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We have previously shown that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in endothelial cells in response to the addition of hydrogen peroxide (H2O2), and this involves, at least in part, the inhibition of AP-1 activity. Thus, the objective of this study was to determine if other cis-element(s) and transcription factor(s) are involved in the oxidant-mediated downregulation of eNOS. Our initial experiments indicated that although H2O2 treatment increased eNOS mRNA levels in ovine pulmonary arterial endothelial cells (OPAECs), there was a significant decrease in the promoter activity of an eNOS promoter construct containing 840bp of upstream sequence. However, a truncated promoter construct that lacked the AP-1 element (650bp) was also inhibited by H2O2. A similar effect was observed when the 650bp human eNOS promoter construct was transfected into human PAECs. We also found that although exposure of the cells to PEG-catalase prevented the inhibitory effect on eNOS promoter activity, the hydroxyl radical scavenger, deferoxamine myslate, did not. Nor could we identify an increase in hydroxyl radical levels in cells exposed to H2O2. Exposure of PAECs caused a significant increase in labile zinc levels in response to H2O2. As the eNOS promoter has a cis-element for Sp1 binding, we evaluated the role of Sp1 in response to H2O2. As previously reported, mutation of the Sp1 consensus lead to the complete loss of eNOS promoter activity, confirming the key role of Sp1 in regulating basal eNOS promoter activity. In addition, we found, using electrophoretic mobility and supershift assays, that H2O2 decreased Sp1 binding. Finally, using chromatin immunoprecipitation analysis, we found a significant decrease in Sp1 binding to the eNOS promoter in vivo in response to treatment with H2O2. Together, these data suggest that the inhibition of Sp1 activity, possibly through loss of zinc in the protein, plays a role in the H2O2-induced inhibition of eNOS promoter activity.
Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining vascular integrity. It is involved in control of blood vessel tone (Huang et al., 1995), remodeling (Rudic et al., 1998), hemostasis (Freedman et al., 1999), and angiogenesis (Lee et al., 1999). Nitric oxide (NO) produced by eNOS is involved in maintaining normal vascular tone and blood pressure and possesses several antiinflammatory properties (Fridovich, 1995). Loss of NO bioavailability causes endothelial dysfunction. Abnormal regulation of endothelial function has been implicated in the pathophysiology of a number of pulmonary hypertensive disorders. For example, in persistent pulmonary hypertension of the newborn (PPHN), pulmonary vascular resistance does not decrease normally at birth, resulting in pulmonary hypertension, right-to-left shunting, and hypoxemia (Steinhorn et al., 1995). Newborns who die of PPHN exhibit an increase both in the thickness of the smooth muscle layer within small pulmonary arteries, extension of this muscle to nonmuscular arteries (Haworth and Reid, 1976), and decreased eNOS expression (Giaid and Saleh, 1995). Surgical ligation of the ductus arterious in the fetal lamb generates a model of PPHN that resembles the human condition (Abman et al., 1989; Wild et al., 1989; Black et al., 1998). Previously, our laboratory has reported an increase in both superoxide (Brennan et al., 2003) and hydrogen peroxide (H2O2) (Wedgwood et al., 2005) in this ovine model of PPHN.
Increased production of reactive oxygen species (ROS) has also been implicated in cardiovascular diseases such as atherosclerosis, restenosis, hypertension, and heart failure (Brandes and Kreuzer, 2005; Cai, 2005). Recent studies indicate that H2O2 affects eNOS activity and NO production. H2O2 treatment of aortic endothelial cells promotes eNOS activity and NO production by inducing changes in eNOS phosphorylation (Thomas et al., 2002), while prolonged H2O2 treatment increased eNOS expression (Drummond et al., 2000). However, we have reported previously that H2O2 treatment in pulmonary arterial endothelial cells (PAECs) isolated from fetal lambs leads to a decrease in eNOS expression and activity (Wedgwood and Black, 2005). Further, we have found that the decrease in expression is due to decreases in eNOS promoter activity (Wedgwood and Black, 2005). We have also shown that H2O2 mediates this decrease in expression, at least in part, by an inhibition of c-Jun activity leading to a decrease in AP-1 transcription factor binding to the eNOS promoter (Kumar et al., 2008). Thus, our goal in this study was to determine if other transcription factors, important for regulating eNOS promoter activity, are inhibited by H2O2. We have found that the binding of Sp1, a key modulator of basal eNOS transcription, is sensitive to inhibition by H2O2. Further, as we also found that the levels of intracellular labile zinc were stimulated by H2O2, we propose that the decrease in binding of Sp1 to the eNOS promoter may be due to the loss of zinc from its zinc finger domain.
