Medical Research Council Group in Lung Development and Lung Biology Research Program, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario, Canada M5G 1X8
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ABSTRACT |
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We have previously
shown that an intermittent mechanical strain regimen (5% elongation,
60 cycles/min, 15 min/h) that simulates fetal breathing movements
stimulated fetal rat lung cell proliferation. Because normal lung
growth requires proper coordination between cell proliferation and
extracellular matrix (ECM) remodeling, we subjected organotypic
cultures of fetal rat lung cells (day 19 of gestation, term = 22 days) to
this strain regimen and examined alterations in ECM gene and protein
expression. Northern analysis revealed that mechanical strain reduced
messages for procollagen-1(I) and biglycan and increased the levels of mRNA for
collagen-
1(IV) and
-
2(IV), whereas laminin
-chain mRNA levels remained constant. Regardless of mRNA changes,
mechanical strain increased the protein content of type I and type IV
collagen as well as of biglycan in the medium. Mechanical strain did
not affect gene expression of several matrix metalloproteinases (MMPs),
such as MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and
MMP-3 (stromelysin-1). Neither collagenase nor gelatinase (A and B)
activities in conditioned medium were affected by mechanical strain.
Tissue inhibitor of metalloproteinase activities in conditioned medium
remained unchanged during the 48-h intermittent mechanical stretching.
These data suggest that an intermittent mechanical strain
differentially regulates gene and protein expression of ECM molecules
in fetal lung cells. The observed increase in matrix accumulation
appears to be mainly a result of an increased synthesis of ECM
molecules and not of decreasing activity of degradative enzymes.
fetal lung development; matrix metalloproteinases; gene expression; protein synthesis
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INTRODUCTION |
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THE LUNG IS CONTINUOUSLY subjected to passive and
active physical forces throughout life. Physical forces have been shown to influence lung function, structure, and metabolism (19). Whole
animal studies have shown that normal fetal breathing movements are
essential for lung development (7, 10). However, the cellular and
spatial complexity of lung tissue makes it difficult to attribute the
effects of mechanical perturbation to individual cells, matrix, or
other structural components (19). Recently, we investigated whether
stretch or distortion directly stimulates fetal lung cell proliferation
using a mechanical strain device for organotypic cell cultures (13). We
observed that a mechanical strain regimen that simulated the reported
frequency, amplitude, and periodicity of normal human fetal breathing
movements in vivo (7) enhanced DNA synthesis and cell division of fetal
rat lung cells (13). Strain-induced fetal lung cell proliferation was mediated through growth factors (15). Both gene expression and protein
synthesis of platelet-derived growth factor (PDGF)-B and -receptor
were stimulated by mechanical stimulation of fetal lung cells (12).
Increasing evidence suggests that cell-matrix interactions play an important role in fetal lung development. Extracellular matrix (ECM) is thought to transmit essential information to pulmonary cells, thereby regulating their proliferation, differentiation, and organization (6). Regulation of ECM dynamics is complicated, involving a balance between synthesis and deposition of ECM molecules as well as their degradation. A family of secreted proteases, matrix metalloproteinases (MMPs), has been implicated in ECM turnover (1, 16). MMP activity is regulated by a variety of mechanisms, including synthesis, secretion, activation, and inhibition by stoichiometric complexing of tissue inhibitors of metalloproteinases (TIMPs) to the activated enzymes (16, 18).
Physical forces have been shown to influence gene expression and protein synthesis of ECM molecules and MMPs in several tissues and cell types (2, 5, 11, 29, 30, 32, 33). In most of these studies, however, single cell types in monolayer (2-dimensional) cultures were subjected to a continuous strain. However, we have recently shown that the proliferative response of mixed fetal lung cells to intermittent mechanical strain requires the presence of a three-dimensional culture environment (14). Also, fetal lung cells are subjected to an intermittent strain in utero. Therefore, we cannot extrapolate the findings obtained with aortic endothelial cells (29) and aortic (30) and bladder (2) smooth muscle cells directly to fetal lung cells. Herein, organotypic cultures of fetal lung cells were subjected to intermittent strain, and alterations in matrix molecule expression were investigated.
