Hypoxia downregulates tropoelastin gene expression in rat lung fibroblasts by pretranslational mechanisms

John L. Berk, Nima Massoomi, Christine Hatch, and Ronald H. Goldstein

Pulmonary Center, Boston University School of Medicine and Boston Veterans Affairs Medical Center, Boston, Massachusetts 02118


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta (TGF-beta ) (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 alpha 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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) - untreated cell supernatant (cpm)]/[treated cell lysate (cpm) + treated cell supernatant (cpm) - untreated cell supernatant (cpm)]} × 100 (4).

Reagents. [alpha -32P]dCTP (3,000 Ci/mmol), [gamma -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 gamma RE2, a rat elastin (43), and palpha 1R1, a rat alpha 1(I) collagen (17), rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (15), and lysyl oxidase (16). The probes were labeled with [alpha -32P]dCTP using a multiprime labeling kit (Amersham). A 20-bp oligonucleotide complementary to 18S ribosomal RNA was end labeled with [gamma -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 [alpha -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 alpha 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Dose-related effect of hypoxia on tropoelastin (ELAST) mRNA levels in lung interstitial cells (LIC). Confluent, quiescent LIC were untreated (21% O2) or treated with 0, 3, or 10% O2 for 36 h. A: equal quantities (10 µg) of total RNA from each experimental condition were electrophoretically separated and transferred to a nylon filter. Radiolabeled elastin cDNA was hybridized to filter; an oligonucleotide probe complementary to 18S ribosomal RNA was used to detect sample loading variation. B: densitometric examination of Northern analysis. Intensities of tropoelastin transcripts were normalized to accompanying 18S ribosomal RNA signal. OD, optical density.

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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Fig. 2.   Recovery of tropoelastin mRNA levels from hypoxia-induced repression. Confluent LIC were made quiescent and cultured in 21% O2 and hypoxic (0% O2) conditions. After 24 h of treatment, total RNA was isolated from representative cultures in 21% O2 (lane 1) and hypoxic (lane 2) groups. Remaining cultures, both 21% (lane 3) and 0% O2 treated (lane 4), were refed and allowed to recover for 96 h in 21% O2 before total RNA was harvested. After electrophoretic separation of different RNAs, resulting filter was hybridized with radiolabeled elastin cDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, and an oligonucleotide probe complementary to 18S ribosomal RNA.

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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Fig. 3.   Effect of hypoxia on LIC proliferation and cell injury. A: cell counts were performed on confluent quiescent LIC after being cultured in 21% O2 or hypoxic conditions (0, 3, and 10% O2) for 36 h. Adherent cells were lifted by trypsin treatment and quantified by Coulter counter analysis. Data are mean values ± SE of 3 separate experiments. B: chromium release experiments were conducted on confluent quiescent LIC. After 51Cr loading overnight (18 h), cells were untreated or treated with varying degrees of hypoxia (0, 3, and 10% O2) for 24 h. Media and cell layers were harvested individually, and amount of 51Cr radioactivity in each sample was determined by gamma counter analysis. The "%specific release" of 51Cr released into media are mean data ± SE from 3 separate experiments.

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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Fig. 4.   Time course of hypoxia-induced inhibition of tropoelastin mRNA. LIC were untreated (21% O2) or cultured under hypoxic conditions (0% O2) for 6-36 h. At specified time points, total RNA from each experimental condition was isolated and 10-µg samples were subjected to Northern analysis. Resulting filter was hybridized with radiolabeled elastin cDNA and an oligonucleotide probe complementary to 18S ribosomal RNA. An autoradiogram was produced. Optical densities of tropoelastin transcript signals were determined by laser densitometry and normalized to the intensity of the accompanying 18S ribosomal RNA signal. Data are means ± SE from 3 separate experiments.

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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Fig. 5.   Newly synthesized proteins do not mediate the downregulation of tropoelastin mRNA by hypoxia. LIC were untreated (21% O2) or cultured in 0% O2 for 24 h in presence and absence of cycloheximide (CHX, 5 µM). Total RNA was isolated from cultures in each experimental group, and 10-µg samples underwent Northern analysis. Radiolabeled elastin cDNA and 18S ribosomal probes were hybridized to the filter, and an autoradiogram was produced.

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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Fig. 6.   Effect of hypoxia on stability of tropoelastin mRNA. Confluent quiescent LIC were cultured under 21% O2 or hypoxic conditions (0% O2) for varying intervals in presence of actinomycin D (5 µM). At appropriate times, total RNA was isolated from 21% O2 and hypoxic groups. Samples (10 µg) from each treatment group were subjected to Northern analysis. Radiolabeled probes for elastin cDNA and 18S ribosomal RNA were hybridized to filter, and an autoradiogram was produced. The intensity of tropoelastin mRNA transcripts was quantified by laser densitometry and normalized to 18S ribosomal RNA signal. Optical density (OD) data represent mean values ± SE from 3 separate experiments.