Primary cultures of PAECs from late-gestation fetal lambs were isolated by the explant technique as we have described previously (Wedgwood et al., 2003). Briefly, the heart and lungs were obtained from fetal (138–140 days gestation) lambs after sacrifice. These fetal lambs had not undergone previous surgery or study. The main and branching pulmonary arteries were removed, and the exterior of the vessels was rinsed with 70% ethanol. The vessel was opened longitudinally, and the interior was rinsed with PBS to remove blood. The endothelium was lightly scraped away, placed in medium DME-H16 (with 10% fetal bovine serum and antibiotics), and incubated at 37°C in 21% O2–5% CO2–balance N2. After 5 days, islands of endothelial cells were cloned to ensure purity. Basic fibroblast growth factor (10ng/mL) was added to the medium every other day. When confluent, the cells were passaged to maintain them in culture or frozen in liquid nitrogen. Endothelial cells' identity was confirmed by the typical cobblestone appearance, contact inhibition, specific uptake of acetylated low-density lipoprotein labeled with 1,1′-dioctadectyl-3,3,3′,3′-tetramethylindocarbocyanine, and positive staining for von Willebrand factor (DAKO, Carpinteria, CA). Cells were studied between passages 12 and 15. Human PAECs (HPAECs) were purchased from Lonza (Allendale, NJ) and grown in six-well plates according to the manufacturer's recommendation.
We generated promoter constructs by polymerase chain reaction (PCR) using human genomic DNA as a template. The reverse primer common to all constructs binds immediately upstream of the ATG initiation codon of eNOS (5′-GTTACTGTG CGT CCA CTCTGCTGCC). Forward primers were 5′-TGTAGTTTCCCTAGTCCCCC (840bp fragment) and 5′-GGTGTGGGGGTTCCAGGAAA (650bp fragment). To determine the effect of Sp1 on the regulation of eNOS promoter activity, we used a promoter construct identical to the −840 eNOS promoter construct except for 3bp mutation in the wild-type Sp1 sequence at −104 (GGGGCGGGG to GGGactGGG). Ovine PAECs (OPAECs) and HPAECs were cotransfected with 1.6μg of test plasmid and 0.4μg of β-gal plasmid (as an internal control to normalize for transfection efficiency) on a 10-cm2 tissue culture plate at 90% confluency with Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Next day, the cells were split onto six-well plates and allowed to adhere for 36h. The culture medium was then replaced with fresh DMEM containing 0–100μM of H2O2 for 16–18h. For the time course experiment, OPAECs were transfected with the 650bp promoter construct and treated with 100μM of H2O2 for 0, 3, 6, 12, 18, and 24h. To check the effect of hydroxyl radical- compared to H2O2 scavenging on eNOS promoter activity, we treated transfected OPAECs with deferoxamine myslate (100μM; Calbiochem, Gibbstown, NJ) or 250U/mL of PEG-Catalase (Sigma-Aldrich, St. Louis, MO) in the presence and absence of H2O2 (100μM). The luciferase activity was determined using a Luciferase assay system (Promega, Madison, WI). In addition, β-galactosidase activity was determined using a β-galactosidase enzyme assay system (Promega) as the transfection efficiency control.