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METHODS |
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Materials. Female (200-250 g) and
male (250-300 g) Wistar rats were purchased from Charles River
(St. Constant, PQ) and bred in our animal facilities. Cell culture
media and trypsin were obtained from GIBCO BRL (Burlington, ON). Fetal
bovine serum (FBS) was from Flow Laboratories (McLean, VA), and DNase
and collagenase were from Worthington (Freehold, NJ). Gelfoam sponges
were from Upjohn (Toronto, ON).
Na235SO4
was from ICN (Irvine, CA), and
[methyl-3H]thymidine
was from Amersham (Oakville, ON).
N-[propionate-2,3-3H]collagen
(rat, type I) was from DuPont (Boston, MA). Rat tail type I collagen
was from Collaborative Biomedical Products (Bedford, MA).
Chondroitinase ABC was from Seikagaku (Rockville, MD). All other
chemicals were from Sigma (St. Louis, MO). The human biglycan cDNA
(insert size 1.69 kb) was from Dr. L. W. Fisher [National Institutes of Health (NIH), Bethesda, MD]. The 0.6-kb murine
TIMP-1 cDNA fragment was provided by Dr. D. T. Denhardt (University of Western Ontario, London, ON), the 1.2-kb rabbit stromelysin-1 (MMP-3)
cDNA was provided by Dr. Z. Werb (University of California, San
Francisco, CA), and the 1.8-kb human
procollagen-1(I) cDNA was
provided by Dr. F. Ramirez (University of New Jersey, Piscataway, NJ).
Interstitial collagenase (MMP-1; insert size 2.05 kb) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; insert size 1.6 kb)
cDNAs were purchased from the American Type Culture Collection (Manassas, VA). Rat
-actin cDNA (0.7 kb) was generated by RT-PCR cloning with rat
-actin primers (Clontech, Palo Alto, CA). The human
72-kDa type IV collagenase (MMP-2; gelatinase A) cDNA (insert size 2.0 kb) was from Dr. P. Huhtala (University of Oulu, Oulu, Finland). Murine
(
1) and
(
2) type IV collagen cDNAs
(insert size 1.8 and 1.1 kb, respectively) and laminin
-chain
(insert 1.1 kb) were supplied by Dr. M. Kurkinen (University of New
Jersey, Piscataway, NJ). Antiserum against human biglycan (LF-15) was a
generous gift from Dr. L. W. Fisher. (NIH). Bovine TIMP-1 antiserum was
from Dr. Y. Declerck (Children's Hospital, Los Angeles, CA). Rabbit
type I and type IV collagen antisera were from Chemicon (El Segundo, CA).
Cell culture. Pregnant rats were
killed by an excess of diethylether on
day
19 of gestation (term = 22 days).
Fetal lungs were pooled (20 lungs) from at least two litters for
each cell isolation. Organotypic and primary cell cultures were
established as previously described (13, 14, 25). Fibroblasts and
epithelial cells seeded on 75-cm2
tissue culture flasks were grown to confluence, washed, and collected with 0.25% (vol/vol) trypsin plus 1 mM EDTA. Trypsin activity was
neutralized with MEM plus 10% (vol/vol) FBS. After centrifugation, cells were washed with MEM. Mixed fetal lung cells and fibroblasts were
resuspended in MEM plus 10% (vol/vol) FBS, whereas epithelial cells
were resuspended in MEM plus 2% (vol/vol) FBS, before being seeded
into organotypic cultures. 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 plus 2-10% (vol/vol) FBS. The sponges were
washed three times with serum-free MEM, and the medium was replaced by
MEM plus 1% (vol/vol) FBS.
Strain of fetal lung cells in organotypic culture. The mechanical strain device used in these studies has been described in detail elsewhere (13, 26). It consisted of a programmable burst timer, a control unit, a DC 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 plastic tubing. The movement of the metal bar and sponges was driven by the magnetic force and recoil property of the sponge. A magnetic force, generated through the solenoid, 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. We have previously shown that such a 48-h intermittent strain regimen optimally enhanced DNA synthesis and cell division without cell injury (13).
