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Am. J. Respir. Cell Mol. Biol., Volume 26, Number 1, January 2002 22-30

Modulation of Inducible Nitric Oxide Synthase by Hypoxia in Pulmonary Artery Endothelial Cells

Javier J. Zulueta, Raneeta Sawhney, Usamah Kayyali, Michael Fogel, Cameron Donaldson, Hailu Huang, Joseph J. Lanzillo, and Paul M. Hassoun

Pulmonary and Critical Care Division, Department of Medicine/Tupper Research Institute, New England Medical Center, Boston, Massachusetts; and Tufts University School of Medicine, Boston, Massachusetts


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The effects of hypoxia on the regulation of inducible nitric oxide synthase (NOS) 2 expression were examined in cultured rat pulmonary microvascular endothelial cells (EC). EC did not express NOS 2 mRNA or protein when exposed to normoxia or hypoxia unless they were pretreated with interleukin (IL)-1beta and/or tumor necrosis factor (TNF)-alpha for 24 h. Induction of NOS 2 by IL-1beta +TNF-alpha was significantly attenuated by concomitant exposure of EC to hypoxia or treatment of EC with antioxidants such as tiron, diphenyliodonium, and catalase, suggesting that NOS 2 expression is dependent on the production of reactive oxygen species. Degradation of Ikappa B and activation of NF-kappa B, which were both induced by treatment of EC with cytokines, were not altered when the cells were exposed to hypoxia, suggesting that the modulation of NOS 2 expression by hypoxia is unrelated to NF-kappa B activation. Following stimulation with IL-1beta +TNF-alpha for 24 h, incubation of EC in normoxia resulted in a progressive decline in NOS 2 expression and a calculated half-life of approximately 6 h for NOS 2 mRNA. Hypoxia significantly prolonged the half-life of NOS 2 mRNA (17 h, P < 0.05 versus normoxic EC). The half-life of NOS 2 mRNA was also prolonged by actinomycin D treatment (19.5 and 29.5 h for normoxic and hypoxic EC, respectively), suggesting that transcription of an RNA destabilizing factor or RNAse contributes to NOS 2 mRNA degradation. In EC transiently transfected with the rat NOS 2 promoter, hypoxia and the combination of IL-1beta +TNF-alpha independently increased promoter activity 2.2- and 3-fold, respectively. As opposed to the attenuating effect that hypoxia had on IL-1beta +TNF-alpha - dependent induction of NOS 2 gene expression, the concomitant treatment with IL-1beta +TNF-alpha and hypoxia synergistically increased NOS 2 promoter activity 17.6-fold. Taken together, these results suggest that hypoxia alone does not induce NOS 2 expression in cultured pulmonary microvascular EC, but may modulate cytokine induction of this enzyme at pretranscriptional, transcriptional, and posttranscriptional levels.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Nitric oxide (NO) is synthesized by a family of enzymes known as the nitric oxide synthases (NOS) and is involved in various physiologic functions, such as regulation of vascular tone, inflammation, and inhibition of platelet aggregation and smooth muscle cell proliferation (1). Two isoforms of NOS, NOS 1 and NOS 3, are constitutively expressed in neuronal and endothelial cells, respectively. The inducible isoform of NOS (i.e., iNOS or NOS 2) is present in many cell types but has been examined mainly in inflammatory cells such as the macrophage. As opposed to NOS 1 and NOS 3, expression of NOS 2 is induced by inflammatory cytokines, and its activity is not calcium dependent. The primary role of NOS 2 in macrophages appears to be as a defense mechanism against microorganisms (1). Several groups have reported expression of NOS 2 in cells other than macrophages, such as vascular smooth muscle cells (2), glomerular mesangial cells (3), microglia (4), and endothelial cells (EC) (5). However, the most important physiologic source of NO in the latter cell type is believed to be the endothelial isoform of NOS (eNOS or NOS 3), which is responsible for regulation of vascular tone (1). NO may also be important in the pathogenesis of some disease processes. For example, the severe hypotension seen in septic shock is believed to be due to an overproduction of NO from NOS 2 expressed in vascular smooth muscle cells in response to circulating cytokines (6). Whether NOS 2 expressed in EC is an additional source of NO in sepsis is unknown.

The present study was conducted to examine the expression of NOS 2 in rat pulmonary microvascular endothelial cells and to determine the potential regulation of this gene by hypoxia. The results indicate that hypoxia alone does not induce NOS 2 gene expression in cultured pulmonary microvascular EC, but may modulate cytokine induction of this gene at various pretranscriptional, transcriptional, and posttranscriptional levels.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reagents

RPMI 1640, penicillin G potassium, streptomycin, amphotericin B, Moloney murine leukemia virus reverse transcriptase (MMLV-RT), RNase inhibitor, and Taq polymerase were obtained from Gibco (Grand Island, NY). 2,3-diaminonaphtalene (DAN), Nomega -nitro-L-arginine methyl esther (L-NAME), ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), pyronin Y, and dithiothreitol were from Sigma (St. Louis, MO). RNAzol B was purchased from Tel-Test (Friensworth, TX). Oligo-d(T)12-18 and pSL1190 were obtained from Pharmacia (Piscataway, NJ). Tumor necrosis factor-alpha (TNF-alpha ) and interleukin (IL)-1beta were obtained from Genzyme (Cambridge, MA). NuSieve and SeaKem were obtained from FMC Bioproducts (Rockland, ME). Goat anti-rabbit IgG-alkaline phosphatase, nitro-block, and CSPD were purchased from Tropix (Bedford, MA).