This procedure was based on previous reports that hydroxyl-free radical (•OH) can react specifically with salicylic acid (SA) to form 2,3- and 2,5-dihydroxybenzoic acid (DHBA) (Floyd et al., 1984; McCabe et al., 1997). Cells were grown on six-well plates to 90% confluency, and then exposed for 4h to serum-free DMEM+ 100μM H2O2+50μM SA. Cell-free reactions were run in parallel to ensure no spontaneous oxidation of SA occurred (data not shown), along with cell-free reactions containing standard concentrations of 2,3- and 2,5-DHBA as positive controls for product detection. After incubation period, supernatants were subjected to electrochemical HPLC analysis for 2,3- and 2,5-DHBA using a DHBA-250 column connected to a Coulochem® III (5200A) analytical system (ESA, Chelmsford, MA). Mobile phase consisted of 50mM sodium acetate, 50mM citric acid, 25% methanol, and 5% isopropanol, pH set to 2.5 using phosphoric acid. Flow rate for detection was set at 0.5mL/min at ambient temperature, and injection volume was 10μL.
Nuclear extracts were prepared using the NE-PER nuclear extraction kit (Pierce Biotechnology, Rockford, IL). Electrophoretic mobility shift assays (EMSAs) were performed using biotinylated double-stranded oligonucleotides corresponding to the Sp1 site at −104 of the eNOS promoter (Marsden et al., 1993). The single-stranded oligonucleotides were biotinylated using Biotin 3′ end DNA Labeling Kit (Pierce Biotechnology) to incorporate 1–3 biotinylated ribonucleotides (Biotin-11-UTP) onto the 3′ end of DNA strands using terminal deoxynucleotidyl transferase and then annealed to make it double stranded. The sequence of the oligonucleotides is Sp1: −119 5′ATT GTG TAT GGG ATA GGG GCG GGG CGA G 3′–92 (IDT Technology, Coralville, IA). Binding reactions involved incubating 10μg of nuclear extract with biotinylated oligonucleotide and 1μg poly(dI.dC) for 20min at room temperature. The DNA–protein complexes were resolved on a 5% nondenaturing polyacrylamide gel in 1× TBE buffer, and then transferred to nylon membrane, and biotinylated oligonucleotide was detected using a LightShift® Chemiluminescent EMSA kit (Pierce Biotechnology). Competition reactions were done with 50- and 100-fold excess of unlabeled Sp1 oligonucleotides. Supershift experiments were conducted by incubating nuclear extracts overnight at 4°C with 4μg of polyclonal antibodies specific to Sp1 (catalog # sc-420×; Santa Cruz Biotechnology, Santa Cruz, CA) followed by the addition of biotinylated oligonucleotide. The complexes were visualized using an Image station 440 CF (Eastman Kodak, New Haven, CT).
Chromatin immunoprecipitation (CHIP) assays were performed essentially following the method of Braunstein et al. (1993). OPAECs (~1.0×106) cultured in 10cm plates were treated with H2O2 (100μM) for 2h and then cross-linked with 1% formaldehyde for 10min, harvested, and sonicated to 200–1000bp fragments. Supernatants were incubated overnight at 4°C with an anti-Sp1 antibody. After ethanol precipitation, DNA was resuspended in 20μL for each 106 cells, and 1μL was used as PCR template. The primer pairs used to amplify the regions containing the Sp1-binding site in the −119/−92 promoter region were 5′-CGTGGAGCTGAGGCTTTAGA/AGCCCTGGCCTTTTCCT TAG (the Sp1-binding site at −104 resulting in a fragment of 153bp). A DNA sample from sonicated lysates that underwent reverse cross-link and phenol/chloroform extraction was used as a positive control (input).
Cells were plated in 24-well plates; treated with 100–200μM of H2O2; washed twice in Zn2+-free DMEM; incubated with 20μM zinquin ethyl ester, ethyl [2-methyl-8-[[(4-methylphenyl)sulfonyl]amino]-6-quinolinyl oxyacetate] (Biotium, Hayward, CA), a live-cell permeant Zn2+-specific fluorophore (Zalewski et al., 1993; Coyle et al., 1994) (20mM in DMSO; diluted 1:1,000 in Dulbecco's PBS [DPBS]) for 30min at room temperature; and washed with fresh DPBS. Zinquin-stained cells were observed at 368nm excitation and 490nm emission, and the average fluorescent intensities were quantified.