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 alpha 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 alpha 1(I) collagen expression to 64% of untreated cultures. Twenty-four hours of 0% O2 marginally inhibited transcription of elastin (77% of control) and alpha 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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Fig. 7.   Effect of hypoxia on transcription rate of extracellular matrix genes. A: nuclei were isolated from confluent, quiescent LIC incubated for 6 h or 24 h under 21 or 0% O2 conditions. Radiolabeled nascent RNA transcripts were hybridized for 48 h to alkali-treated plasmids containing cDNA inserts of various genes or to plasmids without inserts [pBluescript (pBS)] immobilized on nitrocellulose filters. Filters were processed for autoradiography. Displayed data are representative of 2 separate experiments. B: densitometric analysis of mean data from 2 run-on assays. "Percent control" represents transcription rates for elastin, alpha 1(I) collagen, and lysyl oxidase (OX) in 0% O2-treated cells relative to rates in cells kept at 21% O2 for 6, 16, and 24 h.

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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Fig. 8.   Recovery of hypoxia-inhibited elastin gene transcription rates. Nuclei were isolated from LIC cultured under 21% O2 or hypoxic conditions (0% O2) for 24 h and then recovered in 21% O2 for an additional 96 h. Radiolabeled nascent RNA generated in vitro by nuclei isolated from each experimental group were hybridized to filter-fixed plasmids containing cDNA inserts of various extracellular matrix genes or to plasmids without inserts for 48 h. Filters were processed, and autoradiograms were produced.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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 alpha 1(I) collagen production in human cardiac fibroblasts (2). Falanga et al. (13) reported that hypoxia upregulated alpha 1(I) collagen mRNA in explanted human dermal fibroblasts by a transcriptional mechanism apparently dependent on autocrine elaboration of TGF-beta (13). Notably, hypoxia downregulated alpha 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-1beta (6, 34), dexamethasone (25, 42), and retinoic acid (23, 28). In our cultured neonatal rat lung fibroblasts, hypoxia inhibited formation of tropoelastin and alpha 1(I) collagen RNA to a similar degree. This regulation of tropoelastin and alpha 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.


    ACKNOWLEDGEMENTS

We thank J. A. Foster, Boston University School of Medicine, for providing gamma RE2 and D. Rowe, University of Connecticut Medical Center, for providing palpha 1R1.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adamson, I. Y., L. Young, and D. H. Bowden. Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am. J. Pathol. 130: 377-383, 1988[Abstract].

2.   Agocha, A., H. W. Lee, and M. Eghbali-Webb. Hypoxia regulates basal and induced DNA synthesis and collagen type I production in human cardiac fibroblasts: effects of transforming growth factor-beta1, thyroid hormone, angiotensin II and basic fibroblast growth factor. J. Mol. Cell. Cardiol. 29: 2233-2244, 1997[Medline].

3.   Anderson, M. T., F. J. Staal, C. Gitler, and L. A. Herzenberg. Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway. Proc. Natl. Acad. Sci. USA 91: 11527-11531, 1994[Abstract/Free Full Text].

4.   Andreoli, S. P., R. L. Baehner, and J. M. Bergstein. In vitro detection of endothelial cell damage using 2-deoxy-D-3H-glucose: comparison with chromium 51, 3H-leucine, 3H-adenine, and lactate dehydrogenase. J. Lab. Clin. Med. 106: 253-261, 1985[Medline].

5.   Bashir, M. M., Z. Indik, H. Yeh, N. Ornstein-Goldstein, J. C. Rosenbloom, W. Abrams, M. Fazio, J. Uitto, and J. Rosenbloom. Characterization of the complete human elastin gene. Delineation of unusual features in the 5'-flanking region. J. Biol. Chem. 264: 8887-8891, 1989[Abstract/Free Full Text].

6.   Berk, J. L., C. Franzblau, and R. H. Goldstein. Recombinant interleukin-1 beta inhibits elastin formation by a neonatal rat lung fibroblast subtype. J. Biol. Chem. 266: 3192-3197, 1991[Abstract/Free Full Text].

7.   Berk, J. L., N. Massoomi, M. Krupsky, and R. H. Goldstein. Effect of okadaic acid on elastin gene expression in interstitial lung fibroblasts. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L939-L948, 1996[Abstract/Free Full Text].

8.   Bunn, H. F., and R. O. Poyton. Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76: 839-885, 1996[Abstract/Free Full Text].

9.   Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979[Medline].

10.   Czyzyk-Krzeska, M. F., and J. E. Beresh. Characterization of the hypoxia-inducible protein binding site within the pyrimidine-rich tract in the 3'-untranslated region of the tyrosine hydroxylase mRNA. J. Biol. Chem. 271: 3293-3299, 1996[Abstract/Free Full Text].

