Departments of 1 Anesthesiology and 3 Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0010; and 2 Center for Medical Genetics, Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914
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ABSTRACT |
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Type II nitric
oxide synthase (NOS) is upregulated in the pulmonary vasculature in a
chronic hypoxia model of pulmonary hypertension. In situ hybridization
analysis demonstrates that type II NOS RNA is increased in the
endothelium as well as in the vascular smooth muscle in the lung. The
current studies examine the role of hypoxia-inducible factor (HIF)-1 in
regulating type II NOS gene expression in response to hypoxia in
pulmonary artery endothelial cells. Northern blot analyses demonstrate
a twofold increase in HIF-1 but not in HIF-1
RNA with hypoxia in
vivo and in vitro. Electrophoretic mobility shift assays show the
induction of specific DNA binding activity when endothelial cells were
subjected to hypoxia. This DNA binding complex was identified as HIF-1
using antibodies directed against HIF-1
and HIF-1
. Transient
transfection of endothelial cells resulted in a 2.7-fold increase in
type II NOS promoter activity in response to hypoxia compared with
nonhypoxic controls. Mutation or deletion of the HIF-1 site eliminated
the response to hypoxia. These results demonstrate that HIF-1 is
essential for the hypoxic regulation of type II NOS gene transcription
in pulmonary endothelium.
pulmonary hypertension; gene regulation; nitric oxide; nitric oxide synthase; hypoxia-inducible factor-1
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INTRODUCTION |
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CHRONIC HYPOXIA in the pulmonary vasculature is known to result in vascular remodeling characterized by proliferation and migration of smooth muscle cells as well as by an increased accumulation of extracellular matrix. Several factors induced by hypoxia have recently been implicated as modulators or mediators in the vascular remodeling of hypoxia-induced pulmonary hypertension. These include endothelin-1 (15), vascular endothelial growth factor (24), angiotensin II (18), and nitric oxide (NO) (32, 35). Of these, NO is a short-lived inter- and intracellular second messenger generated by a family of enzymes known as nitric oxide synthases (NOSs). All three isoforms of NOS are present in the lung and have been reported to increase in chronic hypoxia-induced models of pulmonary hypertension (12, 22, 32, 33). In the chronic hypoxic rat model, hypoxia was found to increase type II NOS mRNA and protein levels 1.9- and 1.4-fold, respectively (12). NOS expression was induced in the endothelium of pulmonary resistance vessels, in the smooth muscle of large and small pulmonary vessels, and in bronchial smooth muscle (33). Similar results have been observed in the mouse (T. R. Quinlan and R. A. Johns, unpublished observations). Further examination shows that the increase in NOS expression occurs as early as 24 h after hypoxia (32) and that the increased NOS expression continues in a manner that precedes and progresses with the development of pulmonary vascular remodeling. The mechanism of the hypoxia-related upregulation of NOSs in the lung is unknown.
Low O2 tension is known to
regulate the expression of a number of genes, such as growth factors
and cytokines (9, 10, 14), and enzymes, such as NOSs (12, 22, 33). In
addition, the responses of a particular gene to low
O2 tension have also been shown to
be dependent on the cell type (5). The mechanisms by which low
O2 levels regulate gene expression
have only recently been investigated.
Cis-acting sequences responsible for
the induction of gene transcription by hypoxia for the erythropoietin
gene have been identified. The
trans-acting factor hypoxia-inducible
factor (HIF)-1 binds to an enhancer located in the 3'-flanking
region of the erythropoietin gene and is required for induction by
hypoxia (21, 27). This DNA binding protein is a heterodimer composed of
HIF-1 and HIF-1
subunits (25). Both subunits are induced by
hypoxia and rapidly decay on return to normoxia (25). HIF-1 DNA binding
activity has been shown to be phosphorylation and redox dependent (7,
26, 27). Functionally important binding sites for HIF-1 (consensus,
5'-RCGTG-3') have been found in a number of genes known to
be regulated by hypoxia, including those encoding vascular endothelial
growth factor (4); the glycolytic enzymes aldolase A,
enolase-1, lactate dehydrogenase A, and phosphoglycerate kinase-1 (2,
3, 20); and heme oxygenase-1 (13). A putative HIF-1 site in the murine
type II NOS gene was also shown to be required for hypoxia-induced
transcription in a macrophage cell line (17), but the role of HIF-1 was
not definitely established. In this study, the regulation of type II
NOS gene expression by hypoxia and the role that HIF-1 plays in this
regulation were examined in pulmonary artery endothelial cells.
