Pulmonary Center, Boston University School of Medicine and Boston Veterans Affairs Medical Center, Boston, Massachusetts 02118
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ABSTRACT |
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Elastolytic lung injury disrupts cell barriers, flooding alveoli and producing regional hypoxia. Abnormal O2 tensions may alter repair of damaged elastin fibers. To determine the effect of hypoxia on extravascular elastin formation, we isolated rat lung fibroblasts and cultured them under a variety of O2 conditions. Hypoxia downregulated tropoelastin mRNA in a dose- and time-related fashion while upregulating glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The changes in tropoelastin gene expression were not due to cell toxicity as measured by chromium release and cell proliferation studies. Neither cycloheximide nor actinomycin D abrogated this effect. Hypoxia induced early decreases in tropoelastin mRNA stability; minor suppression of gene transcription occurred later. When returned to 21% O2, tropoelastin mRNA recovered to control levels in part by upregulating tropoelastin gene transcription. Taken together, these data indicate that hypoxia regulates tropoelastin gene expression and may alter repair of acutely injured lung.
extracellular matrix; elastin; lung injury
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INTRODUCTION |
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ELASTIN, THE PRINCIPAL structural protein in terminal air spaces, is vital for proper lung function. In animal models of emphysema, intratracheal treatment with elastase disrupts elastin fibers and alveolar architecture, impairing gas exchange (29, 33). Similar events occur in various types of acute lung injury: the initial insult damages type I epithelial cell tight junctions, filling alveoli with inflammatory exudate containing macrophage- and neutrophil-derived metalloproteinases (1, 38). These matrix-degrading enzymes include stromelysins, matrilysin, gelatinases, collagenase, and metalloelastase (37). Extracellular matrix destruction is often accompanied by alveolar collapse, producing regions of tissue hypoxia (38).
Repair of protease-damaged elastin fibers in vitro occurs by two
processes: 1) a salvage mechanism in
which lysyl oxidase enzymatically reestablishes unique cross-links
between dissociated fibers and 2) a
de novo mechanism that requires formation of nascent tropoelastin
molecules, the basic subunit of mature, cross-linked elastin (48). In
extravascular lung tissue, the interstitial fibroblast is the primary
source of tropoelastin (6). Under 21%
O2, various effector molecules
such as insulin-like growth factor I (52), transforming growth
factor- (TGF-
) (36), and retinoic acid (32) upregulate
tropoelastin gene expression in cultured vascular smooth muscle cells
and interstitial lung fibroblasts. However, low ambient
O2 tensions (18-25 mmHg)
occur in models of wound healing (39) and fibrosis (46). It is
unclear whether regional hypoxia following acute lung injury modulates extracellular matrix expression by interstitial lung fibroblasts.
Few studies have examined the effect of hypoxia on matrix formation.
Hypoxia stimulated type I collagen expression in isolated human cardiac
fibroblasts (2) and in explanted human dermal fibroblasts. Hypoxia also
induced production of type IV collagen, laminin, and fibronectin in
renal mesangial cells (26). In the setting of hypoxic pulmonary
hypertension, neonatal calf pulmonary arteries exhibited persistent
fibronectin mRNA expression, reinduction of tropoelastin mRNA, and
upregulation of 1(I) collagen
gene expression (47). Subsequent experiments employing intact vessels and isolated smooth muscle cells determined that vascular pressures and
associated cell stretch, not hypoxia, stimulated tropoelastin production (11, 12, 30).
We hypothesized that hypoxia modifies matrix remodeling in the extravascular lung following tissue injury by affecting the gene expression of certain connective tissue elements. To examine the effect of hypoxia on elastin production, we isolated a pure population of interstitial rat lung fibroblasts and exposed them to varying concentrations and periods of hypoxia. Hypoxia reversibly downregulated elastin gene expression by a complex interplay of transcriptional and posttranscriptional mechanisms.