Isolation and Culture of Cells

Rat pulmonary microvascular EC were a gift from Dr. Una Ryan (Avant Immunotherapeutics, Needham, MA). These cells have been identified as EC by their cobblestone morphology under light and electron microscopy and by the presence of factor VIII antigen (7). EC, which were passaged up to ten times and cultured as previously described (8), maintained their phenotype and endo-thelial characteristics. Culture medium, which consisted of RPMI with 10% fetal calf serum, was changed every other day. Cell counts were monitored routinely by Coulter counter for each control and experimental group. Cell integrity was assessed by morphological examination of the cells under phase-contrast microscopy and trypan blue exclusion as previously described (8, 9).

Exposure of Cells to Oxygen

Cells were placed in humidified airtight incubation chambers (Billups-Rothenberg, Del Mar, CA) and gassed with the desired concentration of O2 (3 or 20% O2), 5% CO2, and balance N2. The chambers were maintained in a New Brunswick incubator (New Brunswick Scientific Co., Inc., New Brunswick, NJ) for the duration of exposure. The percentages of O2 in the chambers were routinely checked using an oxygen analyzer (LB-2; Beckman, Fullerton, CA) and were consistently within 2% of the desired tension. The percentages of CO2 were also monitored (OM-11; Beckman) and were consistently around 4-6%.

Fluorometric Measurement of Nitrite/Nitrate

The cellular release of nitrite in culture medium was determined using a method described by Misko and colleagues (10), with minor modifications (11). This method is based on the conversion, in the presence of nitrite and in acidic condition, of nonfluorescentDAN to the fluorescent compound 1-(H)-naphtotriazole (NT). Briefly, during exposure of EC to the experimental condition (cytokines and/or O2 tension), the cells were incubated in fresh phenol red-free medium devoid of fetal bovine serum, with or without the NOS inhibitor Nomega -nitro-L-arginine methyl esther (L-NAME, 300 µM). At the end of incubation, 850 µl of medium was removed, treated with nitrate reductase to convert nitrate into nitrite, and then mixed with 100 µl DAN (0.05 mg/ml in 0.62 M HCl). The mixture was protected from light and incubated for 10 min at 20°C, after which the reaction was terminated by adding 50 µl of 2.8 N NaOH. Fluorescence of NT was measured using excitation and emission wavelengths of 365 and 450 nm, respectively. NO production is estimated as the L-NAME- inhibitable portion of the total nitrite production of the cells.

Quantitative Polymerase Chain Reaction for Rat NOS 2

RNA isolation and reverse transcription.Total cell RNA was obtained from cultured rat pulmonary microvascular EC using RNAzol B according to the manufacturer's instructions. RNA was serially diluted with diethyl-pyrocarbonate treated water containing 1 unit per µl RNase inhibitor and 3 mM dithiothreitol. RNA was primed with 660 pmol oligo-d(T)12-18 and reverse transcribed by MMLV-RT in a total volume of 30 µl containing 460 µM of each deoxynucleoside triphosphate for 1 h at 37°C as described previously (12). A control was prepared by subjecting RNA to the reverse transcription procedure without MMLV-RT. After reverse transcription, samples were heated at 95°C for 5 min to denature the MMLV-RT and then stored at -40°C until polymerase chain reaction (PCR).

Rat NOS 2 internal standard preparation.A region of plasmid pSL 1190 was amplified and simultaneously extended by PCR with primers such that the fragment had sequences at both ends that were fully homologous with sequences from rat NOS 2 cDNA (13). The resulting fragment was purified on Geneclean (Bio 101, La Jolla, CA) and served as the internal standard (IS) for rat NOS 2. When a mixture of this IS and rat cDNA is subjected to PCR with primers 5'-CTGGGT CAAAGAGGCT-3' (forward) and 5'-ACCCAAA CACCAAG GTCATGC-3' (reverse), a 478-bp fragment is generated from the IS along with a 611-bp fragment of rat NOS 2 cDNA.

PCR. A master PCR reagent mixture was prepared such that each 25 µl contained 0.4 µM of each primer and 52 µM deoxynucleoside triphosphate in 15 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin (pH 8.5) buffer. A constant amount of cDNA was added to the master mix of PCR primers and buffer, and 25 µl aliquots were placed in several tubes. Subsequently, variable amounts of IS were added to each 25-µl PCR reagent mix. Samples were overlaid with light mineral oil and held at 80°C for hot-start PCR. Taq polymerase (1.25 U) in 2 µl water was added to each sample by underlaying, and PCR was performed for 35 cycles with 1 min, 94°C denaturation; 1 min, 60°C annealing; and 1 min, 72°C extension. The last cycle extension was for 10 min. Primer pairs presumably span introns because genomic DNA was not amplified. Controls prepared from RNA without MMLV-RT were negative.

Quantification. After PCR, 10-µl sample aliquots were electrophoresed on 2% NuSieve/1% SeaKem agarose in Tris/acetic acid/ EDTA buffer for 2 h at 80 V, stained with ethidium bromide, and photographed. Densitometry was done with a Millipore Densitometer with Visage v4.6p software (Millipore, Bedford, MA). Because the NOS 2 target is larger than the mutant, target optical density is multiplied by a correction factor of 0.78 to normalize the value before calculating the molar ratio of 473 bp mutant to 611 bp target. For absolute quantification, a fixed amount of cDNA was added to the master mix prior to aliquoting. Subsequently, the IS is serially diluted and added to each sample. When PCR is performed with a fixed amount of cDNA and serial dilutions of IS per sample, optical densities for NOS 2 and IS are plotted versus copies of IS to assess amplification as a function of IS input. The equivalence point, where target and IS copies are equal, is determined from a plot of IS/NOS 2 ratio versus copies of IS. This value was divided by 0.4 to compensate for the less than 100% reverse transcription efficiency, which was presumed to be 40% as determined from the manufacturer's literature and elsewhere (14). Also, the value was multiplied by 2 to compensate for differential amplification during the first PCR cycle where the single-stranded cDNA target is rendered double stranded, whereas the double-stranded IS is amplified geometrically. Corrected values are reported as copies of NOS 2 mRNA per µg of total cellular RNA. To control for potential loading differences, a second quantitative PCR was performed using tubulin and a tubulin internal standard for each sample. Final results were expressed as copies of NOS 2 mRNA per 107 copies of tubulin mRNA.