11.   Durmowicz, A. G., D. B. Badesch, W. C. Parks, R. P. Mecham, and K. R. Stenmark. Hypoxia-induced inhibition of tropoelastin synthesis by neonatal calf pulmonary artery smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 5: 464-469, 1991[Medline].

12.   Durmowicz, A. G., M. G. Frid, J. D. Wohrley, and K. R. Stenmark. Expression and localization of tropoelastin mRNA in the developing bovine pulmonary artery is dependent on vascular cell phenotype. Am. J. Respir. Cell Mol. Biol. 14: 569-576, 1996[Abstract].

13.   Falanga, V., T. A. Martin, H. Takagi, R. S. Kirsner, T. Helfman, J. Pardes, and M. S. Ochoa. Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts. J. Cell. Physiol. 157: 408-412, 1993[Medline].

14.   Forsythe, J. A., B. H. Jiang, N. V. Iyer, F. Agani, S. W. Leung, R. D. Koos, and G. L. Semenza. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16: 4604-4613, 1996[Abstract].

15.   Fort, P., L. Marty, M. Piechaczyk, S. el Sabrouty, C. X. Dani, P. Jeanteur, and J. M. Blanchard. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 13: 1431-1442, 1985[Abstract].

16.   Gacheru, S. N., K. M. Thomas, S. A. Murray, K. Csiszar, L. I. Smith-Mungo, and H. M. Kagan. Transcriptional and post-transcriptional control of lysyl oxidase expression in vascular smooth muscle cells: effects of TGF-beta 1 and serum deprivation. J. Cell. Biochem. 65: 395-407, 1997[Medline].

17.   Genovese, C., D. Rowe, and B. Kream. Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23: 6210-6216, 1984[Medline].

18.   Goldstein, R. H., M. Wall, L. Taylor, M. Cahill, and P. Polgar. Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts. Prostaglandins 28: 717-729, 1984.

19.   Gopfert, T., B. Gess, K. U. Eckardt, and A. Kurtz. Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes. J. Cell. Physiol. 168: 354-361, 1996[Medline].

20.   Graven, K. K., and H. W. Farber. Endothelial hypoxic stress proteins. Kidney Int. 51: 426-437, 1997[Medline].

21.   Graven, K. K., R. F. Troxler, H. Kornfeld, M. V. Panchenko, and H. W. Farber. Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia. J. Biol. Chem. 269: 24446-24453, 1994[Abstract/Free Full Text].

22.   Greenberg, M. E., and E. B. Ziff. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature 311: 433-438, 1984[Medline].

23.   Griffiths, C. E., A. N. Russman, G. Majmudar, R. S. Singer, T. A. Hamilton, and J. J. Voorhees. Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid). N. Engl. J. Med. 329: 530-535, 1993[Abstract/Free Full Text].

24.   Groudine, M., M. Peretz, and H. Weintraub. Transcriptional regulation of hemoglobin switching in chicken embryos. Mol. Cell. Biol. 1: 281-288, 1981[Medline].

25.   Kahari, V. M. Dexamethasone suppresses elastin gene expression in human skin fibroblasts in culture. Biochem. Biophys. Res. Commun. 201: 1189-1196, 1994[Medline].

26.   Kim, S. B., S. A. Kang, J. S. Park, J. S. Lee, and C. D. Hong. Effects of hypoxia on the extracellular matrix production of cultured rat mesangial cells. Nephron 72: 275-280, 1996[Medline].

27.   Kourembanas, S., L. P. McQuillan, G. K. Leung, and D. V. Faller. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J. Clin. Invest. 92: 99-104, 1993[Medline].

28.   Krupsky, M., A. Fine, J. L. Berk, and R. H. Goldstein. Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts. Biochim. Biophys. Acta 1219: 335-341, 1994[Medline].

29.   Kuhn, C., S. Y. Yu, M. Chraplyvy, H. E. Linder, and R. M. Senior. The induction of emphysema with elastase. II. Changes in connective tissue. Lab. Invest. 34: 372-380, 1976[Medline].

30.   Leung, D. Y., S. Glagov, and M. B. Mathews. Cyclic stretching stimulates synthesis of matrix components by arterial smooth muscle cells in vitro. Science 191: 475-477, 1976[Medline].

31.   Levy, A. P., N. S. Levy, S. Wegner, and M. A. Goldberg. Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J. Biol. Chem. 270: 13333-13340, 1995[Abstract/Free Full Text].

32.   Liu, B., C. S. Harvey, and S. E. McGowan. Retinoic acid increases elastin in neonatal rat lung fibroblast cultures. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L430-L437, 1993[Abstract/Free Full Text].