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MATERIALS AND METHODS |
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Isolation of lung interstitial cells. Lung interstitial cells (LIC) were isolated from the lungs of 8-day-old Sprague-Dawley rats (Charles River Breeding Laboratory, Wilmington, MA) as previously described (6). After isolation, LIC were resuspended in MEM containing 10% fetal bovine serum, 0.37 g sodium pyruvate/100 ml, 100 units of penicillin, and 100 µg streptomycin/ml and seeded into a 75-cm2 flask (Falcon Plastics, Los Angeles, CA). Cultures were maintained in a humidified 5% CO2-95% air incubator at 37°C. After 5 days, the cells were passed following treatment with trypsin and grown to confluence in 35-mm or 150-mm plastic dishes (Falcon). The purity of the cultures was assessed with phase microscopy and oil red O staining.
At confluence, the cells were made quiescent by reducing the serum content to 0.4% for 24 h. Cells were then incubated at 37°C in 5% CO2-95% air or in a humidified sealed chamber (Billups-Rothenburg, Del Mar, CA) gassed with 0, 3, or 10% O2 containing 5% CO2-balance N2 mixtures (Medical-Technical Gases). When incubated in a 0% O2 environment, O2 tension in the culture medium falls from 139 ± 11 Torr to a steady state 20-32 Torr over the initial 12 h (27). Parallel LIC cultures were assessed for injury by phase microscopy, adherent cell counts, and 51Cr release studies (4). For 51Cr release experiments, confluent LIC were labeled overnight with 51Cr (30 µCi/ml), washed twice with Hanks' balanced salt solution (GIBCO), and refed MEM containing 0.4% fetal bovine serum. The radiolabeled cells were incubated at 37°C in 0, 3, 10, or 21% O2 with 5% CO2-balance N2 gas mixtures. After 24 h, medium and cell fractions were analyzed for 51Cr by gamma scintillography (Beckman Instruments, Fullerton, CA). The calculation by which 51Cr release was derived was percent specific 51Cr release equals {[treated cell supernatant (counts/min, cpm)Reagents.
[-32P]dCTP (3,000 Ci/mmol), [
-32P]ATP
(3,000 Ci/mmol),
[32P]UTP (3,000 Ci/mmol), and 51Cr (200-500
Ci/g) were purchased from Dupont-New England Nuclear.
RNA isolation and Northern analysis.
Total cellular RNA was isolated using guanidium thiocyanate (American
Bioanalytical, Natick, MA) following the methods of Chirgwin et al.
(9). RNA was quantitated by absorbance at 260 nm. Purity was assessed
by absorbance at 280 and 320 nm. RNA (10 µg) was fractionated by
electrophoresis on a 1% agarose-6% formaldehyde gel, transferred to a
nylon filter (Hybond), and immobilized to the filter by ultraviolet
cross-linking (Stratalinker, Stratagene, CA). Equal loading of RNA
samples was monitored by ethidium bromide staining of ribosomal bands
fractionated on agarose-formaldehyde gels and by radiolabeling the 18S
ribosomal RNA. Hybridization was performed using 2-10 × 106 cpm/lane labeled probe
(specific activity 8-15 × 107 cpm/µg). The filter was
washed by established methods (50). Films were developed after various
exposure times to ensure that densitometric readings were within the
linear range. cDNA probes included RE2, a rat elastin (43), and
p
1R1, a rat
1(I) collagen (17), rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (15), and
lysyl oxidase (16). The probes were labeled with
[
-32P]dCTP using a
multiprime labeling kit (Amersham). A 20-bp oligonucleotide complementary to 18S ribosomal RNA was end labeled with
[
-32P]ATP using T4
polynucleotide kinase (New England Biolabs).
Nuclear run-on analysis.