Quantitative RT-PCR by SYBR green I dye for specific detection of double-stranded DNA was employed to determine eNOS mRNA levels in control and H2O2 (100μM for 0, 3, 6, 12, and 18h)–treated OPAECs. Briefly, total RNA was extracted from the cells using the RNeasy kit (Qiagen), and 1μg total RNA was reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen) in a total volume of 20μL. Primers for eNOS and β-actin were designed by primer 3. The sequences were as follows: eNOS forward, 5′-CCAGC TCAAGACTGGAGACC-3′; eNOS reverse, 5′-TCAATGTCA TGCAGCCTCTC-3′; β-actin forward, 5′-CTCTTCCAGCCT TCCTTCCT-3′; β-actin reverse, 5′-GGGCAGTGATCTCTT TCTGC-3′. Real-time quantitative PCR was conducted on Mx4000 (Stratagene, La Jolla, CA), using 2μL of RT product, 12.5μL of QuantiTect SYBR Green PCR Master Mix (Qiagen), and primers (400nM) in a total volume of 25μL. The following thermocycling conditions were employed: 95°C for 10min, followed by 95°C for 30s, 56°C for 60s, and 72°C 30s for 45 cycles. The threshold cycle (Ct) of a serially diluted control sample was plotted to generate a standard curve. Concentration of eNOS mRNA was calculated by interpolating its Ct on the standard curve and then normalized to β-actin (housekeeping gene) mRNA levels.
OPAECs were treated with 0, 100, or 200μM H2O2 overnight. After incubation, the medium was collected and centrifuged for 5min at 500g, and the supernatant was stored at 4°C until assay. Relative cytotoxicity was quantified by measurement of release of the soluble cytoplasmic enzyme lactate dehydrogenase (LDH). LDH activity in cell-free supernatant was measured using a commercial kit (Roche Applied Science, Indianapolis, IN). After a given time course, 50μL aliquots of cell-free medium were transferred from all wells to a fresh 96-well plate, and 50μL of reconstituted substrate mix were added to each well. The plate was incubated for 30min at room temperature in the dark, and the absorbance was recorded at 490nm. Each culture sample was measured in quadruplicate, with a minimum of three samples per experimental group. Relative cytotoxicity was determined by comparison of absorbance of the experimental group with absorbance of a control cell group treated with 2% Triton X-100 cell lysis buffer according to the manufacturer's protocol.
At high concentrations, H2O2 may be a toxic chemical. Thus, we initially performed LDH-release assays to examine potential cytotoxic effects of H2O2. However, at all doses examined H2O2 did not induce significant cytotoxicity (untreated=0.0%; 100μM for 2h=0.666+0.113%; 200μM for overnight=2.13+0.15%; 2% Triton X-100=100% cell death).
The mean±SEM values were calculated for all samples, and significance was determined either by the unpaired t-test (for two groups) or ANOVA with Newman Kuels post hoc testing (for ≥3 groups). The statistical significance of differences was set at p<0.05. Statistical analysis was performed using GraphPad Prism version 4.01 for Windows (GraphPad Software, San Diego, CA).
We examined the effect of H2O2 on eNOS transcription using several promoter constructs from the human eNOS gene–fused upstream of a luciferase reporter (Fig. 1A). When OPAECs were transfected with constructs containing 840bp of upstream promoter sequence (−840 eNOS promoter construct) and then treated with 100μM H2O2 for 16–18h, there was a significant decrease in eNOS promoter activity (73+10.7%, p<0.05; Fig. 1B). However, when OPAECs were transfected with a promoter construct containing only 650bp of upstream sequence, there was still a significant but reduced decrease in promoter activity (33+7.6%, p<0.05). To check the time-dependent effects of H2O2 on eNOS promoter activity, OPAECs were transfected with the −650 promoter construct then treated with of H2O2 (100μM) for 0, 3, 6, 12, 18, and 24h. We observed a significant decrease in promoter activity as early as 6h, and this decrease was sustained until 24h (Fig. 1C).