For some experiments, a semiquantitative PCR method was used. PCR was conducted as described previously, with the exception of mixing the samples of cDNA with a fixed amount of internal standard. The same process was followed for tubulin, which was used for control of input. The ratio of NOS 2 over IS was used to compare changes in steady-state levels of NOS 2 mRNA between different groups.

Western Blot Analysis

Sample preparation.Cell lysates were obtained by incubating confluent endothelial cells in 100-mm Petri dishes with 1 ml of boiling cell lysis buffer containing 1% sodium dodecyl sulfate (SDS), 1.0 mM sodium vanadate, and 10 mM Tris (pH 7.4). Lysates were then boiled for 5 min and sonicated for 5 s. Insoluble material was removed by centrifugation (14,000 × g for 2 min), and the supernatant was used for analysis. Protein measurements of the supernatant (samples) were made using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Polyacrylamide gel electrophoresis. Samples corresponding to equal amounts of total protein (~ 300 µg) were diluted (1:5 by volume of buffer) in electrophoresis sample buffer (0.2 M EDTA, 40 mM dithiothreitol, 6% SDS, 0.06 mg/ml pyronin [pH 6.8]) and boiled for 3-5 min to denature protein. Samples were then subjected to SDS polyacrylamide gel electrophoresis on a 6% slab gel in a model SE-600 apparatus (Hoefer Scientific, San Francisco, CA) (15).

Immunoblotting. After electrophoresis, gel proteins were electrophoretically transferred to PVDF membrane (Tropifluor; Tropix, Bedford, MA or Immobilon-P; Millipore, Bedford, MA) at 0.5 amperes for 2 1/2 hours in transfer buffer (25 mM Tris, 190 mM glycine, 20% MeOH) (16). The membrane was then placed into blocking buffer containing 5% nonfat dry milk in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 for 1 h at ambient temperature. A 1/1,000 and 1/2,500 dilution of polyclonal Ikappa Balpha and NOS 2 antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA), respectively, in blocking buffer was incubated with the membrane overnight at 4°C with gentle agitation. After washing, the membrane was exposed to the enzyme conjugate goat anti-rabbit IgG- alkaline phosphatase, diluted in blocking buffer for 90 min at ambient temperature. Following washing, detection with the alkaline phosphatase substrate CSPD and film exposure were performed.

Transient Transfections and Luciferase Assay

The 637-bp fragment of the rat NOS 2 gene, which includes 497 bp of the 5'-flanking region as well as 139 of exon 1, inserted into pGL3/ basic (Promega, Madison, WI), upstream of the promoterless luciferase gene (pINOSGEN2) was kindly provided by K-F. Beck (Klinicum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany) (17). Preliminary experiments of transient transfections of rat pulmonary microvascular EC with lipofectamine (Lipofectamin, Gibco) have shown adequate transfection efficiency (70% as determined by a galactosidase assay) of this cell type. The cells are split 16 h before transfection. Plasmids pINOSGEN2 (1 µg) and CMV promoter-driven Renilla luciferase reporter gene (concentration of 1:40 with construct) were cotransfected with lipofectin (2-10 µl/35 mm Petri dish) according to the manufacturer's instructions. Fresh medium was added 4 h after transfection, and the cells were allowed to recover from the procedure for 24 to 48 h before being subjected to the experimental protocol. A dual luciferase assay (Promega Luciferase System Kit; Promega, Madison, WI) was performed at the end of the procedure according to the manufacturer's protocol.

Nuclear Extract Preparation

EC were grown to confluency in 60-mm Petri dishes in RPMI, 10% fetal bovine serum in the presence or absence of interleukin (IL)-1beta (1 ng/ml), or IL-1beta (1 ng/ml)+TNF-alpha (20 ng/ml). The cells were exposed to normoxia (20% O2) or hypoxia (3% O2) for 4 h and then collected in 1 ml phosphate buffered saline (pH 7.4) and centrifuged for 10 s. The cell pellet was resuspended in 0.4 ml 10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 10 mM AEBSF, 8 µM aprotinin, 0.5 mM bestatin, 0.15 mM E-64, 0.2 mM leupeptin, and 0.1 mM pepstatin A and kept on ice for 10 min. The samples were vortexed and centrifuged for 10 s, and nuclear pellets were resuspended in 50 µl 20 mM Hepes-KOH (pH 7.9), 20% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 10 mM AEBSF, 8 µM aprotinin, 0.5 mM bestatin, 0.15 mM E-64, 0.2 mM leupeptin, and 0.1 mM pepstatin A, and incubated on ice for 1 h. Finally, samples were centrifuged for 2 min at 13,000 × g, and supernatants were stored in 10-µl aliquots at -80°C.