Nuclei (~5 × 107
nuclei/sample) were harvested from cultured cells by the procedure of
Greenberg and Ziff (22), resuspended in 200 µl of glycerol buffer
containing 50 mM Tris · HCL, pH 8.3, 5 mM
MgCl2, 0.1 mM EDTA, 40% glycerol,
and frozen in liquid nitrogen. The transcription rates were determined
by published methods (22, 24). Nuclei were incubated in transcription
buffer containing 100 µCi
[-32P]UTP (760 Ci/mmol) at 30°C for 30 min. Labeled RNA transcripts were separated
from DNA and protein by DNase I and proteinase K digestions followed by
phenol-chloroform-isoamyl alcohol extractions. The radiolabeled RNA
(1-5 × 107 cpm/ml) was
hybridized to nitrocellulose-bound samples (10 µg) of empty
pBluescript DNA and plasmid containing cDNA inserts for elastin and
1(I) collagen for 48 h at
42°C. After hybridization, the filters were sequentially washed
with 2× saline-sodium citrate (SSC)-0.1% SDS and 0.1×
SSC-0.1% SDS for 15 min at 37°C. The filters were exposed to X-ray
film at
70°C for 24 h. The autoradiograms were analyzed by densitometry.
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RESULTS |
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To examine the effect of hypoxia on elastin gene expression in cultured
neonatal rat lung fibroblasts, confluent quiescent lipid-containing LIC
were exposed to varying levels of
O2 (0, 3, 10, and 21%
O2). After 36-h treatment, we
isolated total RNA from each culture condition and determined the
steady-state levels of tropoelastin mRNA. Hypoxia suppressed
tropoelastin mRNA levels in a dose-related fashion. Culturing LIC in
0% O2 downregulated the
steady-state levels of tropoelastin mRNA to 21% of control levels,
whereas 3% O2 decreased
tropoelastin mRNA to 66%. In contrast, 10%
O2 did not inhibit tropoelastin
gene expression significantly (88%) (Fig.
1).
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The return of hypoxia-treated cells to 21%
O2 restored elastin gene
expression, indicating that limited periods of hypoxia were not toxic
to the cells (Fig. 2). Immediately
following 24 h of 0% O2,
steady-state levels of tropoelastin mRNA decreased to 54% of untreated
controls. Under the same conditions, GAPDH mRNA levels increased by
334%, demonstrating that hypoxia preferentially downregulated
tropoelastin mRNA. When hypoxic cells were returned to 21%
O2 for 96 h, tropoelastin mRNA
levels recovered to control values (111% of the levels in the
untreated controls). Despite normalization of tropoelastin mRNA levels,
the hypoxia-induced elevation of GAPDH mRNA levels persisted (Fig. 2).
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The ability of cultured lung fibroblasts to tolerate prolonged periods
of hypoxic stimulation was further examined by cell proliferation and
chromium release studies. After a 36-h exposure to varying
concentrations of O2, adherent
cell numbers were measured. All levels of hypoxia induced cell
proliferation. Cell counts in cultures treated with 10, 3, or 0%
O2 increased by 35, 17, and 47%,
respectively, over values in the untreated cells (Fig. 3A).
Chromium release studies, a quantifiable measure of cell injury,
revealed minimal differences between untreated control and
hypoxia-treated cultures. After cells were loaded with radiolabeled chromium, the cultures were exposed to 0, 3, 10, or 21%
O2 concentration for 24 h, and
isotope leakage was determined. Compared with baseline values, exposing
cells to low O2 tensions increased
the amount of 51Cr leak by 0.7 ± 3.8% (mean ± SE) in 10%
O2-, 2.9 ± 5.0% in 3% O2-, and 9.3 ± 8.7% in 0%
O2-treated cells (Fig.
3B). The marginal rise in
51Cr released from LIC cultures
treated with 0% O2 was less than one-half the radiolabel leakage reported in similarly treated human
lung fibroblasts (20), emphasizing the tolerance of rat cells to
hypoxic conditions. The change in specific leak of
51Cr, even at the most extreme
levels of O2 deprivation, was
neither statistically significant nor predictive of cell death as
defined by adherent cell counts and trypan blue exclusion studies (data not shown).
