Division of Pulmonary and Critical Care Medicine, Atlanta Veterans Affairs Medical Center and Emory University School of Medicine, Atlanta, Georgia 30033
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lung injury, characterized by the flooding of interstitial and alveolar spaces with serum proteins, induces the expression of fibronectin (FN). This cell-adhesive extracellular matrix (ECM) glycoprotein is believed to modulate inflammation and wound repair. Murine NIH/3T3 fibroblasts transfected with a 1.2-kb human FN promoter-reporter gene were studied to gain insight into the mechanisms involved in the induction of FN by serum. Transcription of the FN gene, followed by FN protein production, was enhanced by 10% fetal bovine serum. This effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinases. ECMs typically found in injured tissues (i.e., type I collagen, fibrin, and FN) had no effect. Conversely, disruption of actin microfilaments inhibited, whereas disruption of microtubular assembly enhanced, the serum-induced FN response. The stimulatory effects of serum and microtubular disruption on FN gene transcription were related to increased DNA binding of the transcription factor cAMP response element binding protein. The data suggest that regulation of serum-induced FN expression in fibroblasts is dependent on protein kinases and on cytoskeletal integrity.
gene transcription; microtubules; actin microfilaments; murine fibroblasts; cyclic adenosine 5'-monophosphate response element binding protein
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE BIOLOGICAL RESPONSE to lung injury is characterized by a complex set of events that has not yet been entirely elucidated. In many instances of injury, it is unpredictable if the response will be adaptive, perfectly restoring function and form, vs. maladaptive, leading to the replacement of injured tissue with mesenchymal profibrotic components, a process known as the fibroproliferative response (7, 8). Numerous cellular signals are involved in promoting the fibroproliferative response, of which only a fraction has been determined. They include soluble signals found in serum, lung alveolar lining fluid, and tissue such as growth factors and cytokines (13, 26, 35, 67), and solid phase factors such as the extracellular matrix (ECM) components collagens and proteoglycans (reviewed in Refs. 54, 55, and 58).
One ECM component implicated in the pathogenesis of fibroproliferative disorders is fibronectin (FN) (10, 37, 55, 58). This multifunctional cell adhesive glycoprotein can be found in soluble form in plasma and in insoluble form as an integral part of tissues (42, 58). After injury, fibroblasts (among other cell types) residing in or recruited to a wound site synthesize and secrete FN in a soluble monomeric form. The secreted FN is then assembled into an insoluble matrix by poorly understood mechanisms that require the activation of integrins and possibly other cell surface receptors (45, 71, 72). This newly deposited matrix is further supplied with serum FN that, after extravasation from injured vascular structures, gets incorporated into the growing matrix.
In addition to contributing to coagulation (44) and
participating in the architectural changes that result from
fibroproliferation after injury, FN might play other roles in injured
tissues (54). Accurately defining the roles of FN under
these circumstances, however, has proven to be a daunting task, mainly
because there exist no in vivo models by which to study it. The
generation of knockout mice deficient in either FN or one of its
cellular receptors (i.e., the integrin
5
1) results in death during early
embryogenesis (27, 74). Consequently, what is known about
FN function comes from in vitro models with cultured cells and organs.
These studies suggest that during the fibroproliferative response to
tissue injury, FN might exert its influence by providing a provisional substrate on which cells can organize and become activated (16, 28). Once in contact with cells, FN binds integrin surface
receptors capable of signal transduction (15, 28). The
binding of these receptors results in the activation of intracellular
secondary messengers capable of inducing potent transcription factors
which can, in turn, affect the expression of many genes (15,
28). In this fashion, FN can modulate many cellular processes
ranging from adhesion and migration to differentiation and
proliferation, all considered necessary for tissue repair (2, 3,
7, 9, 24, 30, 47, 51). More recently, it has been demonstrated that FN can stimulate the production of proinflammatory cytokines such
as interleukin (IL)-1
(29, 46, 52). In view of this, it
is speculated that increased FN expression and deposition not only may
alter the structure of the lung, with obvious consequences to lung
function, but might modulate (and even amplify) inflammatory and repair
responses elicited in injured tissues.
Because of the many postulated effects on organ structure and function
during fibroproliferation, our work is directed to exploring the
intracellular signaling and transcriptional mechanisms involved in the
control of FN expression in fibroblasts. One factor considered key for
induction of FN expression in injured tissues is the extravasation of
serum proteins with FN-inducing activity (e.g., transforming growth
factor-, platelet-derived growth factor, interleukins) (5, 6,
18, 19, 53). Accordingly, this report explores the intracellular
mechanisms responsible for regulation of FN gene transcription in
fibroblasts exposed to serum and presents data indicating a role for
protein kinases and the state of cytoskeletal organization in
modulation of FN expression.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental reagents. Colchicine, cytochalasin B, taxol, and calphostin C were purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). The MEK1 inhibitor PD-98059 was purchased from New England Biolabs (Beverly, MA). Rat tail type I collagen was purchased from Boehringer Mannheim (Indianapolis, IN) or Upstate Biotechnology (Lake Placid, NY). Fibrin was purchased from American Diagnostica (Greenwich, CT). FN was isolated from bovine plasma by affinity chromatography as previously reported (57).
Plasmid construction.