33.   Lucey, E. C., J. J. O'Brien, Jr., W. Pereira, Jr., and G. L. Snider. Arterial blood gas values in emphysematous hamsters. Am. Rev. Respir. Dis. 121: 83-89, 1980[Medline].

34.   Mauviel, A., Y. Q. Chen, V. M. Kahari, I. Ledo, M. Wu, L. Rudnicka, and J. Uitto. Human recombinant interleukin-1 beta up-regulates elastin gene expression in dermal fibroblasts. Evidence for transcriptional regulation in vitro and in vivo. J. Biol. Chem. 268: 6520-6524, 1993[Abstract/Free Full Text].

35.   McGowan, S. E., R. Liu, C. S. Harvey, and E. C. Jaeckel. Serine proteinase inhibitors influence the stability of tropoelastin mRNA in neonatal rat lung fibroblast cultures. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L376-L385, 1996[Abstract/Free Full Text].

36.   McGowan, S. E., and R. McNamer. Transforming growth factor-beta increases elastin production by neonatal rat lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 3: 369-376, 1990[Medline].

37.   Murphy, G., and A. J. Docherty. The matrix metalloproteinases and their inhibitors. Am. J. Respir. Cell Mol. Biol. 7: 120-125, 1992[Medline].

38.   Myers, J. L., and A. L. Katzenstein. Epithelial necrosis and alveolar collapse in the pathogenesis of usual interstitial pneumonia. Chest 94: 1309-1311, 1988[Abstract].

39.   Niinikoski, J., T. K. Hunt, and J. E. Dunphy. Oxygen supply in healing tissue. Am. J. Surg. 123: 247-252, 1972[Medline].

40.   Parks, W. C., M. E. Kolodziej, and R. A. Pierce. Phorbol ester-mediated downregulation of tropoelastin expression is controlled by a posttranscriptional mechanism. Biochemistry 31: 6639-6645, 1992[Medline].

41.   Pierce, R. A., M. E. Kolodziej, and W. C. Parks. 1,25-Dihydroxyvitamin D3 represses tropoelastin expression by a posttranscriptional mechanism. J. Biol. Chem. 267: 11593-11599, 1992[Abstract/Free Full Text].

42.   Pierce, R. A., W. I. Mariencheck, S. Sandefur, E. C. Crouch, and W. C. Parks. Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L491-L500, 1995[Abstract/Free Full Text].

43.   Rich, C. B., and J. A. Foster. Characterization of rat heart tropoelastin. Arch. Biochem. Biophys. 268: 551-558, 1989[Medline].

44.   Sachs, A. B. Messenger RNA degradation in eukaryotes. Cell 74: 413-421, 1993[Medline].

45.   Scandurro, A. B., I. J. Rondon, R. B. Wilson, S. A. Tenenbaum, R. F. Garry, and B. S. Beckman. Interaction of erythropoietin RNA binding protein with erythropoietin RNA requires an association with heat shock protein 70. Kidney Int. 51: 579-584, 1997[Medline].

46.   Silverstein, J. L., V. D. Steen, T. A. Medsger, Jr., and V. Falanga. Cutaneous hypoxia in patients with systemic sclerosis (scleroderma). Arch. Dermatol. 124: 1379-1382, 1988[Abstract].

47.   Stenmark, K. R., A. G. Durmowicz, J. D. Roby, R. P. Mecham, and W. C. Parks. Persistence of the fetal pattern of tropoelastin gene expression in severe neonatal bovine pulmonary hypertension. J. Clin. Invest. 93: 1234-1242, 1994[Medline].

48.   Stone, P. J., S. M. Morris, K. M. Thomas, K. Schuhwerk, and A. Mitchelson. Repair of elastase-digested elastic fibers in acellular matrices by replating with neonatal rat-lung lipid interstitial fibroblasts or other elastogenic cell types. Am. J. Respir. Cell Mol. Biol. 17: 289-301, 1997[Abstract/Free Full Text].

49.   Swee, M. H., W. C. Parks, and R. A. Pierce. Developmental regulation of elastin production. Expression of tropoelastin pre-mRNA persists after down-regulation of steady-state mRNA levels. J. Biol. Chem. 270: 14899-14906, 1995[Abstract/Free Full Text].

50.   Thomas, P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 100: 255-266, 1983[Medline].

51.   Wang, G. L., and G. L. Semenza. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem. 268: 21513-21518, 1993[Abstract/Free Full Text].

52.   Wolfe, B. L., C. B. Rich, H. D. Goud, A. J. Terpstra, M. Bashir, J. Rosenbloom, G. E. Sonenshein, and J. A. Foster. Insulin-like growth factor-I regulates transcription of the elastin gene. J. Biol. Chem. 268: 12418-12426, 1993[Abstract/Free Full Text].


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