Cell proliferation was assessed for cells cultured for 48 h in either MEM plus 1% (vol/vol) FBS with and without intermittent strain or in MEM plus 10% (vol/vol) FBS without strain. [3H]thymidine incorporation into DNA was determined as described previously (13).
Northern analysis. At different time
intervals (15 min or 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% agarose gel containing 3% (vol/vol) formaldehyde, transferred to Hybond N+ nylon membranes, and immobilized by ultraviolet cross-linking. A random priming kit (Amersham, Arlington
Heights, IL) was used to label the cDNA probes with deoxycytidine
5'-[-32P]triphosphate.
Prehybridization and hybridization were performed in 50% (vol/vol)
formamide, 5× sodium chloride-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 Dupont Cronex intensifying screens were used to expose them to Kodak XAR-5 film. The final wash for
procollagen-
1(I), laminin
-chain, biglycan, GAPDH, and
-actin probes was 0.5×
saline-sodium citrate (SSC) and 0.2% (wt/vol) SDS at 42°C for 10 min. The final wash for type IV collagen
(
1 and
2), MMP-1, MMP-2, MMP-3, and TIMP-1 was 2× SSC and 0.2% (wt/vol) SDS at 42°C for 10 min. To check the evenness of RNA transfer, each membrane was reprobed with either rat
-actin or GAPDH cDNA.
Western immunoblotting. To determine
the effect of strain on the elaboration of ECM proteins, fetal lung
cells were cultured in MEM plus 1% (vol/vol) FBS with or without
intermittent strain. Culture media were collected after 48 h and
concentrated 10 times with Centriprep-10 filters (Amicon, Danvers, MA).
Protein content was determined by a standard protein assay (Bio-Rad
Laboratories, Richmond, CA). Equal amounts of concentrated total
protein (50 µg) from conditioned media were boiled in 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 5% (wt/vol) SDS-PAGE.
Proteins were transferred to nitrocellulose membranes. Nonspecific
binding was blocked by incubation of membranes with 3% (wt/vol)
nonfat milk powder in PBS for 60 min. Blots were then incubated with
the designated polyclonal antibody or antiserum overnight at 4°C,
washed with PBS, and incubated for 60 min at 4°C with horseradish
peroxidase-conjugated goat anti-rabbit IgG. After being washed, blots
were developed with an enhanced chemiluminescence detection kit (Amersham).
Metabolic labeling and biglycan synthesis. To examine the effect of mechanical strain on biglycan synthesis, fetal lung cells were cultured in MEM plus 1% (vol/vol) FBS with or without strain for 48 h. 35SO4 (100 µCi/ml) was added to the medium during the last 24 h of straining. Aliquots of medium containing equal amounts of protein were incubated with or without chondroitinase ABC (2 mU/ml) for 2 h at 37°C and then incubated with rabbit anti-biglycan antiserum (1:100) overnight on an end-over-end rotator at 4°C. Zysorbin was used to collect the immune complex. The immune complex was washed three times with ice-cold PBS containing 1% (vol/vol) Triton X-100, dissociated by boiling for 3 min in SDS sample buffer, and subjected to 5% (wt/vol) SDS-PAGE. Gels were fixed in 10% (vol/vol) acetic acid, prepared for fluorography by soaking in En3Hance (DuPont, Missisauga, ON), dried, and exposed to Kodak XAR-5 film.
Collagenase assay. We measured collagenase activity using soluble [3H]collagen as substrate (8). Aliquots of concentrated conditioned medium from strained or static cell cultures containing 60 µg of total protein were incubated for 1 h in Eppendorf tubes at 35°C with rat tail collagen type I supplemented with a trace amount of type I [3H]collagen. The reaction solution contained 1 mg/ml type I collagen, 25 mM Tris · HCl, pH 7.8, 2.5 mM CaCl2, 0.1 M NaCl, 0.25 M glucose, and 1 mM aminophenylmercuric acetate to maximize the collagenase activity. The reaction was terminated by addition of ice-cold trichloroacetic acid to a final concentration of 10% (wt/vol). After a 30-min incubation on ice, samples were centrifuged at 10,000 g for 10 min to remove precipitated nondegraded collagen, and supernatants were collected and counted in a liquid scintillation spectrometer. Results are expressed as a percentage of total radioactivity released by milligrams per milliliter of bacterial collagenase (8).