The plasmid construct was donated by Dr. Thomas Birkenmeier (Dept. of
Medicine, Washington Univ. School of Medicine, St. Louis, MO). The FN
promoter construct contains ~1,200 base pairs (bp) of the 5'-flanking
region of the human FN gene isolated from the human fibrosarcoma cell
line HT-1080 (20). This construct includes 69 bp of exon
1, a CAAT site located at 150 bp, and the sequence ATATAA at
25 bp
from the transcription start site. The FN promoter also contains
several previously identified regulatory elements, such as three cAMP
response elements located at
415 bp,
260 bp, and
170 bp, and an
SP-1 site at
102 bp from the transcription start site. The FN
promoter was subcloned into the SmaI site of the pGL3
basic luciferase reporter vector (Promega, Madison, WI).
Electroporation and selection of cells. The pFN (1.2 kb) LUC promoter construct was introduced into murine NIH/3T3 fibroblasts from American Type Culture Collection (CRL 1658; Rockville, MD) via electroporation. Briefly, cells were washed with 1× HeBS electroporation media (20 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM dextrose, pH 7.05) and resuspended to a final concentration of 1.5 × 106 cells/ml. NIH/3T3 cells were added to the electroporation cuvettes (0.4-cm electrode gap) along with 40 µg of the pFN (1.2 kb) LUC plasmid DNA and 8 µg of pCL-neomammalian expression vector (Promega) and subjected to 226 V and 500 µF (Gene Pulser II Electroporation System; Bio-Rad, Hercules, CA). Electroporated cells were pooled, plated into 25-cm2 tissue culture flasks, and neomycin phosphotransferyl gene expressing cells were selected by the addition of 400 µg Geneticin antibiotic (G418; Life Technologies, Gaithersburg, MD) for a minimum of 2 wk. To obtain individual clones, cells were serially diluted into 96-well tissue culture plates. Single colonies were subsequently tested for luciferase activity; for this, cells were harvested by cell scraper, washed with PBS, resuspended in 100 µl of cell lysis buffer (Promega), sonicated, and a 10-µl aliquot was tested by adding 25 µl of luciferase assay reagent (Promega). Light intensity was measured using a Dynatech ML3000 microtiter plate luminometer. Results were recorded as normalized luciferase units and adjusted for total protein content, which was measured using the Bradford method (12). On the basis of luciferase expression, one colony (P1d) was selected for all subsequent experiments.
Cell culture and treatment.
Murine NIH/3T3 fibroblasts were maintained in Dulbecco's modified
Eagle's medium with 4.5 g/l glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1%
antibiotic-antimycotic solution (100 U/ml penicillin G sodium, 100 U/ml
streptomycin, and 0.25 µg/ml amphotericin B) and incubated in a
humidified 5% CO2 incubator at 37°C. The cells were
harvested by trypsinization with 2.5× trypsin and 5.3 mM EDTA (Sigma),
washed with PBS, counted, and plated at 1.5 × 105
cells/ml in 12-well tissue culture dishes in 0.4% or 10% FBS. Concurrently, cells were treated with colchicine (50 µM), taxol (20 µM), cytochalasin B (10 µM), calphostin C (1 × 107 M), or mitogen-activated protein kinase (MAPK)
inhibitor (50 µM). The doses of experimental agents were chosen based
on optimal doses reported in the literature. Cells were harvested at
24 h.
Electrophoretic mobility shift assay.
NIH/3T3 cells (3 × 105) were seeded onto
150-mm2 tissue culture flasks and incubated in either 0.4%
or 10% FBS for 24 h with and without concurrent treatment with
either colchicine or cytochalasin B at the doses described in
Cell culture and treatment. Cells were washed with
ice-cold PBS, and nuclear binding proteins were extracted by a
published method (22). Protein concentration was
determined by the Bradford method (12) using Bio-Rad
protein assay reagent. Double-stranded cAMP response element binding
(CREB) protein consensus oligonucleotide
(5'-AGAGATTGCCTGACGTCAGAGAGCTAG) was radiolabeled with
[-32P]ATP using T4 polynucleotide kinase enzyme.
Nuclear protein (5 µg) was incubated with radiolabeled CREB for
30 min at room temperature as described previously (52).
For competition reactions, nonradiolabeled consensus and mutated
CREB double-stranded oligonucleotides (5'-AGAGATTGCCTGTGGTCAGAGAGCTAG) were added to the reaction mixture at 50× molar concentration. DNA-protein complexes were separated on 6% native polyacrylamide gel
(20:1 acrylamide:bis ratio) in low ionic strength buffer (22.25 mM Tris borate, 22.25 mM boric acid, and 500 mM EDTA) for 2-3 h at
4°C at 10 V/cm. Gels were fixed in a 10% acetic acid/10% methanol
solution for 10 min, dried under vacuum, and exposed to X-ray film.
Radiolabeled DNA-protein complexes were extracted from gels and
quantified by scintillation counter.
