levels are induced. The proximal elastin AP-1 sequence of the elastin promoter exhibits increased complex formation with the subconfluent cell extract at times after bFGF addition and no significant change in complex formation with the nuclear extracts from confluent cells. The consensus AP-1 sequence forms a strong complex with the extracts from subconfluent cells that is independent of bFGF addition and little complex formation with the extracts derived from confluent cells. These data are interesting in several respects: 1) the identified bFGF element responds only to bFGF treatment in confluent fibroblasts, and this binding affinity differs from the other two AP-1 sequences examined; and 2) bFGF has little effect on complex formation in subconfluent cells. Together these observations point out that the AP-1 family of transcription factors can bind to a number of different AP-1 sequences depending on their abundance and perhaps modifications. More important for our studies is the finding that the repression of elastin transcription by bFGF is dependent on the state of the fibroblast cell, which includes the endogenous levels of transcription factors and the duration of ERK1/2 activation.


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Fig. 8.   Complex formation among 3 AP-1-like sequences differs with nuclear proteins isolated from bFGF-treated subconfluent and confluent pulmonary fibroblasts cultures. Twenty micrograms of nuclear extract from subconfluent or confluent pulmonary fibroblasts treated with bFGF and 10 µg of poly(dI-dC) were incubated with 1 of the following 32P-labeled double-strand oligodeoxynucleotides for the indicated times: the elastin promoter sequence from the -573- to -546-bp region (5'-GGCAGAACCTGTCTCTAGCCAGACCTG-3'; A), the elastin promoter sequence from the -238- to -212-bp region (5'-TGCGTGTGTTGTGTCAAGAAAAAAGCTC-3'; B), or the AP-1 consensus sequence (5'-CGCTTGATGACGTCAGCCGGAA-3'; C). Underlined nucleotides, AP-1 or AP-1-like site. The resulting complexes were resolved in a 4% native polyacrylamide gel and visualized by autoradiography of the dried gel. Also shown in each gel is the 32P-labeled oligodeoxynucleotide run without the addition of any nuclear extract (F).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neonatal rat pulmonary fibroblast cell cultures represent an in vitro system to investigate injury or repair mechanisms underlying the regulation of elastin gene expression. These cultures elaborate an extracellular matrix rich in insoluble, cross-linked elastin and mimic the cellular environment in lung tissues (6, 13, 21). In the present study, we have pursued the signaling pathway by which bFGF downregulates elastin transcription because understanding its components may shed light on the repair mechanisms spatially restricted to the region of elastase damage (26, 36) as well as the mechanisms underlying developmental regulation of alveogenesis (4, 40).

The data presented demonstrate that the ERK1/2 pathway signals the elastogenic response triggered by the growth factor. Major determinants in the ability of this signaling pathway to repress elastin expression appear to be a sustained activation of ERK1/2 in a cellular environment where there is little to no expression of Fra-1 and where a high basal level of c-Jun expression exists. Under these conditions, bFGF is able to induce Fra-1, which then proceeds to bind the elastin promoter response element as a heterodimer with c-Jun.

The duration of ERK1/2 activation has been shown to be critical to cell signaling decisions. For instance, Raf-activated signaling pathways can elicit either a mitogenic response or a cell cycle arrest in NIH/3T3 cells depending on the level of ERK1/2 activation (42). Other studies have demonstrated that growth factors can stimulate a transient or sustained activation of ERK1/2 that correlates with proliferation or differentiation depending on the magnitude of the activation and the cell type (16, 22, 37). A recent study (11) on the role of sustained ERK1/2 activation on the expression of different AP-1 family members shows that Fra-1, Fra-2, c-Jun, and Jun B are targets for sustained ERK1/2 activation. The data presented here demonstrate that bFGF induces a sustained nuclear activation of ERK1/2 (8-12 h) in confluent cell cultures that leads to a pronounced and lengthy induction of Fra-1.