Zymography assay for gelatinase activity. To examine gelatinase activity released by fetal lung cells, conditioned media were concentrated and subjected to substrate-gel electrophoresis on a 10% (wt/vol) polyacrylamide gel impregnated with 100 µg/ml gelatin as described previously (20). Equal amounts of protein were mixed with sample buffer [10% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.0025% (wt/vol) bromphenol blue, and 0.06 M Tris, pH 8.0] and loaded into wells of a 4% (wt/vol) gel without boiling. After electrophoresis, SDS was eluted from the gel with 2.5% (vol/vol) Triton X-100 washes (2 × 15 min at room temperature), and the gel was incubated overnight at 37°C in 50 mM Tris · HCl, 0.2 M NaCl, 5 mM CaCl2, and 0.5 µg/ml NaN3, pH 7.2, with and without a gelatinase inhibitor (10 mM EDTA). The reaction was stopped by a 3-min immersion in 15% (vol/vol) acetic acid. The gel was stained overnight with 0.1% (wt/vol) Coomassie brilliant blue G-250, 0.2 M H3PO4, and 50 mg/ml ammonium sulfate, pH 2.5, then equilibrated in 25 mg/100 ml ammonium sulfate in 5% (vol/vol) acetic acid to fix the dye and intensify the staining. Gelatinolytic activity from both proenzyme and activated enzyme of the samples was detected as cleared bands against the aqua blue-stained gelatin background.
Reverse zymography for TIMP activity. Reverse zymography was conducted with a 15% (wt/vol) polyacrylamide gel containing 2 mg/ml gelatin and 10% (vol/vol) day 19 rat fetal lung fibroblast conditioned medium. Equal amounts of protein were mixed with sample buffer [4% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.1% (wt/vol) bromphenol blue, and 0.05 M Tris · HCl, pH 6.8], loaded on the gel, and electrophoresed at 100 V for 1.5 h. SDS was eluted from the gel with 2.5% (vol/vol) Triton X-100 in 50 mM Tris · HCl, pH 7.6, for 60 min followed by overnight incubation in developing buffer containing 50 mM Tris · HCl, pH 7.6, 0.2 M NaCl, 5 mM CaCl2, and 0.02% (wt/vol) Brij-35 at 37°C. The gel was stained for 3 h in 30% (vol/vol) methanol containing 10% (vol/vol) glacial acetic acid and 0.5% (wt/vol) Coomassie blue and was destained in 30% (vol/vol) methanol containing 10% (vol/vol) glacial acetic acid. The activity of TIMPs was visualized as an inhibition of gelatinase activity in the gel, and thus positive staining with Coomassie blue could be observed.
Statistical analysis. All experiments were carried out a minimum of three times with materials collected from separate cell cultures. When required, the 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 Duncan's multiple range comparison test, with significance defined as P < 0.05.
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RESULTS |
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Intermittent mechanical strain differentially
regulates gene expression of collagens and biglycan.
Collagenous proteins, laminins, and proteoglycans are major
constituents of ECMs. In addition to their structural roles, collagens,
laminins, and proteoglycans may have numerous developmental and
physiological functions (6). To investigate whether mechanical strain
affects gene expression of these ECM components, the steady-state mRNA
levels of procollagen-1(I), collagen-
1(IV) and
-
2(IV), laminin
-chain, and
biglycan were measured. We found no difference in mRNA levels of these
ECM molecules between static cultured mixed cells and mixed cells
subjected to a 15-min strain (Fig. 1),
suggesting that mechanical strain does not rapidly induce mRNA
expression of these ECM components in mixed fetal lung cells. We then
examined whether a longer duration of mechanical strain influenced the
gene expression of these ECM molecules. Steady-state levels of mRNA for
procollagen-
1(I) and biglycan
increased throughout the 48-h period of static culture. Although
procollagen-
1(I) and biglycan
mRNA also increased in mixed cells subjected to the 48-h period of
intermittent strain, message levels of strained mixed cells were
visibly lower than those of static control cells (Fig.
