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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma (PLC-gamma ) 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-gamma /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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -actin cDNA (0.5 kb) was generated by RT-PCR cloning using rat beta -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) beta -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 [alpha -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 beta -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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (19K):
[in this window]
[in a new window]
 
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.



View larger version (23K):
[in this window]
[in a new window]
 
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.

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.


View larger version (30K):
[in this window]
[in a new window]
 
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.

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 beta -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).



View larger version (74K):
[in this window]
[in a new window]
 
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 beta -actin. A: representative blot. Message RNA for beta -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.

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.


View larger version (46K):
[in this window]
[in a new window]
 
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.

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.


View larger version (29K):
[in this window]
[in a new window]
 
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.

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).


View larger version (61K):
[in this window]
[in a new window]
 
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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adachi, Y., T. Mio, I. Striz, S. Carnevali, D. J. Romberger, J. R. Spurzem, P. Heires, M. G. Illig, R. F. Ertl, and S. I. Rennard. Lipopolysaccharide increases fibronectin production and release from cultured lung fibroblasts partially through proteolytic activity. J. Lab. Clin. Med. 127: 448-455, 1996[Medline].

2.   Bardy, N., G. J. Karillon, R. Merval, J. L. Samuel, and A. Tedgui. Differential effects of pressure and flow on DNA and protein synthesis and on fibronectin expression by arteries in a novel organ culture system. Circ. Res. 77: 684-694, 1995[Abstract/Free Full Text].

3.   Baskin, L., P. S. Howard, and E. Macarak. Effect of physical forces on bladder smooth muscle and urothelium. J. Urol. 150: 601-607, 1993[Medline].

4.   Boudreau, N., N. Clausell, J. Boyle, and M. Rabinovitch. Transforming growth factor-beta regulates increased ductus arteriosus endothelial glycosaminoglycan synthesis and a post-transcriptional mechanism controls increased smooth muscle fibronectin, features associated with intimal proliferation. Lab. Invest. 67: 350-359, 1992[Medline].

5.   Choi, M. G., and R. O. Hynes. Biosynthesis and processing of fibronectin in NIL.8 hamster cells. J. Biol. Chem. 254: 12050-12055, 1979[Abstract].

6.   Dunsmore, S. E., C. Martinez-Williams, R. A. Goodman, and D. E. Rannels. Turnover of fibronectin and laminin by alveolar epithelial cells. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L766-L775, 1995[Abstract/Free Full Text].

7.   Ffrench-Constant, C. Alternative splicing of fibronectin---many different proteins but few different functions. Exp. Cell Res. 221: 261-271, 1995[Medline].

8.   George, E. L., E. N. Georges-Labouesse, R. S. Patel-King, H. Rayburn, and R. O. Hynes. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119: 1079-1091, 1993[Abstract/Free Full Text].

9.   Gorfien, S. F., P. S. Howard, J. C. Myers, and E. J. Macarak. Cyclic biaxial strain of pulmonary artery endothelial cells causes an increase in cell layer-associated fibronectin. Am. J. Respir. Cell Mol. Biol. 3: 421-429, 1990[Medline].

10.   Guzowski, D. E., H. Blau, and R. S. Bienkowski. Extracellular matrix in developing lung. In: Pulmonary Physiology of the Fetus, Newborn, Child and Adolescent, edited by E. Scarpelli. Malvern, PA: Lea & Febiger, 1989, p. 83-105.

11.   Harding, R. Fetal breathing movements. In: The Lung: Scientific Foundations, edited by R. G. Crystal, and J. B. West. New York: Raven, 1991, p. 1655-1664.

12.   Ignotz, R. A., and J. Massague. Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261: 4337-4345, 1986[Abstract/Free Full Text].

13.   Ingber, D. E. Tensegrity: the architectural basis of cellular mechanotransduction. Annu. Rev. Physiol. 59: 575-599, 1997[Medline].

14.   Kitterman, J. A. Physiological factors in fetal lung growth. Can. J. Physiol. Pharmacol. 66: 1122-1128, 1988[Medline].

15.   Liu, M., Y. Qin, J. Liu, A. K. Tanswell, and M. Post. Mechanical strain induces pp60src activation and translocation to cytoskeleton in fetal rat lung cells. J. Biol. Chem. 271: 7066-7071, 1996[Abstract/Free Full Text].

16.   Liu, M., S. J. M. Skinner, J. Xu, R. N. N. Han, A. K. Tanswell, and M. Post. Stimulation of fetal rat lung cell proliferation in vitro by mechanical strain. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L376-L383, 1992[Abstract/Free Full Text].