Electrophoretic Mobility Shift Assay

Equal amounts of nuclear proteins from different samples were incubated with a commercial oligonucleotide probe for a NF-kappa B p65 binding site (Gelshift Plus; Geneka Biotechnology, Montreal, PQ, Canada). The electrophoretic mobility shift assay was performed, with the specific and the mutant oligonucleotides, according to the manufacturer's recommendations. The oligonucleotides were radiolabeled by incubation with [gamma -32P] ATP and T4 polynucleotide kinase as previously described (18). The 32P labeled probe was then incubated with nuclear extract proteins (0.2 mg/ml) with or without 10-fold excess unlabeled probe to test for specificity of binding as described (18). After incubation for 30 min at room temperature, samples (5 µg protein/well) were run on a 5% TBE polyacrylamide gel (12.5 V/cm). The gel was then dried and used for autoradiography or exposed on a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) for quantitation.

Statistical Analysis

All experiments were repeated at least three times with adequate sample numbers for each experimental and control condition (n = >=  3). Values are shown as means ± SD. Unpaired Student's t tests were used for experiments in which two groups were being compared. For all studies involving more than two groups, Fisher's multiple comparison test (Statview 512+; Brainpower, Calabasa, CA) was used to determine significant changes occurring between and within control cells and counterparts exposed to experimental conditions. Significance in all cases was assumed at P < 0.05.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cytokine-Dependent Induction of NOS 2 Expression and Activity in EC

Unstimulated rat pulmonary microvascular EC exposed to normoxia did not express NOS 2 mRNA or protein as determined by PCR and Western blot analysis, respectively (Figures 1A and 1B). However, treatment of these cells for 24 h with IL-1beta (1 ng/ml), TNF-alpha (20 ng/ml), or a combination of these cytokines, resulted in the induction of NOS 2 mRNA (Figure 1A). Using semiquantitative PCR as described in MATERIALS AND METHODS, NOS 2 mRNA expression was greatest after stimulation with the combination of IL-1beta and TNF-alpha (Figure 1A). This combination was therefore used in all subsequent experiments. As shown in Figure 1B, NOS 2 protein expression was also strongly induced by the combination of IL-1beta and TNF-alpha .


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  		Figure 1.   Effect of cytokines on NOS 2 mRNA and protein expression. (A) EC were incubated for 24 h in the absence or presence of IL-1beta , TNF-alpha , or TNF-alpha +IL-1beta . The top panel is a gel electrophoresis of NOS 2 mRNA and NOS 2 IS obtained by semiquantitative RT-PCR (representative of four experiments). The middle panel represents semiquantitative RT-PCR determination of tubulin mRNA, which is used as control for input of cDNA into the PCR mixture. The bottom panel is a bar graph representation of the ratio of NOS 2 mRNA/IS. Results demonstrate maximal induction of NOS 2 mRNA by TNF-alpha +IL-1beta . (B) EC were incubated for 24 h in the absence (lane 1) or presence (lane 2) of TNF-alpha +IL-1beta in the cell media. Following incubation, cells were lysed, and Western blot analysis using polyclonal anti-NOS 2 antibody was performed.

The effect of cytokine stimulation on EC NOS 2 enzymatic activity was evaluated by measuring NO generation, as determined by the L-NAME inhibitable portion of the nitrite/nitrate accumulation in the extracellular media, during a 24-h treatment with the combination of IL-1beta (1 ng/ml) and TNF-alpha (20 ng/ml). As compared with unstimulated EC from which NO release was undetectable, stimulation of EC with the cytokine combination for 24 h resulted in a significant increase in NO release into the cell media (Figure 2, control bar).


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  		Figure 2.   Nitric oxide generation by EC in response to cytokine stimulation and hypoxia. NO generation was measured as the L-NAME-inhibitable portion of the nitrite/nitrate accumulation in the extracellular media. EC were incubated in the absence or presence of the combination of TNF-alpha +IL-1beta for 24 h. As compared with untreated EC, which did not generate any detectable NO, treatment of EC with TNF-alpha +IL-1beta for 24 h resulted in significant NO release (100% control). Following removal of the cytokines from the culture media after 24 h of stimulation, cells were incubated in cytokine-free media and exposed to normoxia (20% O2) or hypoxia (3% O2) for an additional 24 h. The release of NO from EC exposed to an additional 24 h of normoxia remained unchanged (P > 0.5 versus control) but was significantly inhibited in EC exposed to hypoxia (*P < 0.001).

Lack of Induction of NOS 2 Gene Expression by Hypoxia Alone

Hypoxia alone has been shown to activate the murine NOS 2 promoter (19) and to upregulate expression of the enzyme in lung tissue in vivo (20). However, except for studies involving transfection of the NOS 2 promoter (20), the effect of hypoxia on NOS 2 gene expression in isolated EC has never been examined. To evaluate whether acute hypoxia alone induces the expression of NOS 2 in isolated cells, confluent EC were exposed to hypoxia (3% O2) or normoxia (20% O2) for 6-24 h. Hypoxia did not induce the expression of EC NOS 2 at either mRNA or protein levels as determined by RT-PCR and Western blot analysis, respectively (data not shown). To evaluate whether acute hypoxia modulates the induction of EC NOS 2 by IL-1beta and TNF-alpha , confluent EC were stimulated with the cytokine combination for 24 h and simultaneously exposed to either hypoxia (3% O2) or normoxia (20% O2). As compared with EC exposed to normoxia, exposure of EC to hypoxia resulted in a significant attenuation of the induction of NOS 2 mRNA (Figure 3) and protein (Figure 4, C/N and C/H for normoxia and hypoxia, respectively) by the cytokine combination.