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MATERIALS AND METHODS |
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Cell culture. Bovine pulmonary artery endothelial cells were grown in medium 199 supplemented with 10% fetal calf serum and 2.4 µg/ml thymidine and were characterized as previously described (8). Cells were maintained in a humidified 37°C, 5% CO2 incubator and used between passages 7 and 13. For studies involving hypoxic conditions, cells were placed in a modular incubator and purged with 95% N2-5% CO2 for 20 min. The modular incubator was then placed in a 1-2% O2-5% CO2-balance N2 incubator. PO2, PCO2, and pH of the medium were measured in a blood gas analyzer (Corning model 178). Normoxic values were as follows: pH = 7.2 ± 0.1, PCO2 = 39.3 ± 0.6 mmHg, and PO2 = 131.5 ± 0.9 mmHg. Hypoxic values were as follows: pH = 7.2 ± 0.1, PCO2 = 35 ± 1.1 mmHg, and PO2 = 14.9 ± 1.2 mmHg.
Rat protocol. The procedures followed in the care and death of the animals were approved by the Animal Research Committee of the University of Virginia. The protocol for the exposure of rats to hypoxia has been previously described (33). Briefly, male Sprague-Dawley rats (250-300 g) were placed in a Plexiglas chamber maintained at 10% O2 (hypoxic group) or in a chamber open to room air (normoxic group) for 3 wk with a 12:12-h light-dark cycle. Hypoxia was maintained using a Pro:ox model 350 unit (Reming Bioinstruments, Refield, NY), which controlled fractional concentration of O2 in inspired gas by solenoid-controlled infusion of N2 (Roberts Oxygen, Rockville, MD) balanced against an inward leak of air through holes in the chamber. The hypoxic rats were exposed to room air for 10-15 min daily while their cages were changed. CO2, water vapor, and ammonia were removed by pumping the atmosphere of the hypoxia chamber through Bara Lyme (barium hydroxide lime, USP; Chemetron Medical Division, Allied Healthcare Products, St. Louis, MO), Drierite (anhydrous calcium sulfate; Fisher Scientific, Atlanta, GA), and activated carbon (Fisher Scientific).
Constructs.
p1iNOSCAT contains 1,588 base pairs (bp) of the 5'-flanking
region of the murine type II NOS gene and was obtained from Drs. Carl
Nathan and Qiao-wen Xie (31). Constructs containing the HIF-1 mutation
(p209) or the HIF-1 deletion (p220) were obtained from Dr. Giovanni
Melillo (17). The cDNAs for HIF-1 and HIF-1
have been previously
described (25). The construct pBSiNOSprom was generated by inserting
the 1,749-bp Hinc II fragment of
p1iNOSCAT into the Hinc II site
of pBluescript SK (Stratagene, La Jolla, CA).
In situ hybridization.