Western blot. NIH/3T3 cells were treated with 0.4% FBS or 10% FBS for 24, 48, or 72 h, washed with ice-cold PBS, and lysed in 1 ml of homogenization buffer (50 mM NaCl, 50 mM NaF, 50 mM NaP2O7-10 H2O, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 0.01% Triton X-100, 10 µg/ml leupeptin, and 10 mM HEPES, pH 7.4) by repeated passages through a 26-gauge needle. The resulting homogenate was centrifuged at 14,000 rpm for 5 min at 4°C. Protein concentration was determined by the Bradford method (12). The protein (100 µg) was mixed with an equal volume of 2× sample buffer (125 mM Tris · HCl, pH 6.8, 4% SDS, 20% glycerol, 5-10% 2-mercaptoethanol, and 0.004% bromphenol blue), boiled for 5 min, loaded onto a 10% SDS-polyacrylamide gel with a 3.9% stacking gel, and electrophoresed for 1 h at 60 mA. The separated proteins were transferred onto nitrocellulose using a Bio-Rad Trans Blot semidry transfer apparatus for 30 min at 1.0 mA/cm2, blocked with Blotto [1× TBS (10 mM Tris · HCl, pH 8.0, 150 mM NaCl)], 5% nonfat dry milk, and 0.05% Tween 20 for 1 h at room temperature, and washed twice for 5 min with wash buffer (1× TBS and 0.05% Tween 20). Blots were incubated with a polyclonal antibody raised against bovine FN (antibody 1.2; 1:500 dilution) (56) for 2 h at 4°C, washed three times for 5 min with wash buffer, and incubated with a secondary donkey antibody raised against rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution) for 1 h at room temperature. The blots were washed four times in wash buffer, transferred to freshly made ECL solution (Amersham, Arlington, IL) for 1 min, and exposed to X-ray film. Protein bands were quantified by densitometric scanning using a laser densitometer (Molecular Dynamics, Sunnyvale, CA).
Screening for lipopolysaccharide. Experimental reagents were reconstituted in lipopolysaccharide-free water (Sigma). All treatment materials and culture media were screened with a Limulus-based endotoxin assay with a sensitivity of 0.06 ng/ml (Endotect-Schwarz/Mann Biotech, Cleveland, OH) as described (50). Reagents were found to remain endotoxin free throughout all experiments.
Statistics. Data were analyzed using one-way analysis of variance, and differences between groups were identified by Student's t-test. Data are presented as means ± SE of the mean.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum enhances FN gene transcription and protein production.
To characterize what effect serum has on FN expression, NIH/3T3 cells
(clone P1d) permanently transfected with the human FN promoter
connected to the luciferase reporter gene, pFN (1.2 kb) LUC
were cultured in either 0.4% or 10% FBS for 24 h,
harvested, and processed to determine luciferase activity. Figure
1A shows that exposure of
fibroblasts to 10% FBS resulted in a significant increase in FN
expression compared with fibroblasts cultured in 0.4% FBS
(P = 0.0001). This effect was time dependent with the response being noticeable by 6.5 h and achieving a plateau after 18-24 h (Fig. 1B). Cells for all subsequent experiments
were, therefore, harvested between 21 and 24 h. Cells cultured in 0.4% FBS also demonstrated a slight increase in FN expression with time, but
this was not significant. The serum induction of FN gene transcription
was associated with an increase in FN protein production as determined
by Western blotting (Fig. 1C). Note that FN protein
expression was increased at 24 h, with a peak detected at 48 h, followed by a decrease in FN protein production by 72 h. The
variance in the intensity of the serum-induced FN response appeared
dependent on the use of different lots of serum. However, all serum
samples always induced FN expression significantly.
|
Role of ECMs in serum induction of FN gene expression.
Injured tissues are characterized by alterations in ECM expression and
composition and show increased deposition of FN and type I collagen and
fibrin, among other ECMs (54, 55). Because of their
strategic location and their ability to affect intracellular signaling
and gene expression (see DISCUSSION), these ECM molecules could potentially modulate FN expression in injured tissues. To test
this possibility, cells cultured in both 0.4% and 10% FBS were
treated with monomeric soluble ECM components or insoluble ECMs coated
onto culture dishes followed by harvesting at 24 h. Control cells
were grown in serum without additions. FN and type I collagen were
tested at 100 µg/ml, whereas fibrin was tested at 500 µg/ml. We
previously demonstrated that these doses maximally stimulate another
gene (i.e., IL-1) in human monocytic cells (29, 49, 50, 52,
59). The soluble monomeric ECM components tested individually
did not significantly affect FN gene expression compared with controls
(not shown). The results were consistent for both concentrations of
serum. Similar results were obtained when the soluble ECMs
were added simultaneously (Fig.
2A).
|
Role of protein kinases in the serum induction of FN expression.
We next focused on the signal transduction mechanisms
affecting FN expression induced by serum. First, we examined
the possibility that protein kinase C (PKC) could be involved. This
kinase is increased in cells exposed to serum and has been implicated
in the resulting induction of FN and other genes (38).
Consistent with this, we found that exposure of transfected cells with
phorbol ester (25 nM, 24 h), an inducer of PKC, consistently
caused an increase in FN gene transcription in our system (not shown).
We then tested the effects of a potent inhibitor of PKC activation, calphostin C, on FN expression (36). As demonstrated in
Fig. 3A, light-activated
calphostin C not only blocked the serum response (P = 0.00001) but also ablated the constitutive expression of FN, whereas
inactive calphostin C had no effect. We also tested the effects of
another kinase inhibitor, PD-98059. This MEK1 inhibitor blocks the
activation of MAPK (4). Like the PKC inhibitor, PD-98059
also caused a reduction in FN expression, but to a lesser extent than
that seen with calphostin C (P = 0.005; Fig.
3B). It did not affect the constitutive expression of the
gene.
|
Role of cytoskeletal organization in serum-induced FN expression.
The role of cytoskeletal integrity on FN expression was also tested. To
this end, microtubule and actin microfilament structures were
separately disrupted with colchicine or cytochalasin B, respectively, in cells cultured in both 0.4% and 10% FBS. As seen in Fig.
