Mechanical strain-induced posttranscriptional regulation of
fibronectin production in fetal lung cells
Eric
Mourgeon1,
Jing
Xu2,
A. Keith
Tanswell2,
Mingyao
Liu1, and
Martin
Post2,3
Thoracic Surgery Research Laboratory, Toronto General Hospital
Research Institute, The Medical Research Council Group in Lung
Development, Departments of
1 Surgery,
2 Pediatrics, and
3 Physiology, University of
Toronto, Toronto, Ontario, Canada M5G 2C4
 |
ABSTRACT |
We have shown that intermittent mechanical
strain, simulating fetal breathing movements, stimulated fetal rat lung
cell proliferation. Because normal lung growth requires proper
coordination between cell proliferation and extracellular matrix
remodeling, we investigated the effect of strain on fibronectin
metabolism. Organotypic cultures of fetal rat lung cells, subjected to
intermittent strain, showed increased fibronectin content in the
culture media. Fibronectin-degrading activity in media from strained
cells was similar to that of static cultures. Northern analysis
revealed that strain inhibited fibronectin mRNA accumulation seen
during static culture. Synthesis of fibronectin, determined by
metabolic labeling, was increased by strain despite lower mRNA levels
or presence of actinomycin D. This increase was not mediated via a
rapamycin-sensitive mechanism. Strain stimulated prelabeled fibronectin
secretion even in the presence of cycloheximide. These results suggest
that strain differentially regulates fibronectin production of fetal
lung cells at the transcriptional and posttranscriptional levels.
Mechanical strain increases soluble fibronectin content by stimulating
its synthesis and secretion without increasing fibronectin message levels.
fetal lung development; mechanotransduction; gene expression; protein synthesis
 |
INTRODUCTION |
PHYSICAL FORCES play an important role in regulating
proliferation and differentiation of mammalian cells. In many tissues and cell types, physical forces regulate extracellular matrix molecule
(ECM) gene expression and protein synthesis. Reciprocally, ECM
molecules are essential in mediating mechanical force-initiated intracellular signal transduction. ECM-integrin-cytoskeleton
interactions are one of the most commonly studied pathways for
mechanotransduction (13). Interaction between integrins and specific
matrix proteins is, for example, responsible for sensing mechanical
strain in vascular smooth muscle cells (30). Recently, a mechanical
connection between ECM molecules, integrins, cytoskeletal filaments,
and nucleoplasm has been demonstrated that can transmit physical forces from the cytoplasmic membrane to the nucleus (19).
The lung is continuously subjected to passive and active physical
forces throughout life. Physical forces influence lung function, structure, and metabolism (23). Fetal breathing movements are, for
example, essential for lung growth and development (11, 14). Using a
mechanical strain device for organotypic cell cultures, we have
observed that a mechanical strain regimen, which simulated the reported
frequency, amplitude, and periodicity of normal human fetal breathing
movements (11), enhanced DNA synthesis and cell division of fetal rat
lung cells (16). Strain-induced mitogenic activity is primarily
mediated through phospholipase C-
(PLC-
) and the protein kinase C
(PKC) pathway (17) and requires the three-dimensional structure
provided by the organotypic culture technique (18). Cytoskeletal
deformation-induced protein tyrosine kinase activation is an upstream
event of the PLC-
/PKC pathway and is perhaps involved in the
mechanoreception or initiation of mechanical strain-induced
intracellular signaling (15). Cell-matrix interactions play an
important role in fetal lung development. ECM is thought to transmit
essential information to pulmonary cells, thereby regulating their
proliferation, differentiation, and organization (10). Fibronectin
shows a widespread pattern of expression during development. Transgenic
mice lacking fibronectin die during embryonic development (8),
confirming that its expression is essential for normal development (7).