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To determine the time course of hypoxia-induced tropoelastin mRNA
inhibition, LIC were subjected to different periods of hypoxia before
isolation of total RNA. In repeated experiments, 0%
O2 for 4-8 h failed to
modulate tropoelastin mRNA levels (data not shown). Culturing under 0%
O2 for 16-36 h downregulated
elastin gene expression in a time-related fashion. After 16 h of
hypoxia, tropoelastin mRNA levels measured 76% of control levels.
Twenty-four hours of treatment decreased tropoelastin mRNA to 47% of
untreated values, whereas 36 h of exposure inhibited tropoelastin mRNA
to 3% of control levels (Fig. 4). The
degree of hypoxic responsiveness varied somewhat between LIC
isolations, likely reflecting the heterogeneity of primary cells in
culture.
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To examine the requirement for ongoing protein production in the
repression of tropoelastin mRNA levels by hypoxia, LIC were untreated
or exposed to hypoxic conditions in the presence and absence of
cycloheximide. At concentrations that inhibited protein production by
85% (18), cycloheximide (5 µM) significantly increased steady-state
levels of tropoelastin mRNA. The upregulation of tropoelastin mRNA by
cycloheximide, which we have observed previously (6), implies the
presence of a short-lived inhibitory protein acting at the
transcriptional or posttranscriptional level. When combined with 0%
O2 conditions, cycloheximide
failed to block the hypoxia-related decreases in tropoelastin mRNA
(Fig. 5).
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To determine the effect of hypoxia on tropoelastin mRNA stability,
quiescent LIC were cultured under 21 or 0%
O2 conditions for varying periods
of time in the presence of actinomycin D. Although the magnitude of the
response differed between experiments, hypoxia consistently decreased
the stability of tropoelastin mRNA, with significant effects noted
after 4 h of exposure (Fig. 6). In 21%
O2, the half-life of tropoelastin
mRNA measured greater than 32 h by linear regression analysis. Hypoxia
decreased the half-life to less than 18 h. The magnitude of the
decrease in tropoelastin mRNA stability in large measure accounted for
the observed downregulation of elastin gene expression induced by hypoxia.
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To determine whether hypoxia also affected the rate of elastin gene
transcription, nuclei were harvested from cells after being cultured
under standard conditions or for 6, 16, or 24 h in 0%
O2. After in vitro formation of
radiolabeled nascent RNA, the transcripts were hybridized to
filter-bound plasmids containing cDNA of specific genes (Fig.
7). In contrast to the early effects of
hypoxia on tropoelastin mRNA stability, hypoxia appeared to affect
elastin gene transcription at later time points. Hypoxia for 6 h did
not significantly alter elastin or
1(I) collagen gene
transcription (100 and 93% of control values, respectively). After 16 h, hypoxia little affected elastin mRNA formation (82% control) while
decreasing
1(I) collagen
expression to 64% of untreated cultures. Twenty-four hours of 0%
O2 marginally inhibited transcription of elastin (77% of control) and
1(I) collagen genes (74% of
control). Low-level transcription rates of other nonmatrix protein
genes such as lysyl oxidase did not change at any investigated time
point of 0% O2.
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To determine the effect of posthypoxia recovery on matrix gene
transcription, we performed nuclear run-on studies on hypoxia-treated nuclei harvested immediately following stimulation and on nuclei recovered in 21% O2 for 4 days.
As previously noted, 0% O2 mildly decreased the rate of tropoelastin RNA formation. In contrast, hypoxia-treated cells recovered in 21%
O2 expressed rebound increases in
the rate of RNA formation (Fig. 8). Elastin
gene transcription rose to 151% of control; collagen transcription
demonstrated a smaller increase (123%). Therefore, recovery of
tropoelastin mRNA from hypoxia-induced suppression is due at least in
part to upregulation of gene transcription.