In situ hybridization was performed on serial sections of
Formalin-fixed paraffin-embedded lung mounted on
2-aminopropyltriethoxysilane-coated slides. The conditions of target
pretreatment hybridization and probe generation have been extensively
characterized (1, 23). Sense and antisense orientation probes specific
for type II NOS mRNA were generated from pBSiNOSprom. For antisense
riboprobe, pBSiNOSprom was digested with
EcoR I and transcribed using T7 RNA
polymerase; for sense riboprobe, pBSiNOSprom was digested with
Xho I and transcribed using T3 RNA
polymerase. The riboprobes were labeled to a specific activity of 1.1 × 108
disintegrations · min1 · µg
1
using tritiated UTP and CTP and were applied to the sections at a fully
saturating concentration of 0.2 µg · ml
1 · kilobase
1,
followed by stringent washing at 60°C in 0.1× SSC (1×
SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH
7.0). The sections were autoradiographed for 4 wk,
photographically developed, and counterstained with hematoxylin and
eosin before microscopic observation using bright-field and dark-field
optics. The aforementioned conditions were established in a series of
preliminary experiments. In addition, RNA preservation of the specimens
was assessed using a 1.8-kilobase probe directed against a ubiquitously
expressed actin mRNA. The in situ hybridization data were analyzed
using both bright-field morphology as well as dark-field optics to
better visualize the full distribution of the silver grains making up
the autoradiographic signal.
Transient transfections. Bovine pulmonary artery endothelial cells were transfected using LipofectAMINE Reagent (GIBCO BRL, Grand Island, NY) as described by the manufacturer. Briefly, bovine pulmonary artery endothelial cells were seeded at 4 × 104 cells/cm2 onto 100-mm dishes and grown until 50-80% confluent. Sixteen microliters of LipofectAMINE reagent and 3 µg of p1iNOSCAT, p220, p209, or backbone vector (pCAT-Basic) and 7 µg of carrier DNA were used per 100-mm dish. The medium was changed after 16 h, and the cultures were placed into hypoxic or nonhypoxic incubators. Cells were harvested 48 h after transfection. All transfections were performed in triplicate, using at least two plasmid preparations.
Chloramphenicol acetyltransferase assay. Chloramphenicol acetyltransferase (CAT) assays were performed by the method of Gorman et al. (6). Cell lysates were incubated with [14C]chloramphenicol (Amersham Life Science, Arlington Heights, IL) for 2 h at 37°C. The mixture was extracted with ethyl acetate and lyophilized, and the residue was resuspended in 20 µl of ethyl acetate and spotted onto thin-layer chromatography (TLC) plates. The plates were run in a TLC chamber containing 5% methanol-95% chloroform. Percent acetylation was determined by excising the acetylated and unacetylated spots from the TLC plates, followed by analysis in a Beckman LS-6500 liquid scintillation counter. CAT activities were normalized for protein, as determined by the method of Lowry et al. (16), using a bovine serum albumin standard curve.
Nuclear extract preparation.
Nuclear extracts were prepared from bovine pulmonary artery endothelial
cells exposed to normoxia or hypoxia for 48 h as previously described
(29). Briefly, cell pellets were washed twice in cold phosphate-buffered saline and once in 4 packed cell volumes of buffer
A [10 mmol/l
tris(hydroxymethyl)aminomethane hydrochloride (Tris · HCl; pH 7.5), 1.5 mmol/l
MgCl2, 10 mmol/l KCl, 2 mmol/l dithiothreitol (DTT), 0.4 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l
Na3VO4,
2 g/l leupeptin, 2 g/l pepstatin, and 2 g/l aprotinin]. Cell
pellets were resuspended in 4 packed cell volumes of
buffer A, incubated on ice for 10 min, and
homogenized with 50 strokes of a dounce homogenizer. The nuclei were
pelleted and then resuspended in 3 packed nuclear volumes of
buffer
C [0.42 mol/l KCl, 20 mmol/l Tris · HCl (pH 7.5), 1.5 mmol/l
MgCl2, 20% glycerol, 2 mmol/l DTT, 0.4 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l
Na3VO4, 2 g/l leupeptin, 2 g/l pepstatin, and 2 g/l aprotinin]. The
resulting mixture was mixed on a rotator for 30 min, centrifuged to
remove nuclear debris, and then dialyzed with one change of buffer for 4 h at 4°C in buffer
D [20 mmol/l
Tris · HCl (pH 7.5), 0.1 mol/l KCl, 0.2 mmol/l EDTA,
and 20% glycerol]. The extracts were aliquoted and stored at
80°C.