Although several reports have shown that mechanical forces affect
fibronectin production (2, 3, 9, 33), the mechanisms by which
intermittent physical forces such as fetal breathing movements
influence fibronectin formation are unknown. Herein, we investigated
the effect of intermittent mechanical strain on fibronectin metabolism
in fetal lung cell organotypic culture. Mechanical strain significantly
increased fibronectin synthesis, yet, paradoxically, fibronectin mRNA
content in strained cells was lower than in static cultures. Further
studies revealed that the mechanical strain-induced increase in
fibronectin production in fetal lung cells is primarily a
posttranscriptional event.
 |
MATERIALS AND METHODS |
Materials. Female pregnant Wistar rats
(200-250 g) were purchased from Charles River (St. Constant,
Quebec). Cell culture media and trypsin were obtained from GIBCO BRL
(Burlington, Ontario). FBS was from Flow Laboratories (McLean, VA), and
DNase and collagenase were from Worthington (Freehold, NJ). Gelfoam
sponges were from Upjohn (Toronto, Ontario).
Tran35S-label, which
consists of 70%
[35S]methionine and
30% [35S]cysteine,
was from ICN (Irvine, CA). Rabbit antiserum against rat fibronectin was
from Calbiochem (La Jolla, CA). A 0.5-kb rat fibronectin cDNA fragment
was a gift from Dr. R. O. Hynes (Center for Cancer Research, Cambridge,
MA). Rat
-actin cDNA (0.5 kb) was generated by RT-PCR cloning using
rat
-actin primers (Clontech, Palo Alto, CA). All other chemicals
were from Sigma (St. Louis, MO).
Mechanical strain of fetal lung cells in organotypic
culture. Pregnant rats were killed by an excess of
diethylether on day 19 of gestation
(term = 22 days). Fetal lungs were pooled from at least two litters for
each cell isolation. Organotypic cultures of fetal lung cells were
established as previously described (16, 26). Briefly, fetal rat lungs
were dissected out, minced, and resuspended in Hanks' balanced salt
solution. The minced lung tissue was trypsinized [0.125%
(wt/vol) trypsin and 0.4 mg/ml DNase], filtered, and
centrifuged. The cells were inoculated on 2 × 2 × 0.25-cm
Gelfoam sponges at a density of 1.6 × 106 cells/sponge and incubated
overnight in MEM + 10% (vol/vol) FBS. The sponges were washed three
times with serum-free MEM, and the medium was replaced by MEM.
The mechanical strain device used in these studies has been described
in detail elsewhere (16, 27). It consisted of a programmable burst
timer, a control unit, a direct current power supply, and a set of
solenoids. A culture dish with a Gelfoam sponge was placed in front of
each solenoid. One end of each sponge was glued to the bottom of the
dish, and the other end was attached to a movable metal bar, which was
wrapped and sealed in sterile plastic tubing. A magnetic force,
generated through the solenoids, acted on the metal bar to apply strain
to the organotypic cultures. Sponges were subjected to a 5% elongation
from their original length at 60 cycles/min for 15 min/h over 48 h. The
recoil properties of the sponge allow it to return to its normal length
after each episode of elongation. We have previously shown that this
intermittent strain regimen optimally enhanced DNA synthesis and cell
division without cell injury (16). In subsequent experiments, cells
were exposed to various inhibitors before and/or while being subjected to a similar strain protocol. There was no measurable cytotoxic effect
on fetal rat lung cells with the inhibitor concentrations used in the
present study as assessed by
[14C]adenine release
(16).
Western immunoblotting. To determine
fibronectin released by fetal lung cells, culture media were collected
after 48 h of intermittent strain or static culture and were
concentrated 10-fold with Centriprep-10 concentrators (Amicon, Danvers,
MA). Protein content was determined by a standard protein assay
(Bio-Rad Laboratories, Richmond, CA). Equal amounts of total protein
(50 µg) were boiled with SDS sample buffer [10% (vol/vol)
glycerol, 2% (wt/vol) SDS, 5% (vol/vol)
-mercaptoethanol,
0.0025% (wt/vol) bromphenol blue, and 0.06 M Tris, pH 8.0] and
subjected to SDS-PAGE. Proteins were transferred to nitrocellulose
membranes. Nonspecific binding was blocked by incubation of membranes
with 3% (wt/vol) nonfat milk in PBS for 60 min. Blots were incubated
with a polyclonal anti-fibronectin antiserum (1:1,000 dilution)
overnight at 4°C and then washed with PBS and incubated for 60 min
at 4°C with horseradish peroxidase-conjugated goat anti-rabbit IgG
(1:30,000 dilution). After being washed, blots were developed with an
enhanced chemiluminescence detection kit (Amersham, Oakville, Ontario).