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DISCUSSION |
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The effect of hypoxia on lung repair, a process requiring production of extracellular matrix elements by interstitial fibroblasts, is unknown. Our data in a pure population of rat lung interstitial fibroblasts indicate that hypoxia downregulates steady-state levels of tropoelastin mRNA in a dose- and time-related fashion by altering transcript stability and, in part, its rate of formation. In contrast, hypoxia upregulated GAPDH, demonstrating the preferentially suppressive effect of low O2 tensions on extracellular matrix expression. Increased GAPDH levels are not unexpected. As one of five "hypoxia-associated proteins" (HAP) expressed by pulmonary artery endothelial cells, GAPDH may confer tolerance to low O2 conditions (20, 21). Studies to establish its role in the response of lung fibroblasts to hypoxia are ongoing.
Hypoxia did not inhibit tropoelastin mRNA expression through toxic effects on lung fibroblasts. Morphologically, the cells appeared unaffected by the most extreme levels of O2 deprivation (0% O2) conditions which, in culture media, recreate tissue O2 levels (20-32 Torr) measured in experimental models of wound repair (39). Functionally, hypoxia stimulated lung fibroblast replication, with lower O2 tensions inducing greater cell turnover, whereas chromium release studies revealed minor levels of radioactive tracer leak into the culture media. Most importantly, the effects were reversible, as hypoxia-inhibited tropoelastin mRNA levels returned to control levels when LIC were recovered in 21% O2 for 96 h. Although Durmowicz et al. (11) reported that prolonged hypoxic exposure (3% O2 for 120 h) decreased the rate of total protein synthesis in pulmonary vascular smooth muscle cells, hypoxia did not alter tropoelastin expression in lung fibroblasts by inducing nonspecific suppression of protein synthesis. In fact, cycloheximide, a potent inhibitor of protein synthesis, upregulated steady-state levels of tropoelastin mRNA, suggesting the presence of a constitutively expressed repressor protein.
The initial decreases in tropoelastin mRNA levels were primarily due to
transcript destabilization; repression of gene transcription contributed modestly to the later phase of the hypoxic effect. Regulation of other hypoxia-responsive genes such as erythropoietin (19) and vascular endothelial growth factor (14) also involves both
transcriptional and posttranscriptional events. Characteristically, the
effect of hypoxia on target gene transcription is delayed 6-12 h,
as observed with elastin in these lung fibroblasts (8). This delay
implies that indirect signal transduction occurs with hypoxic
stimulation, possibly involving generation of
trans-acting nuclear factors. Wang and
Semenza (51) described a heterodimeric protein complex,
hypoxia-inducible factor-1 (HIF-1), that forms in response to low
O2 tension and binds to an
octanucleotide sequence residing in the 5'- or the
3'-flanking region of hypoxia-responsive genes. However, it is
unlikely that HIF-1 mediates downregulation of elastin gene
transcription in rat lung fibroblasts. Neither cycloheximide nor
actinomycin D blocked hypoxia-induced suppression of tropoelastin mRNA,
indicating that de novo production of a protein mediator was not
required, as in HIF-1-mediated events. Alternative hypoxia signal
transduction could involve changes in either the phosphorylation or
redox state of preformed proteins as occurs with nuclear factor-B
(3). In fact, phosphatase inhibitors downregulate tropoelastin mRNA in
neonatal rat lung fibroblasts (7, 35), supporting protein
phosphorylation as a mechanism by which hypoxia might alter
tropoelastin expression.