It has been reported previously that H2O2 causes an increase in eNOS mRNA (Drummond et al., 2000). To examine the effect of H2O2 exposure on eNOS mRNA level in our OPAECs, we treated PAECs with H2O2 (100μM) for increasing duration (0, 3, 6, 12, and 18h), and eNOS mRNA levels were determined using quantitative RT-PCR. Our data indicate that H2O2 exposure caused a significant increase in eNOS mRNA level after 18h of H2O2 (p<0.05; Fig. 2).
Through Fenton chemistry, H2O2 can lead to the production of the hydroxyl radical. Thus, we next determined if H2O2 was causing decreases in eNOS promoter activity indirectly through hydroxyl radical generation. To study this, we treated OPAECs with H2O2 (100μM) and monitored hydroxyl radical levels through electrochemical detection. We did not observe any hydroxyl radical generation in H2O2-exposed OPAECs although we detected peaks with cell-free–positive control containing 150pg 2,5-DHBA and 40pg 2,3-DHBA as standards (Fig. 3A). We then determined the effect of hydroxyl radical and H2O2 scavenging on the H2O2-mediated decrease in eNOS promoter activity. OPAECs were transfected with the −650 promoter construct and then treated the hydroxyl radical scavenger, deferoxamine myslate (100μM), or the H2O2 scavenger and PEG-catalase (250U/mL) in the presence or absence of H2O2 (100μM). Deferoxamine myslate did not prevent the decrease in promoter activity in the presence of H2O2 although this was prevented in the PEG-catalase exposed cells (Fig. 3B).
To study the effect of H2O2 on the human eNOS promoter in human endothelial cells, HPAECs were transfected with the −650 promoter construct. After 24h, the cells were then exposed to increasing concentrations of H2O2 (0–200μM) for 18h. There was a significant decrease in promoter activity with both 100μM (32+10.7%, p<0.05) and 200μM (35+8%, p<0.05) of H2O2, but no effect was observed with 50μM H2O2 (Fig. 4).
Previously, we have shown that high levels of H2O2 (>250μM) can induce cell death in OPAECs and that this involves, at least in part, the disruption of intracellular zinc homeostasis (Wiseman et al., 2007). To determine if nontoxic doses of H2O2 could also alter zinc homeostasis we utilized the zinc-specific fluorescent indicator, zinquin ethyl ester, to determine the levels of intracellular labile zinc. Our results indicate that H2O2 treatment caused a dose-dependent increase in zinquin fluorescence, indicating that labile zinc levels are increase in H2O2-exposed cells (Fig. 5).
The truncated 650bp construct has an Sp1-binding site at −104. Since it has been reported before that Sp1 transcription factor is required for basal eNOS transcription (Zhang et al., 1995), we transfected OPAECs with a promoter construct that had a 3bp mutation in the wild-type Sp1 sequence at −104 (GGGGCGGGG to GGGactGGG). As expected, the eNOS promoter activity in absence of Sp1-binding site was almost completely ablated (Fig. 1B). Further, the Sp1 mutant eNOS construct did not exhibit any change in promoter activity in response H2O2 treatment (Fig. 1B).
We next examined the effect of H2O2 on Sp1 binding in OPAECs. This was accomplished by EMSA analysis with oligonucleotides corresponding to the −119 to −92 region of the human eNOS promoter as a probe. Nuclear extracts of OPAECs treated with 100μM H2O2 for 2h showed a 24% (p<0.05) decrease in Sp1 binding (Fig. 6A, B), suggesting that H2O2 can inhibit Sp1 activity and reduce its binding to the eNOS promoter, and the specificity of Sp1-DNA binding was confirmed using supershift analysis and a polyclonal Sp1 antibody (Fig. 7 A, B). The specific Sp1 complex was also confirmed using a competition study in which we found that the shifted complex was competed out with 50× and 100× excesses of unlabeled Sp1 oligonucleotide (Fig. 7C).