As a negative control, when the antiserum was omitted from the blotting
procedure, no band was detected (data not shown).
Metabolic labeling and fibronectin
synthesis. To determine the synthesis and secretion of
fibronectin, gelatin-Sepharose beads were used to isolate fibronectin
(12). Cells were washed two times with methionine- and cysteine-free
MEM (Select-Amine Kit; GIBCO BRL), labeled with
35S translabel (25 µCi/ml) in
methionine- and cysteine-free MEM. Cells were then subjected to static
culture or intermittent strain. Culture media were collected and stored
at
70°C as aliquots. Sponges were washed two times with
ice-cold washing buffer (0.15 M NaCl and 25 mM Tris, pH 7.4), incubated
in 1 ml of extraction buffer (1 M urea, 1 mM dithiothreitol, 10 mM
Tris, 10 mM disodium EDTA, and 2 mM phenylmethylsulfonyl fluoride, pH
7.4) at 4°C overnight, spun, and then vortexed and sonicated three
times for 5 s at 4°C. Cell lysates were spun at 12,000 g for 2 min to remove debris, and
supernatants were stored at
70°C. The radioactivity
incorporated into total protein in the medium and cell lysates was
measured after TCA precipitation. No significant difference in
TCA-precipitable counts was observed between control and strained
samples. Aliquots of cell lysates and media from static cultured and
strained cells containing equal counts were adjusted to equal volumes
with MEM (for media) or extraction buffer (for cell lysates).
Gelatin-Sepharose 4B beads (50 µl; Pharmacia, Baie d'Urfé,
Quebec) were added to 0.5 ml of cell lysates with an equal volume of
washing buffer containing 1% (vol/vol) Triton X-100 or to 1 ml of
culture medium, which was also adjusted to contain 0.5% (vol/vol)
Triton X-100, followed by incubation at 4°C with constant mixing
overnight (12). The gelatin-Sepharose beads were recovered by
centrifugation and washed three times with washing buffer containing
0.5% (vol/vol) Triton X-100. Fibronectin was eluted by resuspending
beads in electrophoresis sample buffer and boiling for 3 min and then
was subjected to 6% (wt/vol) SDS-PAGE. Gels were fixed in 10%
(vol/vol) acetic acid, prepared for fluorography by soaking in
En3Hance, dried, and exposed to
Kodak XAR-5 film.
Fibronectin degradation assay. To
examine the degradation of fibronectin, mixed fetal lung cells were
cultured in T75 flasks to subconfluency. Cells were washed with
methionine- and cysteine-free MEM and then incubated with methionine-
and cysteine-free MEM containing Tran35S-label
(85 µCi/ml) for 6 h. Conditioned medium was collected and stored at
70°C. Cold methionine and cysteine were added back to the
conditioned medium, which was then applied to organotypic cultures of
fetal lung cells on Gelfoam sponges (3 ml/sponge). Cells were then
subjected to static culture or mechanical strain for various times (3, 6, 9, and 24 h). Prelabeled fibronectin in the culture medium was
collected on gelatin-Sepharose beads and analyzed by SDS-PAGE.
Northern analysis. At different time
intervals (6, 24, or 48 h), strained and unstrained cells were lysed in
4 M guanidinium thiocyanate, and lysates were centrifuged on a 5.7 M
cesium chloride cushion to pellet RNA. Total RNA (15 µg) was size
fractionated on a 1% (wt/vol) agarose gel containing 3% (vol/vol)
formaldehyde, transferred to Hybond N+ nylon membranes, and immobilized
by ultraviolet cross-linking. cDNA probes were labeled with
[
-32P]dCTP using a
random-priming kit (Amersham, Arlington Heights, IL). Prehybridization
and hybridization were performed in 50% (vol/vol) formamide, 5×
saline-sodium phosphate-EDTA, 0.5% (wt/vol) SDS,
5× Denhardt's solution, and 100 µg/ml denatured salmon sperm DNA at 42°C. After hybridization, blots were washed and exposed to
Kodak XAR-5 film with Dupont Cronex intensifying screens. The final
wash for fibronectin and
-actin probes was 0.5× saline-sodium citrate and 0.2% (wt/vol) SDS at 42°C for 10 min.