1A). Both ECM molecules were predominantly expressed in fetal fibroblasts, and a 48-h strain of
fibroblasts alone decreased
procollagen-
1(I) and biglycan mRNA levels compared with that in static control cells (Fig.
1C). GAPDH and
-actin message
levels were unaffected by mechanical strain (Fig.
1C). The steady-state mRNA levels of
both collagen chains
[
1(IV) and
2(IV)] also increased
during the 48-h static culture period of mixed fetal lung cells. In
contrast to procollagen-
1(I) and biglycan, a 48-h intermittent straining increased
collagen-
1(IV) and
-
2(IV) mRNA expression of mixed
(Fig. 1B) and individual (Fig.
1C) fetal lung cells. Laminin
-chain expression was not altered by straining (Fig. 1,
B and
C). These data suggest that fetal
lung cells exposed to a relatively long period of intermittent strain
differentially regulate mRNA expression of various ECM molecules.
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It has been reported that mechanical strain had opposite effects on
cell proliferation and collagen synthesis in endothelial cells and
smooth muscle cells (28, 29, 30). To determine whether the
strain-induced inhibition of mRNA accumulation for procollagen-1(I) is related to
cell proliferation, we examined mixed fetal lung cell proliferation and
gene expression of
procollagen-
1(I) under three
different conditions. Cells were incubated in MEM plus 1% (vol/vol)
FBS in the presence and absence of mechanical strain or in MEM plus
10% (vol/vol) FBS without strain for 48 h. Compared with cells
cultured in MEM plus 1% FBS, both mechanical strain and 10% FBS
stimulated cell proliferation, increasing DNA synthesis by 64 and
132%, respectively (P < 0.05; Fig.
2A). In contrast, mRNA levels of
procollagen-
1(I) of cells
cultured in MEM plus 10% FBS for 48 h were greater than those of cells
cultured with and without strain in MEM plus 1% FBS (Fig.
2B).
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Intermittent mechanical strain increases soluble
collagen and biglycan content. Regardless of a decrease
in procollagen-1(I) mRNA,
immunoblot analysis revealed that cells subjected to intermittent strain had more type I collagen in their medium (Fig.
3A).
Mechanical strain also increased the content of type IV collagen in the
medium, which is in agreement with its increase in mRNA (Fig.
3A). To measure biglycan, fetal lung
cells were metabolically labeled with
35SO4,
and conditioned medium was analyzed by immunoprecipitation with
biglycan antiserum. A radioactive band corresponding to biglycan (275 kDa) was revealed by SDS-PAGE (Fig.
3B). In accord with previous findings (33), mechanical strain significantly enhanced the intensity
of the band representing biglycan. Chondroitinase ABC treatment almost
completely degraded 35S-labeled
biglycan in conditioned medium of static control cells but only
partially digested the GAG side chains of
35S-labeled biglycan in
conditioned medium of strained cells, further suggesting an increased
accumulation of biglycan in the culture medium. Taken together, these
data indicate that intermittent straining of fetal lung cells increases
soluble ECM protein content.
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Intermittent mechanical strain does not influence
expression and activities of MMP-1, -2, -3, and -9 and TIMP-1 and
-2. ECM remodeling is a dynamic process involving
synthesis and deposition of ECM components as well as their
degradation. The degradation of ECM molecules occurs mainly
extracellularly, involving secreted MMPs and their inhibitors, TIMPs.
Because type I collagen and biglycan increased in the culture medium,
whereas their mRNAs declined relative to that in static control cells,
we speculated that the strain-induced increase in soluble ECM protein
is the result of a change in MMP and/or TIMP activities in the medium. The steady-state mRNA levels of interstitial collagenase (MMP-1), type
IV collagenase/gelatinase A (MMP-2), and stromelysin-1 (MMP-3) were
measured (Fig. 4). MMP-1 and MMP-3 mRNA
levels remained constant, whereas message of MMP-2 increased within the
48-h period of static culture. Neither a short period (15 min) nor a
relatively longer period (48 h) of straining altered the mRNA
expression of these MMPs (Fig. 4). We then measured the
activities of MMPs. Rat tail type I collagen was used as a substrate to
analyze the (interstitial) collagenase activity in the conditioned
medium. The total activity of collagenase was not affected by a 48-h
intermittent strain (24.3 ± 0.3 vs. 27.3 ± 0.8 U/mg protein,
strain vs. static control; n = 3 experiments carried out in triplicate). Gelatinolytic activities in
conditioned medium were analyzed by zymography (Fig.