4, A-C, opposite effects
occurred. Microtubule disruption with colchicine resulted in enhanced
gene expression, particularly when the cells were cultured in 10% FBS
(P = 0.0001; Fig. 4A). Interestingly, when
the microtubule stabilizing chemotherapy drug taxol was used, the serum
induction of FN gene expression was prevented (Fig. 4B). The
effects of these agents were dose dependent (see inset). Disruption of actin microfilament structures with cytochalasin B
produced a marked decrease in expression, to the extent of constitutive inhibition (P = 0.0001; Fig. 4C). Results
were consistent for both concentrations of serum. Cells remained 99%
viable, even with concentrations of cytochalasin B of 30 µM (not
shown).
|
Role of CRE in FN gene transcription.
We found the aforementioned observations regarding the role of
cytoskeletal integrity on FN expression intriguing and decided to
examine them in more detail. The work by Dean and colleagues (20,
21) identified the CRE as the transcription factor predominantly responsible for serum-induced FN gene expression, and, therefore, we
attempted to determine whether the effects of the
cytoskeletal-disrupting drugs were related to alterations in DNA
binding of CREs contained in the FN promoter by CREB. Indeed, we found
this to be the case. Using electrophoretic mobility shift assay, we
demonstrated that serum enhanced DNA binding of CREB (Fig.
5, compare lane 2 vs. lane 5). Colchicine did not inhibit this induction (compare
lanes 2 with 3 and 5 with
6). Conversely, cytochalasin B was found to inhibit DNA
binding of CREB, suggesting that its inhibitory effects on FN induction
by serum are mediated by inhibition of this transcription factor
(compare lanes 2 with 4 and 5 with
7). To show the specificity of the probe-DNA complexes,
nonradiolabeled CREB or mutated CREB oligonucleotides were added to the
mixtures at a 50-fold molar excess concentration. As seen in
lanes 8 and 9, binding was blocked with the
nonradiolabeled probe but was not inhibited by the mutated nonradiolabled oligonucleotide (lanes 10 and 11).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report, we confirmed that serum induces FN gene transcription in a dose- and time-dependent manner using stably transfected murine fibroblasts. As expected, this stimulatory effect was followed by increased production of FN protein. These observations are consistent with those of other investigations (5, 18, 20). The intracellular signals mediating the serum response are not well understood, but the activation of protein kinases such as PKC is thought to be important (38, 39). Herein, we show that serum induction of FN expression in our system does require PKC activation because the inhibition of this protein kinase prevented the serum-induced response. Additionally, we found that serum induction of FN gene transcription is decreased by a potent inhibitor of MEK1 activation, an intermediary step in the MAPK pathway (4).
A role for intracellular protein kinases in the stimulation of genes by
serum is not surprising. Serum contains a number of soluble factors
capable of stimulating signal transduction and FN gene transcription
such as vitamin D, transforming growth factor-, platelet-derived
growth factors, interleukins, and corticosteroids (18-20). In general, these agents have been shown to
induce PKC and MAPK activation, among other effects on signal
transduction (25). Both pathways appear to intersect at
the level of Raf-1, which can be activated by PKC (25).
Also, members of the ribosomal S6 kinase (RSK) protein family (RSK2),
which are substrates of extracellular regulated kinases generated
through the MAPK pathway, can phosphorylate CREB at Ser133,
a critical residue for activation of CREB and for expression of
c-fos (73). The latter pathway might be partially
responsible for the induction of FN by serum.
Another important finding of this report relates to the observation
that the serum-induced FN gene transcription appears to be dependent on
cytoskeletal integrity. This phenomenon is known as mechanotransduction
(31, 32) and has been demonstrated for a variety of genes,
including the ECM molecule osteopontin (11, 48, 50, 62,
68). This process is dependent on the physical and functional
links established between the cytoplasmic domains of ligand-bound
integrin receptors, intracellular cytoskeletal proteins, and secondary
signaling molecules (e.g., kinases) at the cell membrane in structures
termed focal adhesion complexes or FACs (69). This work
begins to define the intracellular signals stimulated during
mechanotransduction that lead to FN expression. First, we found that
microtubule destabilization with colchicine results in further
enhancement of serum-stimulated FN expression, whereas
"stabilization" of microtubules with taxol inhibited the response.
These results suggest that the influence of microtubule integrity on FN
expression runs along a spectrum of stability; the more stable the
microtubule system, the less effect serum has on FN expression. The
observation that a PKC inhibitor could block both the serum- and
colchicine-induced FN expression suggests a role for this kinase in
both processes. Other genes appear to be affected in a similar fashion
(11). Other kinases might contribute to the effects of
colchicine as well (43, 63). Taxol, on the other hand, was
found to inhibit the serum induction of FN, and this effect might be
related to its ability to act on specific signaling and transcriptional
pathways. For example, taxol has been shown to inhibit
microtubule-dependent NF-B activation by phorbol esters
(66), whereas others have shown taxol able to induce
c-jun NH2-terminal kinase signaling pathways
(41).
Together, these studies suggest that, similar to serum treatment,
disruption of microtubules results in the activation of specific
kinase-dependent pathways including PKC, cAMP/protein kinase
A, and MAPK. This is not surprising in view of the reported physical association between certain protein kinases and microtubules for which some investigators have postulated that the microtubule network might act as a "retention receptor for kinases or kinase activators" (43). In this scheme, microtubules serve to
restrain kinases that could become available for the transduction of
signals elicited at the cell surface under the right conditions.