Statistical analysis. All experiments
were carried out two to four times with materials collected from
separate cell cultures. Autoradiographs were quantified with an Imaging
Densitometer (GS-690; Bio-Rad Laboratories, Hercules, CA). The values
of means ± SE from separate experiments were analyzed by Student's
t-test, or, for comparison of more
than two groups, by one-way ANOVA, followed by the Student-Newman-Keuls
test with significance defined as P < 0.05.
 |
RESULTS |
Mechanical strain increased soluble fibronectin
content. To determine the effect of mechanical strain
on fibronectin formation by fetal lung cells, we first assessed the
soluble fibronectin content in the culture media. Immunoblotting
revealed that a 48-h intermittent strain increased fibronectin content
~2.5-fold in culture media (Fig. 1). To
study the effect of mechanical strain on cell-associated and soluble
fibronectin, fetal lung cells were labeled with
Tran35S-label for 24 h, in the presence or
absence of intermittent strain. The amount of TCA-precipitable counts
in the culture media and cell lysates was not affected by mechanical
strain when compared with that in static cultured cells (data not
shown). Mechanical strain increased the accumulation of
[35S]methionine-labeled
fibronectin in the culture media by 6.5-fold (P < 0.001, Fig.
2, B and
C), consistent with the increased
immunoreactive fibronectin content in the media. The amount of
radioactive fibronectin in the cell lysates, however, slightly
decreased compared with that in static cultured controls (Fig. 2,
A and
C).

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Fig. 1.
Mechanical strain increases fibronectin accumulation in culture media.
Fetal lung cells were subjected to intermittent mechanical strain (60 cycles/min, 15 min/h) or static culture for 48 h. Culture media were
collected, concentrated, and analyzed by Western blotting using an
anti-fibronectin antiserum. A:
representative blot. C, control; S, strained.
B: densitometric analysis from 3 separate experiments. * P < 0.05 vs. control.
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Fig. 2.
Mechanical strain increases soluble fibronectin synthesis and
secretion. Fetal lung cells were washed with methionine- and
cysteine-free MEM and incubated with
Tran35S-label for 24 h
with or without mechanical strain. Newly synthesized fibronectin in
cell lysates and culture media was collected using gelatin-Sepharose
beads, resolved with SDS-PAGE, and visualized by autoradiography.
A: cell lysates;
B: culture media;
C: densitometric analysis from 3 separate experiments. Open bars, control; filled bars, strained.
P < 0.0001 among all groups by
one-way ANOVA followed by Student-Newman-Keuls test.
* P < 0.05 vs. all other
groups.
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Strain-induced increase in fibronectin was not due to
inhibition of fibronectin degradation. Synthesis,
secretion, and degradation determine the accumulation of fibronectin.
To investigate whether the increase in soluble fibronectin was due to
an inhibition of fibronectin degradation, we measured the fate of
prelabeled fibronectin. Conditioned culture medium of fetal lung cells
containing 35S-labeled proteins
was applied to organotypic cultures of fetal lung cells, which were
subjected to mechanical strain. At various durations of strain,
prelabeled fibronectin in the culture media was collected on gelatin
beads and analyzed by SDS-PAGE. There was a rapid degradation of
35S-labeled fibronectin within the
first 3 h of static culture, without major further change afterward
(Fig. 3). A similar degradation pattern was
observed for cells subjected to mechanical strain, although the initial
rate of degradation was somewhat greater when compared with that in
static cultured cells (Fig. 3). Thus the increased soluble fibronectin
found in the culture media is not due to the inhibited fibronectin
degradation but to an increased production.

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Fig. 3.