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  		Figure 3.   Effect of hypoxia on the induction of NOS 2 mRNA expression by cytokines. EC were stimulated with TNF-alpha +IL-1beta and concomitantly exposed to either normoxia (20% O2) or hypoxia (3% O2) for 24 h. Exposure to hypoxia resulted in the attenuation of the cytokine-dependent induction of NOS 2 mRNA expression (representative of five experiments) as determined by quantitative RT-PCR.


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  		Figure 4.   Modulation by hypoxia of NOS 2 protein expression. As determined by Western blot analysis (upper panel), concomitant exposure of EC to hypoxia (3% O2) and TNF-alpha  + IL-1beta for 24 h (C/H) resulted in a significant attenuation of the induction of NOS 2 protein as compared with EC treated with TNF-alpha  + IL-1beta but maintained in normoxia (C/N). Following cytokine induction, NOS 2 protein expression significantly declined when cells were subsequently exposed to normoxia (Cright-arrow N) for an additional 24 h in cytokine-free media. The decline in NOS 2 protein levels was abolished when cytokine-stimulated EC were maintained in hypoxia (Cright-arrow H). The bottom graph represents a densitometric analysis of the bands (n = 3 for each group). *P < 0.05 versus C/N; **P < 0.01 versus Cright-arrow N.

Modulation of Cytokine-Induced NOS 2 Expression by Hypoxia

Effect of hypoxia on NOS 2 mRNA. To assess the effects of hypoxia on preexisting NOS 2 mRNA and protein, EC were first treated with IL-1beta and TNF-alpha for 24 h to obtain maximal expression of NOS 2. The cells were then exposed to normoxia or hypoxia for an additional 6-24 h. Following stimulation with IL-1beta +TNF-alpha , incubation of EC in cytokine-free media and normoxia resulted in a progressive decay of NOS 2 mRNA (Figure 5A) with a calculated half-life for NOS 2 mRNA of ~ 6.2 h (95% CI of 5-8 h). This decay in NOS 2 mRNA was significantly prolonged upon exposure of the cells to hypoxia (Figure 5A), with a calculated mRNA half-life of 17 h (95% CI of 11-35 h, P < 0.05 versus NOS 2 mRNA half-life from normoxic cells). To further explore NOS 2 mRNA decay in normoxia and hypoxia, EC were treated as above with IL-1beta and TNF-alpha for 24 h, then washed and exposed to normoxia or hypoxia for an additional 6-24 h in the presence of actinomycin D (1 µg/ml). As shown in Figure 5B, actinomycin treatment resulted in significant stabilization of NOS 2 mRNA rather than the expected increased decay with such treatment. The calculated half-lives for NOS 2 mRNA were 19.5 and 29.5 h, for cells exposed to normoxia and hypoxia, respectively (Figure 5B).


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  		Figure 5.   NOS 2 mRNA decay obtained from cytokine-stimulated EC exposed subsequently to normoxia or hypoxia for 24 h. EC were treated with TNF-alpha +IL-1beta for 24 h and then washed and incubated in cytokine-free media in normoxia or hypoxia for an additional 24 h in the absence (A) or presence (B) of actinomycin D. NOS 2 mRNA levels were assessed at 0, 6, 12, and 24 h. The single-phase exponential decay regression lines are derived from 10 values for each exposure group. The solid line and open circles represent normoxia, and the dashed line and closed diamonds represent hypoxia. Regression curves were obtained from the exponential decay of mRNA using the formula y = ae-bx, where a is the NOS 2 mRNA value at time 0 (100%) and b the decay rate constant for each condition. NOS 2 mRNA half-life for each curve was calculated as t1/2 = [lna - ln50]/b.

Effect of hypoxia on NOS 2 protein and enzymatic activity. As a consequence of increased mRNA stability, NOS 2 mRNA level from EC exposed to hypoxia was 2.6-fold higher than that of EC maintained in normoxia at 24 h of oxygen exposure (Figure 6; P < 0.01). Consistent with this finding, NOS 2 protein level was greater after 24 h of exposure in cells exposed to hypoxia compared with normoxia (Figure 4; Cright-arrow N and Cright-arrow H for normoxia and hypoxia, respectively).


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  		Figure 6.   Effect of hypoxia on NOS 2 mRNA expression following cytokine induction. EC were treated with TNF-alpha +IL-1beta for 24 h, after which the cells were washed and incubated in cytokine-free media in normoxia or hypoxia for an additional 24 h. NOS 2 mRNA levels declined in both exposure groups compared with levels before oxygen exposure (not shown). However, at the end of exposure, NOS 2 mRNA levels in EC exposed to hypoxia were 2.6-fold that of cells maintained in normoxia (mean of five experiments; P < 0.01).

Cellular NOS 2 enzymatic activity was assessed by the L-NAME inhibitable portion of the total nitrite release into the cell media, as explained in MATERIALS AND METHODS. As shown in Figure 2, there was no difference in enzymatic activity in EC exposed to normoxia for 24 h following the initial cytokine stimulation compared with control stimulated EC (125 ± 19% of control, P = 0.32). However, exposure to hypoxia reduced NO release to 20 ± 11% of control (P = 0.0002 versus control release; Figure 3). Therefore, despite an increase in NOS 2 mRNA and protein levels (Figures 4 and 5), NOS 2 activity of EC maintained in hypoxia was significantly reduced, a finding consistent with the requirement of oxygen as a cofactor for NOS 2 activity. However, brief reoxygenation of these hypoxic cells for 4 h resulted in a significant increase in NO release as compared with their counterparts maintained in normoxia (170 ± 8% versus 100 ± 32%, respectively, P = 0.0004; Figure 7), consistent with the higher content of NOS 2 mRNA and protein in these cells.