Colchicine might facilitate the "release" of these kinases by
unfolding the COOH-terminal region of -tubulin (61).
Interestingly, disruption of the cytoskeleton with cytochalasin also resulted in alterations in FN expression; however, in contrast to our observations with colchicine, cytochalasin B inhibited FN expression in response to serum without affecting viability. This suggests a role for the actin microfilament system in this process. As described for the microtubular system, actin microfilament disruption may also affect signaling pathways potentially relevant to FN expression (33). PKC does not appear to mediate the inhibitory role of cytochalasin in our system, but other, less well-defined kinase systems might be involved (1).
The transcriptional mechanisms by which serum induces FN expression
were also examined. The studies performed with cells transfected with
deletion mutation constructs of pFN (1.2 kb) LUC suggest that the
signals induced by serum act directly on the FN gene to promote its
expression. In particular, they point to CREs present within the human
FN promoter as necessary to generate the serum-induced response. A
significant response was observed when all three CREs were present,
whereas the response was prevented in the absence of CREs. A maximal
stimulation required sequences present 5' of the CREs. This
association was further strengthened by the CRE oligonucleotide
cotransfection experiments. This information, together with studies
showing that dibutyryl adenosine 3',5'-cyclic monophosphate (an analog
of cAMP) and forskolin (an inducer of adenyl cyclase) stimulate FN gene
transcription in our system (unpublished observations), implicate CREB
in the serum-induced FN response. This is consistent with our findings
showing that DNA binding by CREB was indeed stimulated by both serum
and colchicine, but inhibited by cytochalasin. Therefore, it appears
that the signals elicited at the cell membrane by serum ultimately
result in the induction of CREB, which, in turn, is responsible for the observed effects on FN gene transcription. Furthermore, the work suggests that microtubular disruption might act by stimulating common
downstream signals that lead to CREB induction. Similarly, others have
demonstrated alterations in transcription factor production in response
to cytoskeletal disruption, including activator protein-1 and
NF-B (40, 48, 60, 64). More relevant to our work, colchicine has been shown to increase cAMP in S49 lymphoma cells (33). Interestingly, taxol has been shown to antagonize
the increase in cAMP induced by colchicine, and this might explain its
inhibitory effect in our system (70).
Finally, we tested the effects of ECMs on FN gene expression. Injured tissues are characterized by increased deposition of fibrin, FN, type I collagen, and other ECMs. These ECM components can trigger PKC-dependent signals as well as induce transcription factors involved in the regulation of gene expression. Moreover, intracellular events elicited by ECMs also appear to be modulated by cytoskeletal organization (15, 30, 34, 46, 49, 50, 52, 54, 59). Because of the possibility that a change in ECM composition might feedback to either stimulate or inhibit FN production, we tested the effects of some ECMs in our in vitro system. Our results suggest that, at least in vitro, FN and type I collagen display little, if any, feedback control on the constitutive expression of FN or on its response to serum. Fibrin, however, tended to stimulate gene transcription above control, but only when presented polymerized onto a substrate. Although this effect did not reach statistical significance in our system, it might be relevant in vivo because fibrinogen is present in serum and can be incorporated into newly formed insoluble matrices in injured tissues (37, 50, 65). Also, we have shown previously that fibrinogen can stimulate DNA binding by CREB in U-937 cells (49).
Implications to lung injury. In view of the above, we postulate that FN expression in injured lungs is stimulated via at least two mechanisms. The first relates to the stimulation induced by serum and the injurious agent. Acute lung injury is characterized by increased vascular permeability and the flooding of alveoli with proteinaceous material from serum (14). Under these circumstances, FN expression might be stimulated by the injurious agent directly, as demonstrated for paraquat and other agents (54, 55), or indirectly by the serum that floods the alveoli, which contains FN-stimulating factors. These factors stimulate PKC- and MAPK-dependent signals in interstitial and alveolar fibroblasts, resulting in increased FN expression. This process could be amplified by the release of chemotactic growth factors that promote the recruitment and proliferation of fibroblasts into the affected site.
Mechanical ventilation is yet another mechanism likely to be involved in the processes that lead to increased FN expression in acute lung injury. During mechanical ventilation, regional overinflation can result in cellular stretch and cytoarchitectural distortion that compromises cytoskeletal integrity (23). On the basis of our observations with agents capable of cytoskeletal disruption, it is conceivable that such an effect could also result in enhanced FN expression. If one of the consequences of ventilator-induced lung injury is microtubule destabilization, this would enhance the stimulatory effect of serum and, consequently, FN expression and deposition. Consistent with this, Berg and colleagues (6) found that inflation of rabbit lungs with high levels of positive end-expiratory pressure (PEEP) increased the expression of FN mRNA twofold compared with low PEEP ventilation. In summary, we have demonstrated that serum induces the transcription of the FN gene in cultured fibroblasts and that this effect is mediated via the activation of specific protein kinases and the induction of the transcription factor CREB. Serum-induced FN expression was altered by disruption of the cytoskeleton with microtubule destabilization, resulting in further induction of FN, and microfilament depolymerization, resulting in inhibition of the response. Further work will be required to determine the true relevance of these mechanisms in the ability of the lung to heal after injury. ![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank William Schuyler and Susanne Roser for excellent technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by an Established Investigator Award from the American Heart Association (to J. Roman).
