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-1α but not in HIF-1β 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-1α and HIF-1β. 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
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. Thetrans-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-1α and HIF-1β 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.
MATERIALS AND METHODS
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 betweenpassages 7 and13. 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. Po 2, Pco 2, 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, Pco 2 = 39.3 ± 0.6 mmHg, and Po 2 = 131.5 ± 0.9 mmHg. Hypoxic values were as follows: pH = 7.2 ± 0.1, Pco 2 = 35 ± 1.1 mmHg, and Po 2 = 14.9 ± 1.2 mmHg.
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).
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-1α and HIF-1β 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 withEcoR I and transcribed using T7 RNA polymerase; for sense riboprobe, pBSiNOSprom was digested withXho I and transcribed using T3 RNA polymerase. The riboprobes were labeled to a specific activity of 1.1 × 108disintegrations ⋅ min−1 ⋅ μg−1using 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.
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 × 104cells/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 ofbuffer 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 ofbuffer 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 ofbuffer 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-1α and HIF-1β 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-1α, HIF-1β, and β-actin were labeled with [α-32P]dCTP using the RTS RadPrime DNA labeling system (GIBCO BRL). The 24-bp oligonucleotide for 18S RNA, 5′-ACGGTATCTGATCGTCTTCGAACC-3′, was labeled with [α-32P]dCTP by terminal deoxynucleotide transferase (GIBCO BRL). Hybridizations were performed using Rapid-hyb buffer (Amersham) as described by the manufacturer.
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.
Hypoxia induces HIF-1α but not HIF-1β 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-1α and HIF-1β cDNAs (Fig.2). In addition, Northern blots containing the RNA obtained from the lungs of rats exposed to hypoxia were probed with cDNA for β-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-1α and HIF-1β RNA present in the rat lung samples or the bovine pulmonary artery endothelial cells were normalized to β-actin or 18S RNA, respectively. RNA isolated from both normoxic rat lung and pulmonary artery endothelial cells cultured under nonhypoxic conditions contained HIF-1α and HIF-1β mRNA. In rat lung, hypoxia increased HIF-1α mRNA levels 2.2-fold (P < 0.0044, Student’s t-test,n = 8), whereas HIF-1β mRNA levels were not significantly increased. Similar results were observed in the cultured endothelial cells (Fig.2 B).
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.3 B). 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.3 A, 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. 3 A,lanes 9–11). Furthermore, an oligonucleotide containing a mutation in the HIF-1 site did not compete (Fig. 3 A, 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-1α and HIF-1β 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.
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).
To determine whether the HIF-1 binding site is functionally required for hypoxic induction of the murine type II NOS gene, transient transfections were performed using constructs in which the HIF-1 binding site was mutated (p209) or deleted (p220) (Fig. 5). Removal of the HIF-1 binding site had two effects. First, the basal activity of the mutated or deleted HIF-1 construct was less than that of the full-length construct, suggesting that the deletion or mutation disrupted binding of a factor involved in determining basal promoter activity. Second, both the mutation as well as the deletion of the HIF-1 binding site eliminated the increase in promoter activity seen in response to hypoxia, confirming that the HIF-1 binding site is necessary for transcriptional activation of the type II NOS gene in hypoxic pulmonary artery endothelial cells.
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-1α and HIF-1β. 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-1α and HIF-1β 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-1β protein is present in these cells under nonhypoxic conditions, whereas both HIF-1α and HIF-1β 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-1β is present in excess and DNA binding activity and transcriptional activity are primarily determined by the steady-state level of the HIF-1α 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-γ (17). Our results demonstrate that type II NOS expression is induced in hypoxic pulmonary artery endothelial cells in the absence of interferon-γ. 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-γ 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-κB 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 O2tension 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.
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.
Address for reprint requests: L. A. Palmer, Dept. of Anesthesiology, Univ. of Virginia Health Sciences Center, PO Box 10010, Charlottesville, VA 22906-0010.
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).
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