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  		Figure 7.   NO release from cytokine-stimulated EC exposed to normoxia or hypoxia. EC previously stimulated with TNF-alpha  + IL-1beta for 24 h were incubated in cytokine-free media and exposed to normoxia (control) or hypoxia (H/R) for an additional 24 h. Following exposure, EC from both groups were exposed to normoxia (reoxygenation) for an additional 4 h, during which the generation of NO was measured as the L-NAME-inhibitable accumulation of nitrite/nitrate in the extracellular media. Reoxygenation of EC previously exposed to hypoxia resulted in a significant increase in NO release as compared with EC previously exposed to normoxia.

Taken together, these studies indicate that (i) hypoxia alone does not stimulate NOS 2 expression, (ii) hypoxia and actinomycin D treatment stabilize preformed NOS 2 mRNA and protein, (iii) hypoxia decreases NOS 2 activity, and (iv) reoxygenation of previously hypoxic EC upregulates NOS 2 activity in cytokine-stimulated cells.

Effect of Antioxidants on the Cytokine-Dependent Induction of EC NOS 2 mRNA

We speculated that downregulation of NOS 2 gene expression in cytokine-stimulated EC concomitantly exposed to hypoxia could be related to decreased production of reactive oxygen species (ROS) by EC (21, 22). Therefore, the possibility that the induction of NOS 2 by IL-1beta and TNF-alpha might be dependent on the generation of ROS was first tested. EC were treated with the combination of IL-1beta and TNF-alpha for 24 h in the absence or presence of several antioxidants. Treatment included tiron (1 mM); a scavenger of superoxide, allopurinol (100 µM), which inhibits the superoxide-producing enzyme xanthine oxidase; catalase (3,000 U/ml), which detoxifies hydrogen peroxide; or diphenyliodonium (1 µM), a nonspecific inhibitor of flavoprotein enzymes such as the NAD(P)H oxidase enzyme. As shown in Figure 8, all antioxidants, with the exception of allopurinol, attenuated the induction of NOS 2 mRNA by the cytokine combination. These experiments support the hypothesis that cytokine induction of NOS 2 occurs through ROS.


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  		Figure 8.   Effects of antioxidants on the induction of NOS 2 mRNA by IL-1beta and TNF-alpha . EC were treated for 24 h with TNF-alpha  + IL-1beta alone (control) or in combination with tiron (1 mM), allopurinol (100 µM), catalase (3,000 U/ml), or DPI (10 µM). NOS 2 mRNA was determined by semiquantitative RT-PCR (representative of three experiments). Except for allopurinol, all other antioxidant treatments decreased NOS 2 mRNA levels as compared with control EC.

Activation of NF-kappa B by Cytokines

The induction of NOS 2 by certain cytokines occurs through degradation of Ikappa B and activation of NF-kappa B (23). This process may be dependent on the generation of ROS (24, 25). Because the latter may be decreased in hypoxia (21, 22), we tested the possibility that hypoxia might alter either the degradation of Ikappa B or the activation of NF-kappa B. EC were treated with IL-1beta (1 ng/ml) and TNF-alpha (20 ng/ml) and exposed to hypoxia or normoxia for periods of 5 min to 4 h. After exposure, Western blot analysis and electrophoretic mobility shift assays were performed to assess Ikappa B degradation and NF-kappa B activation, respectively. Hypoxia did not alter the degradation of Ikappa B, which was evident 15 min after EC were exposed to cytokines (Figure 9A). As shown in Figure 9B, there was no NF-kappa B-DNA binding in unstimulated EC exposed to normoxia or hypoxia. Binding of NF-kappa B-DNA was detected at 30 min (results not shown) and maximal at 1 h (Figure 9B) in normoxic EC treated with IL-1beta and TNF-alpha , with no alteration when the cells were exposed to hypoxia. The binding was specific for the labeled oligonucleotide containing the NF-kappa B site because excess unlabeled oligonucleotide (cold probe) competed with the labeled probe, whereas a mutant oligonucleotide did not (Figure 9B). These studies suggest that cytokine induction of NOS 2 occurs through NF-kappa B activation, a phenomenon that is not prevented by hypoxia.


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  		Figure 9.   Lack of modulation by hypoxia of I-kappa B degradation and NF-kappa B activation. EC were treated with TNF-alpha +IL-1beta and exposed to hypoxia and normoxia for 5 min to 4 h. (A) As shown by Western blot analysis, degradation of I-kappa B was evident 15 min after exposure of EC to cytokines and hypoxia had no effect on this degradation (H15'). (B) Nuclear extracts were obtained, and electrophoretic mobility shift assays were performed as described in MATERIALS AND METHODS. The oligonucleotide probe alone (with no nuclear extracts) and unstimulated hypoxic (H) or normoxic (N) EC resulted in no NF-kappa B-DNA binding (lanes 1-3). There was significant NF-kappa B-DNA binding in normoxic EC treated with TNF-alpha +IL-1beta (lane 5), with no alteration of this binding when the cells were exposed to hypoxia (lane 4). Positive controls include TNF-alpha with hot oligonucleotide probe, cold competitor or mutant competitor (lanes 6, 7, and 8, respectively).

