Differential regulation of extracellular matrix molecules by mechanical strain of fetal lung cells

Jing Xu, Mingyao Liu, and Martin Post

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 1(I) and biglycan and increased the levels of mRNA for collagen-alpha 1(IV) and -alpha 2(IV), whereas laminin beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    METHODS
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INTRODUCTION
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-alpha 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 beta -actin cDNA (0.7 kb) was generated by RT-PCR cloning with rat beta -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 (alpha 1) and (alpha 2) type IV collagen cDNAs (insert size 1.8 and 1.1 kb, respectively) and laminin beta -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 (approx 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'-[alpha -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-alpha 1(I), laminin beta -chain, biglycan, GAPDH, and beta -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 (alpha 1 and alpha 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 beta -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) beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

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-alpha 1(I), collagen-alpha 1(IV) and -alpha 2(IV), laminin beta -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-alpha 1(I) and biglycan increased throughout the 48-h period of static culture. Although procollagen-alpha 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-alpha 1(I) and biglycan mRNA levels compared with that in static control cells (Fig. 1C). GAPDH and beta -actin message levels were unaffected by mechanical strain (Fig. 1C). The steady-state mRNA levels of both collagen chains [alpha 1(IV) and alpha 2(IV)] also increased during the 48-h static culture period of mixed fetal lung cells. In contrast to procollagen-alpha 1(I) and biglycan, a 48-h intermittent straining increased collagen-alpha 1(IV) and -alpha 2(IV) mRNA expression of mixed (Fig. 1B) and individual (Fig. 1C) fetal lung cells. Laminin beta -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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Fig. 1.   Mechanical strain differentially affects mRNA expression of extracellular matrix proteins. Mixed fetal rat lung cells (A and B) and epithelial cells (Epi) and fibroblasts (Fib) alone (C) were subjected for various time periods (15 min to 48 h in A and B and 48 h in C) to intermittent mechanical strain regimen (60 cycles/min, 15 min/h). Equal amounts of total RNA (15 µg) isolated from both strained (S) and unstrained (control; C) cells were subjected to Northern blot hybridization with 32P-labeled cDNA probes. GADPH, glyceraldehyde-3-phosphate dehydrogenase.

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-alpha 1(I) is related to cell proliferation, we examined mixed fetal lung cell proliferation and gene expression of procollagen-alpha 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-alpha 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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Fig. 2.   Comparison of effects of fetal bovine serum (FBS) and mechanical strain on DNA synthesis and gene expression of procollagen-alpha 1(I). Mixed fetal rat lung cells were cultured in either 1% FBS with and without strain or 10% FBS without strain for 48 h. A: DNA synthesis was determined as [3H]thymidine incorporation into DNA during 48-h culture period. * P < 0.05 compared with cells cultured in 1% FBS without strain. B: equal amounts of total cellular RNA (15 µg) were subjected to Northern blot hybridization with 32P-labeled cDNA probes encoding for procollagen-alpha 1(I) and beta -actin.

Intermittent mechanical strain increases soluble collagen and biglycan content. Regardless of a decrease in procollagen-alpha 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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Fig. 3.   Mechanical strain increases soluble protein content of type I and IV collagens and biglycan. A: mixed fetal lung cells were subjected to mechanical strain (S) or static culture (C) for 48 h. Culture media were collected, concentrated, and analyzed by Western blotting with antibodies against type I (alpha 1) and type IV (alpha 1 and alpha 2) collagen. B: to study synthesis of biglycan, fetal lung cells were metabolically labeled with 35SO4, and secreted proteoglycans were immunoprecipitated using anti-biglycan antibodies and analyzed by SDS-PAGE. Intensity of 275-kDa band, corresponding to biglycan, was significantly enhanced in mechanical strained samples. Nature of biglycan was further confirmed by enzymatic treatment of samples with chondroitinase ABC, which removed most chondroitin sulfate/dermatan sulfate side chains of biglycan. +, Chondroitinase ABC-treated sample; -, untreated sample.

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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Fig. 4.   Mechanical strain does not alter gene expression of matrix metalloproteinases (MMP). Mixed fetal rat lung cells were subjected to strain for either 15 min (left) or 48 h (right). Equal amounts of total RNA (15 µg) extracted from both strained and unstrained cells were hybridized with 32P-labeled cDNA probes, encoding for interstitial collagenase (MMP-1), gelatinase A (type IV collagenase; MMP-2), or stromelysin-1 (MMP-3).



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Fig. 5.   Mechanical strain does not alter gelatinase activity. Mixed fetal rat lung cells were subjected to intermittent strain or static culture for 48 h. Culture media were collected and concentrated, and samples containing equal amounts of protein were analyzed by zymography. Gelatinolytic activities appeared as zones of substrate clearing with 2 major areas of lysis at 92 and 72 kDa (arrows), representing gelatinases B and A, respectively. Without EDTA, no difference was observed between strained samples and static controls (lanes 1 and 2). In presence of 10 mM EDTA, a chelating agent that inhibits MMPs, cleared zones in substrate gel were not observed (lanes 3 and 4).



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Fig. 6.   Mechanical strain does not alter gene and protein expression of tissue inhibitor of metalloproteinase (TIMP)-1. A: mixed fetal rat lung cells were subjected to strain for either 15 min or 48 h. Equal amounts of total RNA (15 µg) extracted from both strained and unstrained cells were hybridized with 32P-labeled cDNA probe encoding for TIMP-1. B: culture media were concentrated, and proteins were resolved by SDS-PAGE and immunoblotted with antiserum to TIMP-1.



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Fig. 7.   Mechanical strain does not alter TIMP activities. Mixed fetal rat lung cells were subjected to intermittent strain or static culture for 48 h. Culture media were collected and concentrated, and samples containing equal amounts of protein were analyzed by reverse zymography. Gelatinolytic inhibitory activities appeared as zones of positive staining with Coomassie blue with 2 major areas at 28 and 21 kDa, representing TIMP-1 and TIMP-2, respectively. No difference was observed between strained samples and static controls. Experiment was performed with 3 separate sets of conditioned medium.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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-alpha 1(I) and biglycan with duration of culture, which was dampened by mechanical strain. However, mechanical strain increased the accumulation of mRNA for collagen-alpha 1(IV) and -alpha 2(IV) in fetal lung cells, whereas laminin beta -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-alpha 1(I) but increased that of collagen-alpha 1(IV) and-alpha 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-alpha 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-alpha 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-alpha 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.


    ACKNOWLEDGEMENTS

We are grateful to Jason Liu and Yi Qin for technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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