Mechanical strain does not affect fibronectin degradation. Mixed fetal
lung cells in monolayer culture were prelabeled with
Tran35S-label, and the medium was
collected. Conditioned medium was applied to organotypic cultures of
fetal lung cells, which were then subjected to mechanical strain or
static control for various times (3, 6, 9, and 24 h). Fibronectin in
the medium was collected on gelatin beads, resolved by SDS-PAGE, and
visualized by autoradiography. A:
representative blot. B: densitometric
analysis of 3 blots.
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Mechanical strain inhibited fibronectin mRNA
accumulation. In rat glomerular mesangial cells, a
mechanical force-induced increase in fibronectin synthesis has been
reported to be associated with increased message levels (33).
Organotypic cultures of fetal lung cells were subjected to either
static culture or an intermittent strain regimen. At various time
intervals (6, 24, or 48 h), total RNA was isolated and measured by
spectrophotometry. Total RNA content was not affected by mechanical
strain. The mRNA levels of fibronectin were analyzed by Northern
hybridization. As we have recently reported elsewhere (31),
steady-state levels of
-actin mRNA were not affected by mechanical
strain (Fig.
4A), allowing their use to normalize the relative densitometric intensities of other mRNAs. During static culture, fibronectin mRNA levels increased with time. Compared with the level at 6 h of culture, it
increased 1.3- and 2.0-fold at 24 and 48 h, respectively (Fig. 4B). This increment, however, was
not observed in strained cells, suggesting that mechanical strain
prevented the increase in mRNA levels of fibronectin (Fig. 4,
A and
B).


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Fig. 4.
Mechanical strain inhibits accumulation of fibronectin mRNA. Fetal rat
lung cells were subjected to mechanical strain or static culture for 6, 24, and 48 h. Total RNA (15 µg) isolated from strained or control
cells was subjected to Northern blot analysis with
[32P]cDNA probes
encoding fibronectin and -actin. A:
representative blot. Message RNA for -actin was not influenced by
static culture or mechanical strain and was used as a reference for
equal loading of the same blot. B:
densitometric analysis from 3 separate experiments. Filled bars,
control; open bars, strained. P < 0.001 among all groups by one-way ANOVA followed by
Student-Newman-Keuls test. * P < 0.05 vs. strained groups.
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Mechanical strain increased fibronectin
synthesis. The discrepancy between the increment of
fibronectin protein in the culture media and reduced mRNA level in the
cells suggests that mechanical strain primarily affects the synthesis
and/or secretion of fibronectin. To determine the impact of the
relatively lower fibronectin mRNA levels, cells were strained (15 min/h) for 2, 6, 24, or 48 h. During the last 2 h and 15 min (3 strain
periods of 15 min separated by 2 resting periods of 45 min), cells were
labeled with Tran35S-label.
The
[35S]methionine-labeled
fibronectin released into the culture media was collected using gelatin
beads and analyzed by SDS-PAGE. Mechanical strain increased the
fibronectin synthesis immediately after the first three bursts of
strain (designated as 2 h in Fig.
5A).
Although the mRNA levels of fibronectin were downregulated by
mechanical strain (Fig. 4), the amount of
[35S]methionine-labeled
fibronectin synthesis released into the medium increased consistently
after various periods of mechanical strain (Fig.
5A). To further elucidate the
relationship between fibronectin gene transcription and protein
synthesis, cells were cultured for 2 h with or without an inhibitor of
gene transcription, actinomycin D (8 µM). In our previous study,
using the same model, this concentration of actinomycin D inhibited
strain-induced cell proliferation (16). Cells were washed two times
with methionine- and cysteine-free MEM, incubated with MEM containing
Tran35S-label with or without
actinomycin D, and then subjected to mechanical strain or static
culture for another 2 h and 15 min. A mechanical strain-induced
increase in fibronectin synthesis was still observed in the presence of
actinomycin D (Fig. 5B), albeit
fibronectin synthesis was markedly decreased compared with cells
cultured in the absence of actinomycin D. This suggests that
strain-initiated intracellular signals may directly stimulate
fibronectin protein synthesis and/or secretion.