Address for reprint requests and other correspondence: J. Roman, Atlanta Veterans Affairs Medical Center, Pulmonary Section, Rm. 12C191, 1670 Clairmont Road, Decatur, GA 30033 (E-mail: jesse.roman{at}med.va.gov).
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.
10.1152/ajplung.00445.2000
Received 6 December 2000; accepted in final form 17 October 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abedi, H,
and
Zachary I.
Cytochalasin D stimulation of tyrosine phosphorylation and phosphotyrosine-associated kinase activity in vascular smooth muscle cells.
Biochem Biophys Res Commun
245:
646-650,
1998[ISI][Medline].
2.
Adams, JC,
and
Watt FM.
Fibronectin inhibits the terminal differentiation of human keratinocytes.
Nature
340:
307-309,
1989[ISI][Medline].
3.
Akiyama, K,
Yamada SS,
Chen WY,
and
Yamada KM.
Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.
J Cell Biol
109:
863-875,
1989[Abstract].
4.
Alessi, DR,
Cuenda A,
Cohen P,
Dyudley DT,
and
Saltiel AR.
PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo.
J Biol Chem
270:
27489-27494,
1995
5.
Asselot-Chapel, C,
Combacau L,
Labat-Robert J,
and
Kern P.
Expression of fibronectin and interstitial collagen genes in smooth muscle cells: modulation by low molecular weight heparin fragments and serum.
Biochem Pharmacol
49:
653-659,
1995[ISI][Medline].
6.
Berg, JT,
Fu X,
Breen EC,
Tran H-C,
Mathieu-Costello O,
and
West JB.
High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma.
J Appl Physiol
83:
120-128,
1997
7.
Bitterman, PB.
Pathogenesis of fibrosis in acute lung injury.
Am J Med
92:
39S-43S,
1992[Medline].
8.
Bitterman, PB,
and
Henke CA.
Fibroproliferative disorders.
Chest
99:
81S-84S,
1991[Medline].
9.
Bitterman, PB,
Rennard SI,
Adelberg S,
and
Crystal RG.
Role of fibronectin as a growth factor for fibroblasts.
J Cell Biol
97:
1925-1932,
1983[Abstract].
10.
Bitterman, PB,
Rennard S,
Adelberg S,
and
Crystal RG.
Role of fibronectin in fibrotic lung disease. A growth factor for human lung fibroblasts.
Chest
83:
96S,
1983[Medline].
11.
Botteri, FM,
Ballmer-Hofer K,
Rajput B,
and
Nagamine Y.
Disruption of cytoskeletal structures results in the induction of the urokinase-type plasminogen activator gene expression.
J Biol Chem
265:
13327-13334,
1990
12.
Bradford, MM.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
13.
Broekelmann, TJ,
Limper AH,
Colby TV,
and
McDonald JA.
Transforming growth factor 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis.
Proc Natl Acad Sci USA
88:
6642-6646,
1991[Abstract].
14.
Burkhardt, A.
Alveolitis and collapse in the pathogenesis of pulmonary fibrosis.
Am Rev Respir Dis
140:
513-524,
1989[ISI][Medline].
15.
Clark, EA,
and
Brugge JS.
Integrins and signal transduction: the road taken.
Science
268:
233-239,
1995[ISI][Medline].
16.
Clark, RAF,
Lanigan JM,
DellaPelle P,
Manseau E,
Dvorak HF,
and
Colvin RB.
Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization.
J Invest Dermatol
79:
264-269,
1982[Abstract].
17.
Davis, LS,
Oppenheimer-Marks N,
McIntyre BW,
and
Lipsky PE.
Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules.
J Immunol
145:
785-793,
1990
18.
Dean, DC.
Expression of the fibronectin gene.
Am J Respir Cell Mol Biol
1:
5-10,
1989[ISI][Medline].
19.
Dean, DC,
Birkenmeier TM,
Rosen GD,
and
Wientraub SJ.
Glycoprotein synthesis and secretion. Expression of fibronectin and its cell surface receptors.
Am Rev Respir Dis
144:
S25-S28,
1991[ISI][Medline].
20.
Dean, DC,
Bowlus CL,
and
Bourgeois S.
Cloning and analysis of the promoter region of the human fibronectin gene.
Proc Natl Acad Sci USA
84:
1876-1880,
1987[Abstract].
21.
Dean, DC,
McQuillan J,
and
Weintraub S.
Serum stimulation of fibronectin gene expression appears to result from rapid serum-induced binding of nuclear proteins to a cAMP response element.
J Biol Chem
265:
3522-3527,
1990
22.
Dignam, JD,
Lebovitz RM,
and
Roeder RG.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res
11:
1475-1489,
1983[Abstract].
23.
Dreyfuss, D,
and
Saumon G.
Ventilator-induced lung injury.
Am J Respir Crit Care Med
157:
294-323,
1998
24.
Dufour, S,
Duband JL,
Humphries MJ,
Obara M,
Yamada KM,
and
Thiery JP.
Attachment, spreading, and locomotion of avian neural crest cells are mediated by multiple adhesion sites on fibronectin molecules.
EMBO J
7:
2661-2671,
1988[Abstract].
25.
Force, T,
and
Bonventre JV.
Growth factors and mitogen activated protein kinases.
Hypertension
31:
152-161,
1998
26.
Fowler, AA,
Hyers TM,
Fisher BJ,
Bechard DE,
Centor RM,
and
Webster RO.
The adult respiratory distress syndrome: cell populations and soluble mediators in the air spaces of patients at high risk.