Electrophoretic mobility shift assay.
Nuclear extracts (3 µg) prepared from bovine pulmonary artery
endothelial cells were preincubated in binding buffer (10 mmol/l Tris,
50 mmol/l KCl, 50 mmol/l NaCl, 1 mmol/l
MgCl2, 1 mmol/l EDTA, 5 mmol/l
DTT, and 5% glycerol) for 5 min at 4°C. The radiolabeled oligonucleotide probe (1.5 fmol) was added and incubated for 15 min.
The mixture was loaded on a 4% nondenaturing polyacrylamide gel, and
electrophoresis was performed in 0.3 × TBE [1× TBE is 89 mmol/l Tris-borate and 20 mmol/l EDTA (pH 8.0)] at 4°C.
The gel was dried, and autoradiography was performed. When used,
competitor oligonucleotides were added at the start of the 5-min
preincubation period. For experiments in which antisera were used,
nuclear extract was incubated with probe for 10 min before the addition
of antiserum. The mixture was incubated on ice for 20 min before being
loaded onto the gel. Preimmune and immune antisera for HIF-1 and
HIF-1
have been previously described (25).
Isolation of RNA and Northern analysis.
Total RNA was purified from hypoxic and normoxic cells using TRIREAGENT
(Molecular Research Center, Cincinnati, OH) as described by the
manufacturer. Animals were exposed to 3 wk of hypoxia or normoxia, and
poly(A)+ RNA was isolated from
normoxic and hypoxic rat lungs as previously described (12). Aliquots
of RNA [20 µg of poly(A)+
RNA from rat lung samples and 10 µg of total RNA from cultured endothelial cells] were fractionated by glyoxyl-agarose gel
electrophoresis and transferred to Hybond-N+ nylon membrane
(Amersham). cDNA probes for HIF-1, HIF-1
, and
-actin were
labeled with
[
-32P]dCTP using
the RTS RadPrime DNA labeling system (GIBCO BRL). The 24-bp
oligonucleotide for 18S RNA,
5'-ACGGTATCTGATCGTCTTCGAACC-3', was labeled with
[
-32P]dCTP by terminal
deoxynucleotide transferase (GIBCO BRL). Hybridizations were performed
using Rapid-hyb buffer (Amersham) as described by the manufacturer.
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RESULTS |
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Hypoxia induces type II NOS RNA in the endothelium and vascular smooth muscle of the lungs of rats exposed to chronic hypoxia. In situ hybridization was used to determine the cellular location of type II NOS RNA expression in the lungs of rats subjected to 3 wk of chronic hypoxia. Type II NOS RNA was increased in the endothelium and vascular smooth muscle of lungs from rats exposed to chronic hypoxia compared with the nonhypoxic controls (Fig. 1). In addition, increases in type II NOS RNA were detected in the bronchial epithelium and alveolar lining cells.
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Hypoxia induces HIF-1 but not
HIF-1
mRNA expression in the lungs of rats subjected to
chronic hypoxia and in pulmonary artery endothelial cells.
To determine whether expression of mRNAs encoding HIF-1 increased in
response to hypoxia in cells and tissues, Northern blots containing RNA
obtained from the lungs of rats subjected to hypoxia for 3 wk or from
pulmonary artery endothelial cells subjected to hypoxia for 48 h were
probed with HIF-1
and HIF-1
cDNAs (Fig. 2). In addition, Northern blots containing
the RNA obtained from the lungs of rats exposed to hypoxia were probed
with cDNA for
-actin, and the Northern blots containing the RNA from
the bovine pulmonary artery endothelial cells were probed with an
oligonucleotide directed against 18S RNA. The levels of HIF-1
and
HIF-1
RNA present in the rat lung samples or the bovine pulmonary
artery endothelial cells were normalized to
-actin or 18S RNA,
respectively. RNA isolated from both normoxic rat lung and pulmonary
artery endothelial cells cultured under nonhypoxic conditions contained HIF-1
and HIF-1
mRNA. In rat lung, hypoxia increased HIF-1
mRNA levels 2.2-fold (P < 0.0044, Student's t-test,
n = 8), whereas HIF-1
mRNA levels
were not significantly increased. Similar results were observed in the
cultured endothelial cells (Fig.