In 21% O2, posttranscriptional events often regulate extravascular tropoelastin mRNA expression as predicted by the genomic composition of the tropoelastin promoter. Similar to constitutively expressed housekeeping genes with unvarying rates of transcription, the 5'-flanking region of the tropoelastin gene has no canonical TATA box, is filled with GC-rich domains, and has multiple transcriptional start sites (5). Posttranscriptional events are critical to postnatal rat lung development. Using RT-PCR to quantitate pre-mRNA, Swee et al. (49) demonstrated that tropoelastin mRNA falls to nearly undetectable levels in the adult animal despite persistently high rates of gene transcription. The importance of posttranscriptional mechanisms in downregulating tropoelastin expression is further illustrated by 12-O-tetradecanoylphorbol 13-acetate (40) and 1,25-dihydroxyvitamin D3 (41), which destabilize mRNA transcripts in rat lung fibroblasts and bovine chondrocytes. Transcript stability is generally conferred by the size of its poly(A) tail or by AU-rich cis-acting sequences located in flanking untranslated regions (UTR) or open reading frames (44). Tropoelastin mRNA, however, does not contain AU-rich motifs, suggesting that less well-described motifs dictate transcript stability.
Hypoxia affects stability of certain mRNA transcripts (10) by inducing nuclear factor binding to either AUUUA or polypyrimidine sequence motifs (31) present in the 3'-UTR of the target molecule. Specifically, hypoxia prolongs the half-life of human tyrosine hydroxylase mRNA by promoting interaction of trans-acting protein(s) with a pyrimidine-rich domain positioned less than 50 bp downstream from the translation stop codon (10). Analysis of reported human and bovine elastin 3'-UTR sequences reveals several potential hypoxia-inducible protein binding sites (UCCCCCU) closely linked to the translational stop and surrounded by pyrimidine-rich regions. Although most stabilizing mechanisms require binding of newly synthesized protein(s) to specific RNA elements, hypoxia-induced suppression of tropoelastin expression is cycloheximide insensitive. Recently, Scandurro et al. (45) reported that stabilization of EPO mRNA by hypoxia involved preformed ligand(s). Alternatively, hypoxia could promote dissociation of trans-acting proteins bound to RNA-stabilizing motifs.
In cultured rat kidney mesangial fibroblasts, hypoxia stimulated the
accumulation of laminin, fibronectin, and type IV collagen (26).
Similarly, hypoxia induced 1(I)
collagen production in human cardiac fibroblasts (2). Falanga et al.
(13) reported that hypoxia upregulated
1(I) collagen mRNA in explanted
human dermal fibroblasts by a transcriptional mechanism apparently
dependent on autocrine elaboration of TGF-
(13). Notably, hypoxia
downregulated
1(I) collagen
gene transcription in our rat lung fibroblasts. It is not unusual for
specific matrix elements in lung and dermal fibroblasts to have
divergent responses to a variety of perturbations including
interleukin-1
(6, 34), dexamethasone (25, 42), and retinoic acid
(23, 28). In our cultured neonatal rat lung fibroblasts, hypoxia
inhibited formation of tropoelastin and
1(I) collagen RNA to a similar
degree. This regulation of tropoelastin and
1(I) collagen expression by
hypoxia may alter repair of extravascular lung following elastolytic injury.
In summary, hypoxia downregulates tropoelastin mRNA levels in rat lung fibroblasts by a complex interplay of transcriptional and posttranscriptional mechanisms. The inability of cycloheximide or actinomycin D treatments to block the suppression of tropoelastin mRNA distinguishes this hypoxic effect from HIF-1-mediated gene regulation. Taken together, these data indicate that hypoxia may suppress the expression of elastin and collagen following acute lung injury, impacting organ repair. Further studies are needed to localize the hypoxia-responsive motifs altering tropoelastin mRNA stability and repressing elastin promoter function.
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ACKNOWLEDGEMENTS |
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We thank J. A. Foster, Boston University School of Medicine, for
providing RE2 and D. Rowe, University of Connecticut Medical Center,
for providing p
1R1.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. L. Berk, Pulmonary Center, 80 East Concord St., R-304, Boston, MA 02118 (E-mail: jberk{at}lung.bumc.bu.edu).
Received 29 December 1997; accepted in final form 14 May 1999.
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