5). Gelatinolytic activities appeared as
zones of substrate clearing. The cleared zones in the substrate gel
were not observed in the presence of EDTA, a chelating agent that
inhibits metalloproteinases. Two major areas of lysis were observed at
72 and 92 kDa, representing type IV collagenase/gelatinase A (MMP-2)
and type IV collagenase/gelatinase B (MMP-9), respectively.
The MMP-9 zone of lysis was significantly more pronounced than that of
MMP-2. In addition to the major 72- and 92-kDa gelatinolytic bands,
minor bands were observed at 64 and 84 kDa, representing the activated
forms of MMP-2 and MMP-9, respectively. Gelatinolytic activities in
conditioned medium were not affected by mechanical strain (Fig. 5). We
also measured the effect of mechanical strain on TIMP-1 gene and
protein expression. The mRNA levels of TIMP-1 increased during the 48-h
static culture period, but TIMP-1 mRNA expression was not influenced by
either short (15-min) or long (48-h) periods of mechanical straining (Fig.
6A). In
agreement with this observation, a 48-h intermittent strain did not
change the TIMP-1 protein content of conditioned medium compared with
that of static control cells (Fig.
6B). To determine whether other
TIMPs were affected by mechanical strain, we conducted reverse
zymography (Fig. 7). Two major
gelatinolytic inhibitory areas were visualized at 28 and 21 kDa, most
likely representing TIMP-1 and TIMP-2, respectively. Both TIMP
activities in conditioned medium were not affected by mechanical
strain. Taken together, these results suggest that the mechanical
strain-induced increase of ECM protein in the culture medium is likely
not a result of alterations in MMP and/or TIMP activities.
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DISCUSSION |
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The present study demonstrates that an intermittent mechanical strain selectively regulates mRNA expression of various ECM molecules in fetal lung cells. Differential regulation of ECM molecules has been reported for a variety of cell types subjected to a continuous mechanical strain. A continuous cyclic uniaxial compression stimulated synthesis of large proteoglycan and biglycan but had no effect on decorin in fetal bovine deep flexor tendon explants (5). Mechanical strain increased type III collagen and fibronectin synthesis but decreased type I collagen synthesis of bladder smooth muscle cells (2). In contrast, bladder urothelial cells responded to strain with an increase in type I collagen and fibronectin synthesis, whereas type III collagen remained unchanged (2). Thus it appears that mechanical force-induced ECM remodeling is cell-type and tissue specific.
Sadoshima and Izumo (23) demonstrated that stretching cardiac muscle
cells induced the expression of immediate-early genes such as
c-fos,
c-jun,
c-myc,
JE, and
Egr-1 within 30 min. We have recently
demonstrated that mechanical strain induced mRNA expression of PDGF-B
and PDGF -receptor in fetal lung cells within 5-15 min (12).
The genes encoding for ECM and cytoskeletal proteins such as
fibronectin and actin have also been suggested to belong to the
immediate-early genes because of the rapid response to serum
stimulation in NIH/3T3 cells (22). In the present study, no rapid
changes of mRNA expression of ECM molecules were observed. In contrast,
we found a gradual accumulation of mRNAs of
procollagen-
1(I) and biglycan
with duration of culture, which was dampened by mechanical strain.
However, mechanical strain increased the accumulation of mRNA for
collagen-
1(IV) and
-
2(IV) in fetal lung cells,
whereas laminin
-chain mRNA expression was unaffected. The effect of strain on collagen production by vascular smooth muscle and endothelial cells has also been shown to be time dependent. The effect was greater
at day
5 than at
day
3, whereas 1-day strain showed the least effect (29, 30). Taken together, these observations indicate that
mechanical strain may affect the expression of a variety of genes in a
time-dependent manner.