Regulation of the 5'-Flanking Region of the Rat NOS 2 Gene by Hypoxia

To determine whether the attenuation in the cytokine-dependent induction of NOS 2 expression by hypoxia might be associated with similar changes in the activity of the NOS 2 promoter, transient transfection of rat pulmonary microvascular EC was performed using piNOSGEN2, as detailed in MATERIALS AND METHODS. This DNA fragment contains 497 bp of the 5'-flanking region of the rat NOS 2 gene linked to a luciferase reporter gene. When compared with control transfected EC (unstimulated cells exposed to normoxia), treatment of cells with either the combination of IL-1beta and TNF-alpha for 24 h or hypoxia for 24 h resulted in a 3- (P < 0.001) and 2.2-fold (P < 0.001) increase, respectively, in NOS 2 promoter activity (Figure 10). Concomitant treatment of transfected EC with the cytokine combination and hypoxia for 24 h resulted in a 17.6-fold increase in NOS 2 promoter activity (P < 0.001).


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  		Figure 10.   NOS 2 promoter studies. EC transiently transfected with plasmid pINOSGEN2, which contains 497 bp of the rat NOS 2 5' flanking region inserted upstream of the promoter-less luciferase gene, were treated for 24 h with or without TNF-alpha  + IL-1beta and concomitantly exposed to normoxia or hypoxia. At the end of exposure, EC were lysed, and luciferase activity was measured by chemiluminescence. The data represent means and SD with n = 4 for each experimental condition. *P < 0.001 versus control.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Hypoxia has been shown to modulate the expression of several endothelial genes, including vascular endothelial growth factor (26), endothelin-1 (27), platelet-derived growth factor (28), and the endothelial isoform of NOS (i.e., NOS 3) (11, 29). Using in situ hybridization, Palmer and colleagues recently demonstrated that hypoxia induced type 2 NOS mRNA in pulmonary endothelial as well as vascular smooth muscle cells in a chronic hypoxia model of pulmonary hypertension (20). Upregulation of NOS 2 in this model appears to occur through induction of the hypoxia-inducible factor (HIF)-1. Furthermore, using transient transfection of EC, the authors demonstrated a 2.7-fold increase in NOS 2 promoter activity in hypoxic transfected cells as compared with nonhypoxic controls. This increase was completely abrogated with deletion or mutation of the HIF-1 binding site, further supporting an essential role for HIF-1 in the hypoxic regulation of NOS 2 transcription in pulmonary endothelium. However, whereas increased NOS 2 mRNA transcription was demonstrated in the hypoxic lung in that study, evidence of increased transcription of NOS 2 in cultured EC in response to hypoxia was not provided beyond the aforementioned effect on the promoter activity (20).

The present study was undertaken to specifically test the hypothesis that hypoxia might regulate NOS 2 expression in cultured endothelial cells. Also, it became apparent from preliminary experiments on the regulation of NOS 2 that mRNA expression, which was upregulated in the rat lung in response to acute hypoxia (i.e., 24 h), was undetectable when cultured pulmonary EC were exposed to hypoxia for up to 48 h (30). Furthermore, it appears that gene regulation by hypoxia, in general, might differ between in vivo and in vitro systems. For example, whereas Le Cras and colleagues demonstrated an upregulation of endothelial cell NOS (i.e., NOS 3) in response to hypoxia in the rat lung (29), we (11) and others (31) have shown a downregulation of the expression of the same gene in cultured EC. Therefore, we reasoned that, in relation to the regulation of NOS 2, factors other than the direct effect of hypoxia might be operative in vivo, perhaps explaining the discrepancies observed between in vitro and in vivo systems.

The present data indicate that exposure of cultured pulmonary microvascular EC to hypoxia alone does not result in the induction of NOS 2 mRNA or protein expression. This is consistent with the observation by Melillo and colleagues that hypoxia alone does not induce the expression of NOS 2 mRNA in isolated murine macrophages (19). However, like murine brain endothelial cells (5), pulmonary microvascular EC respond to stimulation by cytokines with strong induction of NOS 2 mRNA, protein, and activity. To our knowledge, this is the first report of induction of NOS 2 expression in cultured pulmonary microvascular EC. As is the case for other cell types, such as the macrophage (1) or the smooth muscle cell (2), stimulation by more than one cytokine appears to have synergistic effects.

Whereas hypoxia alone did not induce NOS 2 expression in cultured pulmonary microvascular EC, this factor had significant modulating effects on the regulation of the NOS gene. Concomitant exposure of EC to hypoxia and IL-1beta /TNF-alpha resulted in significant attenuation of NOS 2 mRNA, protein expression, and activity, as compared with normoxic cytokine-stimulated EC. Several lines of evidence indicate that decreased production of ROS in hypoxia could have been responsible for this modulating effect of hypoxia on gene expression. We have previously shown that hypoxia significantly inhibits the intra- and extracellular generation of ROS from bovine pulmonary artery EC (21, 22). A similar decrease in ROS production was found in rat pulmonary microvascular EC exposed to hypoxia (data not shown). Furthermore, the present experiments demonstrate that treatment with various antioxidants reduces the induction of NOS 2 mRNA expression by IL-1beta and TNF-alpha , suggesting that ROS are necessary signals for this induction. Finally, it is known that induction of NOS 2 expression is dependent on activation of transcription factors such as NF-kappa B (17). Because the latter is redox sensitive (25, 32), we postulated that hypoxia might alter NF-kappa B activation. We first demonstrated that the combination of IL-1beta and TNF-alpha caused the degradation of Ikappa B followed by activation of NF-kappa B binding. However, contrary to our expectations, exposure of cytokine-stimulated EC to hypoxia did not prevent the degradation of Ikappa B or alter the binding of NF-kappa B, suggesting that transcriptional modulation of NOS 2 by hypoxia involves mechanisms other than NF-kappa B activation. In this respect, our results differ from the attenuation of IL-1beta -dependent induction of NOS 2 and activation of NF-kappa B observed in cardiac myocytes exposed to hypoxia for 48 h (33).