17.   Liu, M., J. Xu, J. Liu, M. E. Kraw, A. K. Tanswell, and M. Post. Mechanical strain-enhanced fetal lung cell proliferation is mediated by phospholipase C and D and protein kinase C. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L729-L738, 1995[Abstract/Free Full Text].

18.   Liu, M., J. Xu, P. Souza, B. Tanswell, A. K. Tanswell, and M. Post. The effect of mechanical strain on fetal rat lung cell proliferation: comparison of two- and three-dimensional culture systems. In Vitro Cell. Dev. Biol. 31: 858-866, 1995.

19.   Maniotis, A. J., C. S. Chen, and D. E. Ingber. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc. Natl. Acad. Sci. USA 94: 849-854, 1997[Abstract/Free Full Text].

20.   Maniscalco, W. M., R. A. Sinkin, R. H. Watkins, and M. H. Campbell. Transforming growth factor-beta 1 modulates type II cell fibronectin and surfactant protein C expression. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L569-L577, 1994[Abstract/Free Full Text].

21.   Pullen, N., P. B. Dennis, M. Andjelkovic, A. Dufner, S. C. Kozma, B. A. Hemmings, and G. Thomas. Phosphorylation and activation of p70s6k by PDK1. Science 279: 707-710, 1998[Abstract/Free Full Text].

22.   Rasoamanantena, P., R. Thweatt, J. Labat-Robert, and S. Goldstein. Altered regulation of fibronectin gene expression in Werner syndrome fibroblasts. Exp. Cell Res. 213: 121-127, 1994[Medline].

23.   Riley, D. J., D. E. Rannels, R. B. Low, L. Jensen, and T. P. Jacobs. Effect of physical forces on lung structure, function and metabolism. Am. Rev. Respir. Dis. 142: 910-914, 1990[Medline].

24.   Rolland, G., J. Xu, A. K. Tanswell, and M. Post. Ontogeny of extracellular matrix gene expression by rat lung cells at late fetal gestation. Biol. Neonate 73: 112-120, 1998[Medline].

25.   Sadoshima, J., and S. Izumo. Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J. 12: 1681-1692, 1993[Abstract].

26.   Simpson, L. L., A. K. Tanswell, and M. G. Joneja. Epithelial cell differentiation in organotypic cultures of fetal rat lung. Am. J. Anat. 172: 31-40, 1985[Medline].

27.   Skinner, S. J. M. Fetal breathing movements: a mechanical stimulus for fetal lung cell growth and differentiation. In: Advances in Fetal Physiology, edited by B. M. Johnston, and P. D. Gluckman. Ithaca, NY: Perinatology, 1989, p. 133-141.

28.   Terada, N., J. J. Lucas, A. Szepesi, R. A. Franklin, K. Takase, and E. W. Gelfand. Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells. Biochem. Biophys. Res. Commun. 186: 1315-1321, 1992[Medline].

29.   Vincent, P. A., R. A. Rebres, E. P. Lewis, V. T. Hurst, and T. M. Saba. Release of ED1 fibronectin from matrix of perfused lungs after vascular injury is independent of protein synthesis. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L485-L492, 1993[Abstract/Free Full Text].

30.   Wilson, E., K. Sudhir, and H. E. Ives. Mechanical strain of rat vascular smooth muscle cells is sensed by specific extracellular matrix/integrin interactions. J. Clin. Invest. 96: 2364-2372, 1995[Medline].

31.   Xu, J., M. Liu, J. Liu, I. Caniggia, and M. Post. Mechanical strain induces constitutive and regulated secretion of glycosaminoglycans and proteoglycans in fetal lung cells. J. Cell Sci. 109: 1605-1613, 1996[Abstract/Free Full Text].

32.   Yamazaki, T., K. Tobe, E. Hoh, K. Maemura, T. Kaida, I. Komuro, H. Tamemoto, T. Kadowaki, R. Nagai, and Y. Yazaki. Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes. J. Biol. Chem. 268: 12069-12076, 1993[Abstract/Free Full Text].

33.   Yasuda, T., S. Kondo, T. Homma, and R. C. Harris. Regulation of extracellular matrix by mechanical stress in rat glomerular mesangial cells. J. Clin. Invest. 98: 1991-2000, 1996[Abstract/Free Full Text].

34.   Zhou, B., N. Boudreau, C. Coulber, J. Hammarback, and M. Rabinovitch. Microtubule-associated protein 1 light chain 3 is a fibronectin mRNA-binding protein linked to mRNA translation in lamb vascular smooth muscle cells. J. Clin. Invest. 100: 3070-3082, 1997[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 277(1):L142-L149
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society