Growth factors have been widely implicated in the regulation of gene expression of ECM components and related enzymes (4, 9, 21, 31). Because intermittent straining of fetal lung cells resulted in an increased elaboration of growth factors (12, 15), it is plausible that the observed changes in steady-state mRNA levels of ECM molecules are partly a result of changes in growth factors.
Sumpio and co-workers (29) have reported that a cyclic strain regimen
stimulated aortic endothelial cell proliferation (28) and inhibited
collagen production. However, the proliferation of smooth cells
isolated from the same tissue was inhibited by cyclic strain (27),
whereas collagen production was increased (30). In the present study,
we found that intermittent mechanical strain stimulated fetal lung cell
proliferation, decreased the levels of mRNA for
procollagen-1(I) but increased
that of collagen-
1(IV) and-
2(IV) and increased both
collagen I and IV protein production. In contrast, skin fibroblasts
maintained under tension in a collagen gel (bound lattices) increased
their expression of ECM components compared with fibroblasts with free
retracting lattices, and this increase coincided with a higher cell
proliferation (11). To determine the relationship between cell
proliferation and collagen I formation, we compared the effects of
mechanical strain with those triggered by a mitogenic stimulus, FBS, in
static cultures. We found that both mechanical strain and FBS
stimulated fetal lung cell proliferation, but they had opposite effects
on procollagen-
1(I) mRNA
expression. Thus the responses triggered by mechanical strain appeared
to be different from those induced by mitogenic stimuli in fetal lung cells.
Regardless of strain-induced decreases in message levels of
procollagen-1(I) and biglycan,
mechanical strain increased their soluble protein content in the
medium. Although we cannot explain the uncoordinated
alteration in mRNA and protein, it is possible that mechanical
strain-initiated signals bypass gene transcription and enhance
translational activity of mRNAs encoding for
procollagen-
1(I) and biglycan.
It is also plausible that these increases in protein mass result from
an alteration of ECM degradation. Interstitial collagenase (MMP-1)
specifically degrades fibrillar collagens (16). Stromelysin-1 (MMP-3)
is able to degrade many ECM proteins, including proteoglycans (16).
Type IV collagenases, also known as gelatinases A and B, degrade type
IV collagen and possess high activity against denatured collagen
(gelatin) (3, 16, 17, 20). Both enzymes also degrade other ECM
molecules such as collagen V and elastin (17, 24). In the present
study, we found that the steady-state mRNA levels for these three MMPs
as well as the activities of collagenase and gelatinases remained
unchanged by mechanical strain. The expression of TIMP-1, an inhibitor
of metalloproteinases, was also not altered by mechanical stretching of
fetal lung cells. Reverse zymography showed that fetal lung cells
elaborated mainly TIMP-1 and TIMP-2 activities in their media, and both
activities were not affected by mechanical strain. Therefore,
mechanical strain-induced increase in soluble ECM content is likely not
brought about by a decrease in ECM degradation. Recently, we observed that a similar intermittent mechanical strain increased biglycan synthesis and sulfation. Strain also increased secretion of biglycan and glycosaminoglycans through both constitutive and regulatory pathways (33). Although the exact mechanism remains an enigma, these
latter findings suggest that mechanical strain-induced ECM accumulation
is in part a result of alterations in ECM protein synthesis and
intracellular transportation.
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ACKNOWLEDGEMENTS |
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We are grateful to Jason Liu and Yi Qin for technical assistance.
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FOOTNOTES |
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This work was supported by group and operating grants from the Medical Research Council of Canada (to M. Post and M. Liu, respectively), Grant R01-HL-43416 from the National Heart, Lung, and Blood Institute (to M. Post), equipment grants from the Ontario Thoracic Society (to M. Post and M. Liu), and a Dean's Fund from the Faculty of Medicine, University of Toronto (Toronto, ON) (to M. Liu).
M. Liu is a recipient of a scholarship from 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. Post, Lung Biology Research Program, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: mppm{at}sickkids.on.ca).
Received 13 May 1998; accepted in final form 19 January 1999.
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REFERENCES |
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