1 Department of Biochemistry and 2 The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 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 [-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.
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RESULTS |
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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We acknowledge the superb technical assistance of Valerie Verbitzki and Daniel Pine in isolating and maintaining the pulmonary fibroblasts.
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FOOTNOTES |
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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.
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