2B).
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Hypoxia induces a protein that binds to the HIF-1
sequence in the type II NOS promoter.
The 5'-flanking region of the murine type II NOS gene contains a
putative binding site for HIF-1 (17). To determine whether exposure of
pulmonary artery endothelial cells to hypoxia induces proteins that
could bind to this site, electrophoretic mobility shift assays were
performed using nuclear extracts prepared from cells cultured under
hypoxic or nonhypoxic conditions and a 30-bp wild-type (WT)
oligonucleotide containing the putative HIF-1 binding site found in the
5'-flanking region of the murine type II NOS gene (Fig.
3B). Two
constitutively expressed DNA binding activities were present in
extracts made from cells cultured under hypoxic or nonhypoxic
conditions. In addition to these constitutively expressed factors, a
DNA binding activity was specifically induced by hypoxia (Fig.
3A, arrowhead). This DNA binding
activity could be detected after cells were subjected to hypoxia for 4 h (data not shown) and was present for up to 48 h of continuous
hypoxia. The binding of this HIF was specific for the WT
oligonucleotide because incubation with excess unlabeled WT
oligonucleotide effectively competed with the probe for formation of
this complex (Fig. 3A, lanes
9-11). Furthermore, an
oligonucleotide containing a mutation in the HIF-1 site did not
compete (Fig. 3A, lanes 12-14),
demonstrating that the induced protein specifically recognized the
HIF-1 binding site. To determine whether the hypoxia-induced DNA
binding activity contained HIF-1, antisera raised against recombinant
HIF-1 and HIF-1
were utilized (Fig.
4). Both antisera supershifted the hypoxia-induced complex, whereas the respective preimmune sera did not,
demonstrating that the DNA binding activity induced by hypoxia is in
fact HIF-1.
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Hypoxia increases transcriptional activity of the type II NOS promoter in pulmonary artery endothelial cells via HIF-1. To determine whether type II NOS promoter activity was affected by hypoxia, transient transfections were performed using p1iNOSCAT, which contains 1,588 bp of 5'-flanking DNA from the murine type II NOS gene linked to the CAT gene, or pCAT-Basic, the promoterless vector. Hypoxia was found to increase type II NOS promoter activity 2.7-fold in hypoxic compared with the nonhypoxic bovine pulmonary artery endothelial cells (Fig. 5).
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DISCUSSION |
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These studies were designed to investigate the mechanism of hypoxia-induced upregulation of type II NOS gene expression observed in the in vivo model of chronic hypoxia-induced pulmonary hypertension (12). In situ analysis of the lungs of rats exposed to chronic hypoxia indicates that type II NOS RNA is increased in the endothelium as well as in the vascular smooth muscle. The studies presented address the mechanism by which type II NOS is regulated in the vascular endothelium.
One mechanism by which hypoxia may increase type II NOS gene expression
is through the induction of transcription factors such as HIF-1. In the
in vitro studies presented, the 5'-flanking region of the murine
type II NOS gene was shown to contain a DNA sequence that was
functionally essential for hypoxia-induced transcriptional activation
in pulmonary artery endothelial cells. This 9-bp sequence, 5'-CTACGTGCT-3', was identical to the binding site for
HIF-1 identified in the human erythropoietin gene (21). In addition, a
nuclear factor was induced in hypoxic pulmonary artery endothelial
cells that bound to an oligonucleotide containing the putative HIF-1 binding site, and mutation of this site eliminated binding activity. Furthermore, the proteins binding to this site were identified using
antibodies against HIF-1 and HIF-1
. Together, the studies presented here demonstrate that HIF-1 is induced by hypoxia in pulmonary artery endothelial cells and its binding site is required for
hypoxic induction of type II NOS gene expression.