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Fig. 5.
Mechanical strain-induced fibronectin synthesis and secretion is
independent from its mRNA levels. A:
fetal lung cells were subjected to intermittent strain (15 min/h) or
static cultures for 2, 6, 24, or 48 h. During the last 2 h and 15 min,
cells washed with methionine- and cysteine-free MEM were labeled with
Tran35S-label.
B: fetal lung cells were preincubated
with or without actinomycin D (8 µM) for 2 h, washed with methionine-
and cysteine-free MEM, and then subjected to intermittent mechanical
strain or static culture for an additional 2 h and 15 min in
methionine- and cysteine-free MEM containing
Tran35S-label in the presence or
absence of actinomycin D. Newly synthesized fibronectin in culture
media was collected using gelatin-Sepharose beads, resolved with
SDS-PAGE, and visualized by autoradiography.
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Mechanical strain stimulated fibronectin
secretion. To determine whether strain affected
fibronectin secretion, cells were incubated with
Tran35S-label for 4 h to label newly
synthesized proteins, washed, and incubated for 30 min in MEM with or
without cycloheximide [10 µg/ml, a concentration shown to
inhibit strain-induced cell proliferation (16)]. Cells were then
subjected to mechanical strain or static culture for 2 h and 15 min.
Because cycloheximide blocks new protein synthesis by inhibiting
translation, any change in
[35S]methionine-labeled
fibronectin in the medium would mainly be due to an altered release of
prelabeled intracellular fibronectin. As can be seen in Fig.
6, mechanical strain-induced increase in [35S]methionine-labeled
fibronectin in the medium was not inhibited by cycloheximide,
suggesting that, in addition to an increase in fibronectin synthesis,
strain also increased the release of fibronectin.

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Fig. 6.
Mechanical strain increases fibronectin release. Fetal lung cells were
washed with methionine- and cysteine-free MEM and labeled with
Tran35S-label for 4 h. They were then
thoroughly washed, preincubated in regular MEM with or without
cycloheximide (10 µg/ml) for 30 min, and then subjected to
intermittent mechanical strain or static culture for an additional 2 h
and 15 min. Radiolabeled fibronectin in culture media was collected
using gelatin-Sepharose beads, resolved with SDS-PAGE, and visualized
by autoradiography. Cycloheximide did not block strain-induced release
of fibronectin.
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Mechanical strain-induced fibronectin synthesis was
not mediated through a rapamycin-sensitive mechanism.
Translation from mRNA to protein is a complex and highly regulated
process. It has been reported (25, 32) that mechanical loading can
activate protein p70 S6 kinase. Activation of p70 S6 kinase may result in an increased translation of a family of mRNAs essential to the
protein synthetic apparatus (21) and increased protein synthesis. Rapamycin has been demonstrated to specifically inhibit the activity of
p70 S6 kinase, subsequent phosphorylation of ribosomal S6 protein, and
protein synthesis (28). Therefore, we tested whether this p70 S6
kinase-related pathway was responsible for mechanical strain-induced fibronectin synthesis. Fetal lung cells were preincubated with or
without rapamycin (20 ng/ml) for 30 min, washed two times, and then
subjected to mechanical strain or static culture for 2 h and 15 min in
methionine- and cysteine-free MEM containing Tran35S-label with or without rapamycin
(20 ng/ml). Rapamycin did not affect the strain-induced increase of
labeled fibronectin in the medium (Fig. 7).

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Fig. 7.
Mechanical strain-induced fibronectin synthesis is not mediated through
a rapamycin-sensitive pathway. Fetal lung cells were preincubated with
or without rapamycin (20 ng/ml) for 30 min, washed with methionine- and
cysteine-free MEM, and then subjected to intermittent mechanical strain
or static culture for an additional 2 h and 15 min in methionine- and
cysteine-free MEM containing
Tran35S-label with or without
rapamycin. Newly synthesized fibronectin in culture media was collected
using gelatin-Sepharose beads, resolved with SDS-PAGE, and visualized
by autoradiography.