Am Rev Respir Dis
136:
1225-1231,
1987[ISI][Medline].
27.
George, EL,
Georges-Labouesse EN,
Patel-King RS,
Rayburn H,
and
Hynes RO.
Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin.
Development
119:
1079-1091,
1993
28.
Giancotti, FG,
and
Ruoslahti E.
Integrin signaling.
Science
285:
1028-1032,
1999
29.
Graves, KL,
and
Roman J.
Fibronectin modulates expression of interleukin-1 and its receptor antagonist in human mononuclear cells.
Am J Physiol Lung Cell Mol Physiol
271:
L61-L69,
1996
30.
Hynes, RO.
Integrins: versatility, modulation, and signaling in cell adhesion.
Cell
69:
11-25,
1992[ISI][Medline].
31.
Ingber, DE.
Integrins as mechanochemical transducers.
Curr Opin Cell Biol
3:
841-848,
1991[Medline].
32.
Janmey, P.
The cytoskeleton and cell signaling: component localization and mechanical coupling.
Physiol Rev
78:
763-781,
1998
33.
Jasper, JR,
Post SR,
Desai SR,
Insel PA,
and
Bernstein D.
Colchicine and cytochalasin B enhance cAMP accumulation via post receptor actions.
J Pharmacol Exp Ther
274:
937-942,
1995[Abstract].
34.
Juliano, RL,
and
Haskill S.
Signal transduction from the extracellular matrix.
J Cell Biol
120:
577-585,
1993[ISI][Medline].
35.
Kelly, J.
Cytokines of the lung.
Am Rev Respir Dis
141:
765-788,
1990[ISI][Medline].
36.
Kobayashi, E,
Nakano H,
Morimoto M,
and
Tamaoki T.
Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.
Biochem Biophys Res Commun
159:
548-553,
1989[ISI][Medline].
37.
Kuhn, C,
Boldt J,
King T,
Crouch E,
and
McDonald JA.
An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis.
Am Rev Respir Dis
140:
1693-1703,
1989[ISI][Medline].
38.
Lee, B,
Park R,
Choi J,
Ryoo H,
Sohn K,
and
Kim I.
Stimulation of fibronectin synthesis through protein kinase C signaling pathway in normal and transformed human lung fibroblasts.
Biochem Mol Biol Int
39:
895-904,
1996[ISI][Medline].
39.
Lee, B,
Park R,
Sohn K,
and
Kim I.
Antagonistic regulation of protein kinase C-induced stimulation of fibronectin synthesis by cyclic AMP in human lung fibroblasts.
Biochem Mol Biol Int
43:
383-390,
1997[ISI][Medline].
40.
Lee, JS,
von der Ahe D,
Kiefer B,
and
Nagamine Y.
Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells.
Nucleic Acids Res
21:
3365-3372,
1993[Abstract].
41.
Lee, LF,
Li G,
Templeton DJ,
and
Ting JP.
Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK).
J Biol Chem
273:
28253-28260,
1998
42.
Limper, AH,
and
Roman J.
Fibronectin: a versatile matrix protein with roles in thoracic development, repair, and infection.
Chest
101:
1663-1673,
1992[Abstract].
43.
Manie, S,
Schmid-Alliana A,
Kubar J,
Ferrua B,
and
Rossi B.
Disruption of microtubule network in human monocytes induces expression of interleukin-1 but not that of interleukin-6 nor tumor necrosis factor-.
J Biol Chem
268:
13675-13681,
1993
44.
McDonagh, J,
Hada M,
and
Kaminski M.
Plasma fibronectin and fibrin formation.
In: Plasma Fibronectin. Structure and Function, edited by McDonagh J.. New York: Marcel Dekker, 1985, p. 121-148.
45.
McDonald, JA.
Extracellular matrix assembly.
Annu Rev Cell Biol
4:
183-207,
1988[ISI].
46.
Pacifici, R,
Roman J,
Kimble R,
Civitelli R,
Brownfield CM,
and
Bizzarri C.
Ligand binding to monocyte 5
1-integrin activates the
2
1 receptor via the
5 subunit cytoplasmic domain and protein kinase C.
J Immunol
153:
2222-2233,
1994
47.
Patel, VP,
and
Lodish HF.
A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.
J Cell Biol
105:
3105-3118,
1988[Abstract].
48.
Pavlath, GK,
Shimizu Y,
and
Shimizu N.
Cytoskeletal active drugs modulate signal transduction in the protein kinase C pathway.
Cell Struct Funct
18:
151-160,
1993[ISI][Medline].
49.
Perez, RL,
Ritzenthaler J,
and
Roman J.
Transcriptional regulation of the interleukin-1 promoter via fibrinogen engagement of the CD18 integrin receptor.
Am J Respir Cell Mol Biol
20:
1059-1066,
1999
50.
Perez, RL,
and
Roman J.
Fibrin matrices enhance the expression of interleukin-1 by human peripheral blood mononuclear cells. Implications for lung granulomatous inflammation.
J Immunol
154:
1879-1887,
1995
51.
Rickard, KA,
Taylor J,
Rennard SI,
and
Spurzem JR.
Migration of bronchial epithelial cells to extracellular matrix components.
Am J Respir Cell Mol Biol
8:
63-68,
1993[ISI][Medline].
52.
Ritzenthaler, J,
and
Roman J.