Results from this study show that AP-1 family members play an important role in conveying the bFGF-dependent repression of elastin expression. Components of the AP-1 family and ERK1/2 cascades are ubiquitous transcription factors and enzymes that participate in an array of cellular programs ranging from proliferation to differentiation (1, 8, 22). We have previously hypothesized that the level, modification, and/or sequestration of ubiquitous transcription factors endogenous to the cell control elastin transcriptional regulation (17). This hypothesis was based on the "housekeeping" features of the elastin promoter (3, 25). In the present study, we show that elastin expression and bFGF signaling are different between confluent and subconfluent fibroblast cultures. Specifically, the level of elastin mRNA in subconfluent cells is not detectable and the binding to the elastin promoter bFGF response element is negligible. These observations suggest that the lack of elastin expression in proliferating cells is not due to bFGF or its cognate response element but involves some other cis element(s) and trans-acting factor(s). Therefore, it appears that bFGF is a repressor of elastin transcription rather than a true inhibitor (all or none). This hypothesis is consistent with the proposal that the repression of elastin expression is critical during secondary septation of the developing lung and is regulated by bFGF (40).

The data obtained from comparative studies of subconfluent and confluent fibroblast cultures are interesting in several respects. Many of the published studies of bFGF-dependent effects on cell proliferation, growth, and differentiation were performed in transformed cell lines (31-33). Fewer reports address the response of primary cell cultures to bFGF. In these latter situations, cells were isolated at a committed phenotype in vivo yet were capable of changing phenotypic expression within different culture conditions (2, 6, 13). Consequently, primary cell cultures allow the possibility of viewing the effect of bFGF in a situation in which the cells are flexible in phenotypic expression and where entry of the cells into the cell cycle is dependent on cell-matrix communication and cell-cell contact. In the comparative studies reported here, the data show that not only the duration of bFGF-dependent ERK1/2 signaling but also the endogenous levels of AP-1 family members differ significantly between the two cell culture conditions. Furthermore, the comparative gel shift analyses of two AP-1-like sites in the elastin promoter and the AP-1 consensus sequence demonstrate the unique binding affinities of these elements. A major difference in binding potential is illustrated by the fact that the consensus AP-1 sequence and the elastin promoter -229- to -223-bp sequence are able to bind homodimers of c-Jun, whereas the -564- to -558-bp sequence binds only heterodimers (26). Although not shown, we have found that the -229- to -223-bp sequence does not compete effectively with protein complexes formed by the upstream -564- to -558-bp sequence, suggesting that the elastin promoter is poised to respond differentially to cellular levels and/or modifications of AP-1 proteins (19, 26). Physiologically, these observations may be very important because they suggest that the impact of bFGF in vivo will differentially affect cells depending on their proliferative potential and subsequent basal levels of transcription factors.

In conclusion, the data presented in this study identify the functional components of the bFGF signaling pathway that lead to elastin repression in confluent pulmonary fibroblast cultures. Comparative analyses of confluent and subconfluent fibroblast cultures reveal significant differences in elastin mRNA levels and AP-1 protein factors involved in the regulation of elastin transcription. Significantly, bFGF stimulation of ERK1/2 activation and induction of Fra-1 differ between these culture conditions. These findings suggest that bFGF modulates specific cellular events that are dependent on the state of the cell and provide a rationale for differential responses that can be expected in development and injury or repair situations. Current studies are now focused on the elucidation of an elastase-initiated signaling pathway in confluent fibroblast cultures. Preliminary data (12) show that elastase itself activates ERK1/2 independent of bFGF and that activation results in a number of different downstream targets that can modulate elastin expression.


    ACKNOWLEDGEMENTS

We acknowledge the superb technical assistance of Valerie Verbitzki and Daniel Pine in isolating and maintaining the pulmonary fibroblasts.


    FOOTNOTES

This work was supported by the National Heart, Lung, and Blood Institute Grant HL-46902.

S. DiCamillo and I. Carreras were supported by National Institute on Aging Grant AG-00115 and National Heart, Lung, and Blood Institute Training Grant HL-07035.

Address for reprint requests and other correspondence: J. A. Foster, Dept. of Biochemistry, Boston Univ. School of Medicine, 80 East Concord St., Boston, MA 02118 (E-mail: jfoster{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.

Received 14 February 2001; accepted in final form 16 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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