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 |
DISCUSSION |
Regulation of ECM dynamics is a complex process involving a balance
between synthesis, deposition, and degradation of ECM molecules. In the
present study, we observed that mechanical strain increased soluble
fibronectin in the culture media. This was not due to decreased
fibronectin degradation but to an increase in fibronectin synthesis and
secretion by fetal lung cells. In this organotypic model, epithelial
cells and fibroblasts represent the main cell populations; both cell
types have been shown to express fibronectin (24). Which cell type
responded to mechanical strain with increased fibronectin production is
not studied. It is possible that different subpopulations have
different responses. However, the interaction between different cell
types, which is maintained in organotypic cultures, is a prerequisite
to extrapolate the responses of lung cells in vitro to those in vivo.
Physical force-induced fibronectin production has been reported for
several other cell types normally subject to physical forces in vivo
(2, 3, 9, 33). However, the mechanisms by which physical forces result
in increased fibronectin production have not been well defined. In
contrast to other observations, which showed coordinated increases in
both fibronectin mRNA and protein, we found that mechanical strain
inhibited the fibronectin mRNA accumulation seen in static culture but
enhanced fibronectin synthesis and release.
Inhibition of fibronectin mRNA accumulation by
mechanical strain. Steady-state levels of fibronectin
mRNA increased during organotypic culture under static conditions. This
may reflect the dynamic reorganization of fetal lung cells into
"alveolar-like structures" in the sponge (16, 18, 26). Gelfoam
sponges are made with denatured collagen, which may be better suited
for cell attachment than plastic. The architecture of sponges provides a three-dimensional space for cells to migrate, to recognize each other, and to reaggregate themselves. The accumulation of fibronectin mRNA during static culture is consistent with a rapid turnover of ECM
molecules during these dynamic changes of cell rearrangement. Mechanical strain increases fetal lung cell proliferation (16), which
may require an even greater turnover of ECM molecules to match
increased cell number and surface area. Although the increase in
fibronectin mRNA is inhibited by mechanical strain, the increased accumulation of soluble fibronectin suggests an enhanced fibronectin synthesis due to a more efficient translation and/or an increase in
fibronectin release into the medium. This may be a compensatory mechanism, allowing cells to economically utilize both transcriptional and translational machinery to best meet the increased demands for both
cell proliferation and ECM remodeling. Whether the mechanical strain-induced decrease in fibronectin mRNA accumulation is due to a
decreased rate of transcription and/or decreased RNA stability needs to
be elucidated.
Posttranscriptional regulation of mechanical
strain-induced fibronectin production. Given the many
roles of fibronectin in cell adhesion, migration, proliferation, and
differentiation, its production needs to be tightly regulated. In
response to many stimuli, fibronectin mRNA and protein increase in
parallel. For example, transforming growth factor-
1-increased
fibronectin production by adult type II alveolar epithelial cells was
accompanied by an increase in fibronectin mRNA (20). Serum can
stimulate both mRNA and protein expression of fibronectin in human
fibroblasts isolated from individuals affected by Werner syndrome (22). Mechanical strain increased both fibronectin mRNA and protein in
glomerular mesangial cells (33). However, there are a few reports
suggesting that fibronectin mRNA and protein synthesis could be
differentially regulated. Cultured smooth muscle cells (SMC) of ductus
arteriosus produced up to threefold more fibronectin compared with SMC
of aorta after serum stimulation (4). However, the steady-state mRNA
levels for fibronectin were 50% less in SMC from ductus arteriosus
relative to SMC from aorta (4). Recently, we reported that mechanical
strain-induced proteoglycan production was not associated with an
increase in mRNAs for core proteins of proteoglycans (31). In the
present study, we found that mechanical strain-induced fibronectin
synthesis took place in the presence of the transcriptional inhibitor
actinomycin D. These latter observations are compatible with mechanical
strain-initiated signals bypassing gene transcription and enhancing
translational activity of mRNAs encoding for fibronectin and other ECM
molecules. We found that mechanical strain-induced fibronectin
synthesis was not mediated via a rapamycin-sensitive translational
mechanism. Recently, an AU-rich element (ARE) in the
3'-untranslated region of fibronectin mRNA has been identified
that is responsible for its translational control (34). In addition,
Zhou et al. (34) identified an ARE-binding protein that regulates the
translation of fibronectin of SMC from ductus arteriosus and aorta.