Finally, to gain further insight into the effect of H2O2 on the binding of Sp1 to the eNOS promoter, OPAECs were treated with H2O2 (100μM, 2h), and the cells subjected to CHIP analysis. Our data indicate that H2O2 also caused a significant decrease in the binding of Sp1 to the endogenous eNOS promoter in vivo (Fig. 8).
NO is an endothelium-derived relaxing factor synthesized and released from the endothelial cell by the activation of eNOS (Rubanyi et al., 1986; Black and Fineman, 2006). NO is one of the primary components for blood flow and pressure regulation, and loss of NO bioavailability is a key feature of endothelial dysfunction. Multiple pathways can reduce NO bioavailability by altering its synthesis or biodegradation, but excessive production of ROS, including H2O2, seems to be a major mechanism of reduced vascular NO levels (Guzik and Harrison, 2006). In many cardiovascular diseases, such as atherosclerosis, restenosis, hypertension, diabetic vascular complications, and heart failure, there is an increase in the production of ROS (Li and Shah, 2004; Touyz and Schiffrin, 2004; Brandes and Kreuzer, 2005; Cai, 2005). Also, in PPHN, there is a decrease in eNOS expression (Giaid and Saleh, 1995). An animal model of human PPHN has been generated using the surgical ligation of the ductus arterious in the fetal lamb (Abman et al., 1989; Wild et al., 1989; Black et al., 1998). Previously, our laboratory has reported a decrease in eNOS mRNA and protein in this ovine model of PPHN (Black et al., 1998). These changes in gene expression were associated with an increase in both superoxide (Brennan et al., 2003) and H2O2 (Wedgwood et al., 2005). Further, our previous in vitro studies have demonstrated that eNOS expression and promoter activity are decreased when PAECs isolated from fetal lambs were treated with H2O2 (Wedgwood and Black, 2005). The eNOS promoter also shares many features with other endothelial-specific promoters. The eNOS is a TATA-less promoter, which has binding sites for a variety of transcription factors, including Sp1, Ets, GATA, NF-1, AP-1, KLF-2, MTF-1, and shear-stress response element (Marsden et al., 1993). These transcription factors are involved in the regulation of eNOS expression basally or in response to exogenous stimuli (Zhang et al., 1995; Wedgwood et al., 2003; Grumbach et al., 2005). Of the above-mentioned factors, AP-1 and Sp1 are redox-sensitive transcription factors (Meyer et al., 1993, 1994; Shi et al., 2004). Sp1 was initially considered a basal transcription factor, but now it is known that it plays an important role in a wide range of cellular processes, including cell cycle regulation, hormonal activation, apoptosis, angiogenesis, and regulation of redox homeostatis (Ammendola et al., 1994; Grinstein et al., 2002; Kavurma and Khachigian, 2003; Josko and Mazurek, 2004). Previously, we found that the AP-1 cis-element plays a key role in regulating eNOS expression and promoter activity in response to shear stress (Wedgwood et al., 2003) and antioxidants (Kumar et al., 2007). Further, we have recently found that eNOS promoter activity is decreased by H2O2 exposure in OPAECs transfected with a 840bp upstream promoter construct (Kumar et al., 2008). The 840bp construct contains an AP-1–binding site at −661 and mutation of two base pairs within the consensus AP-1–binding sequence specifically disrupts DNA binding (Bohmann et al., 1987; Kaluzova et al., 2001). Previously, we have shown that introduction of the same mutations within the AP-1 sequence (AP-1m) prevented shear-induced increase in promoter activity in OPAECs (Wedgwood et al., 2003). We found that the AP-1m had an attenuated H2O2-mediated decrease in eNOS promoter activity, indicating that a loss of AP-1–binding activity (made up of c-Jun and FosB), in addition to Sp1, is involved in the redox regulation of eNOS transcription (Kumar et al., 2008). However, it is worth noting that in this study we found that although there was a modest trend downward, the −650bp mutant construct was not significantly decreased in the presence of H2O2. It is unclear why a more detailed analysis of the −650bp promoter construct in this study exhibits decreased activity toward H2O2. However, it is worthwhile noting that we have used higher passage cells in this study than in the previous study, suggesting that as the OPAECs age in culture they become more sensitive to H2O2-mediated effects.