HIF-1 activates transcription of a number of genes in hypoxic cells.
Both HIF-1 mRNA and protein are rapidly induced by hypoxia in a variety
of cell types and rapidly decay on return to nonhypoxic conditions (25,
27, 28). In our in vivo studies, both HIF-1 and HIF-1
mRNAs were
detected in the lungs of rats exposed to normoxia, consistent with
reports that HIF-1 mRNAs are expressed in all organs of humans and
rodents (30). In pulmonary artery endothelial cells, mRNA for both
subunits of HIF-1 was detected under nonhypoxic conditions. HIF-1
protein is present in these cells under nonhypoxic conditions, whereas
both HIF-1
and HIF-1
proteins were induced on exposure to 1%
O2 (A. Y. Yu and G. L. Semenza,
unpublished data). However, HIF-1 DNA binding activity was only
detected in nuclear extracts prepared from cells grown under hypoxia.
These results are consistent with the observations that HIF-1
is
present in excess and DNA binding activity and transcriptional activity
are primarily determined by the steady-state level of the HIF-1
protein (7, 20, 25).
The response of a particular gene to hypoxia is dependent on cell type
(5). Type II NOS gene expression was previously shown to be induced by
hypoxia in a macrophage cell line only when cells were costimulated
with interferon- (17). Our results demonstrate that type II NOS
expression is induced in hypoxic pulmonary artery endothelial cells in
the absence of interferon-
. However, additional studies performed in
our laboratory show that type II NOS in rat aortic smooth muscle cells
does not respond to hypoxia in the same manner as in endothelial cells
(Palmer and Johns, unpublished observations). Yet HIF-1 is present in hypoxic rat aortic smooth muscle cells (13), indicating that other cell
type-specific transcription factors may mediate the response to
hypoxia.
The role HIF-1 plays in the transcriptional regulation of gene
expression in response to hypoxia may be both cell type and gene
specific. For instance, in the human hepatoblastoma cell line Hep 3B,
transcriptional activation mediated by HIF-1 requires the binding of a
second unidentified factor at site 2 of the erythropoietin gene
enhancer (21). In the murine macrophage line ANA-1, the effects of
hypoxia on type II NOS transcription that require the HIF-1 binding
site are augmented by interferon- treatment (17). Regulation of the
lactate dehydrogenase A gene by hypoxia in the human cervical carcinoma
cell line HeLa is augmented by forskolin and is dependent on the HIF-1
binding site and an adenosine 3',5'-cyclic monophosphate
response element (3). So far, the requirement for additional factors
for transcriptional activation of the type II NOS gene in hypoxic
pulmonary artery endothelial cells is not known. Comparison of
sequences around the HIF-1 site present in the 5'-flanking region
of the type II NOS gene and the 3' enhancer of the erythropoietin
gene shows a region of similarity 10 bp downstream from the HIF-1 site.
This 5-bp sequence, 5'-CACTG-3', is similar to site 2, 5'-CACAG-3', of the erythropoietin gene enhancer. Mutations
of the 5'-CACAG-3' sequence eliminated the ability of the
erythropoietin enhancer to activate transcription in response to
hypoxia (21). Thus it is possible that the 5'-CACTG-3' sequence in the NOS gene may also be involved in the hypoxic response.