Differential effects of protein kinase C inhibitors on fibronectin-induced interleukin-1 gene transcription, protein synthesis and secretion in human monocytic cells.
Immunology
95:
264-271,
1998[ISI][Medline].
53.
Roberts, CR,
Birkenmeier TM,
McQuillan JJ,
Akiyama SK,
Yamada SS,
Chen WT,
Yamada KM,
and
McDonald JA.
Transforming growth factor stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.
J Biol Chem
263:
4586-4592,
1988
54.
Roman, J.
Extracellular matrices and lung inflammation.
Immunol Res
15:
163-178,
1996[ISI][Medline].
55.
Roman, J.
Extracellular matrices in the pathogenesis of lung injury and repair.
In: Interstitial Lung Disease, edited by Schwarz M,
and King T.. London: Decker, 1998, p. 207-227.
56.
Roman, J,
Jeon YJ,
Gal A,
and
Perez RL.
Distribution of extracellular matrices, matrix receptors, and transforming growth factor 1 in human and experimental lung granulomatous inflammation.
Am J Med Sci
309:
124-133,
1995[ISI][Medline].
57.
Roman, J,
LaChance RM,
Broekelmann TJ,
Kennedy CJR,
Wayner EA,
Carter WJ,
and
McDonald JA.
The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices.
J Cell Biol
108:
2529-2543,
1989[Abstract].
58.
Roman, J,
and
McDonald JA.
Fibronectins and fibronectin receptors in lung development, injury and repair.
In: The Lung: Scientific Foundations, edited by Crystal RG,
West JB,
Weibel ER,
and Barnes PJ.. Philadelphia, PA: Lippincott-Raven, 1997, p. 737-756.
59.
Roman, J,
Ritzenthaler JD,
Perez RM,
and
Roser S.
Differential modes of regulation of interleukin-1 expression by extracellular matrices.
Immunology
98:
228-237,
1999[ISI][Medline].
60.
Rosette, C,
and
Karin M.
Cytoskeletal control of gene expression: depolymerization of microtubule activates NF-kB.
J Cell Biol
128:
1111-1119,
1995[Abstract].
61.
Sackett, DL,
and
Varma JK.
Molecular mechanism of colchicine action: induced local unfolding of -tubulin.
Biochemistry
32:
13560-13565,
1993[ISI][Medline].
62.
Santell, L,
Marotti K,
Bartfeld N,
Baynham P,
and
Levin E.
Disruption of microtubules inhibits the stimulation of tissue plasminogen activator expression and promotes plasminogen activator inhibitor type I expression in human endothelial cells.
Exp Cell Res
201:
358-365,
1992[ISI][Medline].
63.
Schmid-Alliana, A,
Menou L,
Manie S,
Schmid-Antomarchi H,
Millet MA,
Giuriato S,
Ferrua B,
and
Rossi B.
Microtubule integrity regulates src-like and extracellular signal-regulated kinase activities in human pro-monocytic cells. Importance for interleukin-1 production.
J Biol Chem
273:
3394-3400,
1998
64.
Shaw, RJ,
Doherty DE,
Ritter AG,
Benedict SH,
and
Clark RA.
Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA.
J Cell Biol
111:
2139-2148,
1990[Abstract].
65.
Sitrin, RG,
Brubaker PG,
and
Fantone JC.
Tissue fibrin deposition during acute lung injury in rabbits and its relationship to local expression of procoagulant and fibrinolytic activities.
Am Rev Respir Dis
135:
930-936,
1987[ISI][Medline].
66.
Spencer, W,
Kwon H,
Crepieux P,
Leclerc N,
Lin R,
and
Hiscott J.
Taxol selectively blocks microtubule dependent NF-B activation by phorbol ester via inhibition of I
B
phosphorylation and degradation.
Oncogene
18:
495-505,
1999[ISI][Medline].
67.
Strieter, RM,
and
Kunkel SL.
Acute lung injury: the role of cytokines in the elicitation of neutrophils.
J Investig Med
42:
640-651,
1994[ISI][Medline].
68.
Toma, C,
Ashkar S,
Gray M,
Schaffer J,
and
Gerstenfeld L.
Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts.
J Bone Miner Res
12:
1626-1636,
1997[ISI][Medline].
69.
Wang, N,
Butler JP,
and
Ingber DE.
Mechanotransduction across the cell surface and through the cytoskeleton.
Science
260:
1124-1127,
1993[ISI][Medline].
70.
Wolberg, G,
Stopford CR,
and
Zimmerman TP.
Antagonism by taxol of effects of microtubule-disrupting agents on lymphocyte cAMP metabolism and cell function.
Proc Natl Acad Sci USA
81:
3496-3500,
1984[Abstract].
71.
Wu, C,
Fields AJ,
Kapteijn BA,
and
McDonald JA.
The role of 4
1 integrin in cell motility and fibronectin matrix assembly.
J Cell Sci
108:
821-829,
1995
72.
Wu, C,
Keivins VM,
O'Toole TE,
McDonald JA,
and
Gingsberg MH.
Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix.
Cell
83:
715-724,
1995[ISI][Medline].
73.
Xing, J,
Ginty DD,
and
Greenberg ME.
Coupling of the RAS-MAPK pathways to gene activation by RSK2, a growth factor-regulated CREB kinase.
Science
273:
959-960,
1996[Abstract].
74.
Yang, JT,
Rayburn H,
and
Hynes RO.
Embryonic mesodermal defects in 5 deficient mice.
Development
119:
1093-1105,
1993