Similar mechanisms may also be involved in mechanical strain-induced
fibronectin synthesis of fetal lung cells. The increased translation of
fibronectin mRNA may enhance mRNA turnover. Thus the strain-induced
increase in fibronectin mRNA degradation may offset the increase in
fibronectin mRNA transcription, allowing the steady-state level of
fibronectin mRNA to remain constant.
Mechanical strain-induced fibronectin
release. Our results suggest that the increased release
of fibronectin in the medium by mechanically strained cells is likely
due to an enhanced translation of fibronectin mRNA, as well as an
increased secretion of newly synthesized fibronectin. We found that
mechanical strain increased the release of prelabeled fibronectin by
fetal lung cells even in the presence of cycloheximide. An increased
release of fibronectin in the presence of cycloheximide has also been
observed after H2O2
challenge in a rabbit model of vascular lung injury (29). Similarly,
lipopolysaccharide-stimulated fibronectin release in the culture medium
of human fibroblasts was not attenuated by cycloheximide (1).
Fibronectin is present in three compartments of the culture system:
within the cell, associated with cell layer or matrix, and in the
medium. Intracellular storage of fibronectin has been reported to be
limited (5). Therefore, a plausible explanation of strain-induced
fibronectin release is redistribution of fibronectin from the cell
layer to the medium. Such a phenomenon has been described for primary
cultures of adult rat type II pneumocytes (6).
As mentioned earlier, most reports showed a coordinated alteration in
both fibronectin mRNA and protein (2, 3, 9, 33). It is possible that
proliferating and partially differentiated fetal lung cells differ from
the fully differentiated and growth-arrested adult cells used in most
studies in their response to mechanical strain. The discrepancy may
also be due to differences in spatial culture environment. In the
present study, we strained three-dimensional organotypic cultures,
whereas in previously reported studies, cells were strained in
two-dimensional culture systems. Previously, we have shown that
mechanical strain-induced fetal lung cell proliferation and
intracellular signal transduction are highly dependent on the
architecture of culture environment (18). Another striking difference
is that we employed an intermittent strain regimen with a relatively
small amplitude (5% elongation) to mimic fetal breathing movements; in
previous studies, cells were subjected to a continuous strain using
greater amplitudes. We have previously shown that such periodic
mechanical straining of fetal lung cells stimulated cell proliferation,
whereas mechanical straining with a larger amplitude (10% elongation)
as well as a continuous strain induced cell injury and inhibited cell
growth (16). The pattern of fetal breathing movements is intermittent
during late fetal gestation. It is possible that the responsiveness of
cells to stimuli, the intracellular signal transduction mechanisms, and subsequent cellular functioning are different between the breathing and
relaxation cycles. This may be required to coordinate cell proliferation and remodeling of ECM during late fetal lung development.
 |
ACKNOWLEDGEMENTS |
We are grateful to Xiaoming Zhang and Jason Liu for technical assistance.
 |
FOOTNOTES |
This work was supported by an operating grant (MT-13270, M. Liu) and a
group grant (M. Post and A. K. Tanswell) from the Medical Research
Council of Canada, grants from James H. Cumming's Foundation of USA
(M. Liu); National Heart, Lung, and Blood Institute Grant R01HL-43416
(M. Post); and equipment grants from the Ontario Thoracic Society. E. Mourgeon is a recipient of a Fellowship from Société Française d'Anesthésie et de Réanimation (French
Anesthesiology and Critical Care Society) and from the Dean's Office,
Faculty of Medicine, University of Toronto. M. Liu is a Scholar of the Medical Research Council of Canada.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Liu, Toronto
General Hospital, Room: CCRW 1-821, 200 Elizabeth St., Toronto,
Ontario, Canada M5G 2C4 (E-mail: mingyao.liu{at}utoronto.ca).
Received 27 August 1998; accepted in final form 16 March 1999.
 |
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