It has been reported that activating transcription factor (ATF)-1 and adenosine 3',5'-cyclic monophosphate response element binding protein (CREB)-1 constitutively bind to the HIF-1 consensus sequence (11). However, it is unclear whether HIF-1 and ATF-1/CREB-1 bind simultaneously or whether ATF-1/CREB-1 binding is replaced by HIF-1 binding during hypoxia. Basal activity of the full-length type II NOS gene reporter construct was higher compared with the HIF-1 deletion or mutation constructs p220 and p209. This reduction in basal activity with a deletion or mutation of the HIF-1 site has not been reported for other hypoxia-inducible genes. This may indicate the presence of other transcription factors, the binding sites of which are close to or overlap with the HIF-1 consensus site (e.g., ATF-1/CREB-1) and are involved in regulating basal type II NOS gene expression in pulmonary endothelium.
HIF-1 is one transcription factor known to play a role in
O2 regulation of gene expression.
For the murine type II NOS gene, HIF-1 is essential to the hypoxia
response, since mutation or deletion of the HIF-1 binding site
abolishes the hypoxic induction of type II NOS. The 5'-flanking
region of the rat type II NOS gene is 85% homologous to that of the
murine sequence (19). Furthermore, the HIF-1 consensus site is intact,
suggesting that the rat type II NOS gene is regulated in a similar
manner. There is no known HIF-1 binding site contained within the
published sequence for the human type II NOS gene, and it is unknown
whether this transcription factor plays a role. It is possible that
this site is present upstream from the known published sequence and may
be involved in the hypoxic regulation of the human type II NOS gene.
Alternately, the human type II NOS gene may not be regulated by hypoxia
via HIF-1. Other factors such as activator protein-1 and nuclear
factor-B have been implicated in the regulation of gene expression by O2 tension. The
putative binding sites for these transcription factors are present in
the human type II NOS gene and may be involved in the regulation of
this gene by low O2 tension.
The regulation of NOS gene expression in response to low O2 tension may be important in several physiological and pathological conditions in which O2 availability is compromised. In chronic hypoxia-induced models of pulmonary hypertension, increases in NOS expression have been shown to correlate with the development of the remodeling process (32). This increase in NOS has been proposed both to modulate and to stimulate vascular remodeling (32). The mechanism by which NOS is increased in chronic hypoxia-induced models of pulmonary hypertension is unknown. Our studies indicate that hypoxia induces HIF-1 in this lung model and that the increased expression of HIF-1 results in the transcriptional activation of type II NOS gene expression. This mechanism of hypoxic upregulation of NOS may also be physiologically relevant in the transition of the fetal circulation to that of the newborn, in which a marked upregulation of NOS in the hypoxic fetal lung has been demonstrated. It has been proposed that this upregulated NOS is inactive until O2 substrate becomes available with the first breath, allowing for NO production and subsequent pulmonary vascular and bronchial smooth muscle relaxation (34). Changes in NOS expression with low O2 tension have also been implicated in the pathophysiology of ischemia-reperfusion injury, adult respiratory distress syndrome, and hypoxic brain injury. Thus understanding the mechanism of hypoxic-induced upregulation of type II NOS is therefore of critical importance and may lead to novel therapeutic interventions.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. C. Nathan and Q.-W. Xie for providing the plasmid p1iNOSCAT and Dr. G. Melillo for the plasmids p220 and p209. We also thank A. Tichotsky and N. Zhou for technical assistance. We thank A. Y. Yu for sharing unpublished data.
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FOOTNOTES |
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This work was supported by a Research and Development Award from the University of Virginia and a Virginia Thoracic Society Grant (to L. A. Palmer) and National Institutes of Health Grants RO1-HL-39706 and RO1-GM-49111 (to R. A. Johns). G. L. Semenza is an Established Investigator of the American Heart Association and was supported in part by grants from the American Heart Association and the National Heart, Lung, and Blood Institute (RO1-HL-55338).
Address for reprint requests: L. A. Palmer, Dept. of Anesthesiology, Univ. of Virginia Health Sciences Center, PO Box 10010, Charlottesville, VA 22906-0010.
Received 24 June 1997; accepted in final form 10 November 1997.
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