Hypoxia was found to have other important modulating effects on NOS 2 transcriptional and post-translational expression. Hypoxic exposure of EC transfected with the murine NOS 2 promoter resulted in a 2-fold increase in activity of this promoter, a finding comparable to the 2.7-fold increase described by Palmer and colleagues in similar experiments (20). This effect most likely involves activation and binding of HIF-1, as elegantly demonstrated by these investigators (20). However, as shown in the present study, activation of the promoter by hypoxia does not result in transcription and translation of the message in cultured EC. Such a discrepancy between activation of the promoter and lack of increase of NOS mRNA may be explained by the lack of inclusion of all regulatory elements in the promoter used for the present experiments. It is also possible that synthesis of an active lung NOS 2 protein may occur in vivo secondary to other parallel events such as activation of cytokines by hypoxia. Hypoxia alone has been shown to stimulate cytokine production (such as IL-1, IL-6, and TNF) from human tissues (34, 35). Also, increased IL-1 and IL-6 expression in response to hypoxia has been demonstrated in cardiac myocytes (36), human umbilical vein EC (37), and alveolar macrophages (38). Another interaction between hypoxia and cytokines that is relevant to the present study is the finding that IL-1beta and TNF-alpha strongly stimulate DNA binding of HIF-1 above and beyond the effect of hypoxia alone on this interaction (39). It is noteworthy that, whereas hypoxia and stimulation by IL-1beta /TNF-alpha had independent modest effects on NOS promoter activity (i.e., 2-fold increase) in the present study, the combination of these stimuli resulted in a significantly larger increase (i.e., 17.6-fold) in that activity. A similar synergistic activation by hypoxia and interferon-gamma has been demonstrated by Melillo and colleagues in transfected murine macrophages (19).

Finally, hypoxia was demonstrated to have posttranscriptional effects. As observed with other cell types exposed to cytokines, there was decay of NOS 2 after removal of IL-1beta and TNF-alpha from the cell culture medium in EC exposed to normoxia, resulting in a calculated NOS 2 mRNA half-life of 6 h. However, the decay in mRNA and protein levels was significantly prolonged in hypoxia, resulting in higher mRNA and protein levels in cells maintained in hypoxia for 24 h after cytokine stimulation and a significant increase in NOS 2 mRNA half-life (17 h). There was significantly more release of NO from these cells after reoxygenation compared with cells maintained in normoxia, consistent with the higher levels of NOS 2 protein in the hypoxic cells. Because hypoxia does not induce NOS 2 transcription in rat pulmonary microvascular EC, the observed increased mRNA and protein expression, conferred by exposure of the cells to hypoxia after initial stimulation with cytokines, is likely due to stabilization of NOS mRNA. We performed experiments with actinomycin D treatment to further assess NOS 2 stability. Actinomycin D treatment resulted in significant stabilization of NOS 2 mRNA, with half-lives of 19.5 and 29.5 h for cells exposed to normoxia and hypoxia, respectively. This effect of a transcription inhibitor, although not entirely explained, is likely to be attributed to inhibition of a labile NOS 2-specific RNAse, as previously suggested (40). It appears that NOS 2 mRNA destabilization (e.g., after induction with cytokines) depends on several cis determinants such as the AU-rich element, which contains one or more AUUUA motifs in the 3'-untranslated region (41). The AU-rich element is a target for specific AU-binding factors (42, 43), which confer stability to mRNA. One such factor is AU-A, a 34-kD protein that is constitutively expressed and whose cytoplasmic content is increased following inhibition of transcription (44). Also of note is that hypoxia has been shown to increase the stability of VEGF mRNA through activation of another RNA-binding protein, namely HuR (45). The latter has been demonstrated to confer stability to NOS 2 MRNA in response to cytokine induction in human intestinal epithelial cells (43). Whether hypoxia could prevent putative NOS mRNAse(s) or activate stabilizing factors, such as AU-A or HuR, was not addressed by the present study and remains speculative.

In summary, this study shows that NOS 2 is not expressed in unstimulated cultured pulmonary microvascular EC. However, this gene can be induced in response to a combination of IL-1beta and TNF-alpha . Hypoxia appears to have significant transcriptional (at the NOS 2 promoter level), posttranscriptional (mRNA stabilization), and post-translational (NOS 2 activity) effects on the regulation of this gene. On the basis of these results, we speculate that the pulmonary microvascular EC may actively participate in inflammatory processes and that tissue hypoxia may create a favorable environment for prolonged NOS action.

    Footnotes

Address correspondence to: Paul M. Hassoun, M.D., Associate Professor of Medicine, New England Medical Center, Pulmonary and Critical Care Division, 750 Washington Street, NEMC 257 Boston, MA 02111. E-mail: phassoun{at}lifespan.org

(Received in original form January 24, 2001 and in revised form August 16, 2001).

Abbreviations: diaminonaphtalene, DAN; endothelial cell, EC; ethylenediaminetetraacetic acid, EDTA; hypoxia-inducible factor, HIF; interleukin, IL; inducible nitric oxide synthase, iNOS; internal standard, IS; Nomega -nitro-L -arginine methyl esther, L-NAME; Moloney murine leukemia virus reverse transcriptase, MMLV-RT; nitric oxide, NO; nitric oxide synthase, NOS; 1-(H)-naphtotriazole, NT; polymerase chain reaction, PCR; reactive oxygen species, ROS; tumor necrosis factor, TNF; tumor necrosis factor-alpha , TNF-alpha .

Acknowledgments: This work was supported by grants from the NIH (HL49441) (P.M.H.), American Lung Association (U.K.), and the Massachusetts Tobacco Control Program (U.K.).
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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