H2O2 is an injurious by-product of cellular metabolism, but it also participants in cell signaling and has been shown to be regulator of vascular tone (Forman and Torres, 2002; Suvorava et al., 2005). Thus, H2O2 can exhibit vasoconstrictive or vasorelaxant properties depending on the concentration. We have previously reported that high concentrations of H2O2 (≥250μM) cause an increase in intracellular-free zinc, and this was associated with increased cell death (Wiseman et al., 2007). In this study, we have used 100–200μM of H2O2, which did not cause significant cell death, but resulted altered zinc homeostasis as we observed an increase in intracellular labile zinc levels. Zinc is an important component of number of cellular proteins and perhaps 30–50% of proteins in a given proteome of a cell contain zinc-binding domains. Zinc finger proteins are one of the largest classes of DNA-binding proteins (Berg, 1992). They are prime targets for redox regulation because they all contain essential cysteine residues as part of the metal-binding, DNA-intercalating fingers (Desjarlais and Berg, 1992). These zinc finger transcription factors regulate expression of many genes in response to a stimulus or basially or both. The best-characterized zinc finger transcription factor is Sp1 (Courey et al., 1989). Sp1 is a member of an extended family of DNA-binding proteins that have three zinc finger motifs and bind to GC-rich DNA (Courey et al., 1989; Berg, 1992). In this study we have found, using luciferase reporter constructs, that the promoter activity of eNOS could be significantly decreased with H2O2 treatment even in the absence of the AP-1 site. Further, we found that this was associated with an increase in the levels of labile zinc in the cells and a reduction in the activity of the Sp1 transcription factor. Sp1 is ubiquitously expressed in mammalian tissues and was one of the first transcription factors to be cloned (Dynan and Tjian, 1983, 1985; Briggs et al., 1986). It binds to GC-rich DNA and is involved in transcriptional regulation of genes that lack a TATA or CAAT box element in their proximal promoter. Studies show that besides being a basal transcription factor, it is also an important modulator of tissue-specific transcription (Zhang et al., 1997). Oxidative stress, translational modifications such as glycosylation, phosphorylation, and proteolytic cleavage, and other transcription factors are involved in regulating activity of Sp1. It contains three zinc finger motifs that are not only essential for its DNA-binding activity but also provide a structural basis for redox regulation (Courey et al., 1989; Berg, 1992). Redox imbalances cause lipid peroxidation and oxidation of proteins. Oxidation of cellular thiols can trigger intracellular zinc release. Under physiological conditions, the concentration of intracellular labile zinc ions is low (Krezel et al., 2007; Cortese et al., 2008). However, we have shown, both in this study and previously (Wiseman et al., 2007), that H2O2 can cause increases in labile zinc, likely from oxidation-mediated modification of proteins that contain zinc. As zinc provides critical structural stabilization to a multitude of proteins, including the zinc-finger transcription factors (Rana et al., 2008) such as Sp1, GATA, and p53, we propose that the loss of Sp1 activity in H2O2 challenged OPAECs is due to the loss of the zinc atom from one or more of the three zinc-fingers in the protein. It has also been previously reported that 68% of eNOS promoter activity is regulated by −166/−1 eNOS promoter. This region contains an Sp1-binding site at −104 (Xing et al., 2006), and previously published studies have reported that Sp1 is involved in the basal transcription of the eNOS gene (Tang et al., 1995; Zhang et al., 1995). Further, Sp1 has been shown to be involved in the upregulation of eNOS expression by insulin (Fisslthaler et al., 2003), lysophosphatidylcholine (Cieslik et al., 1998; Xing et al., 2006), and cyanidin-3-glucoside (Xu et al., 2004). We also found that mutating Sp1 consensus sequence at −104 of the eNOS promoter almost completely abolished basal eNOS promoter activity. Further, Sp1 showed a decrease in the binding to its element on exposure to H2O2. Other studies have shown similar findings in nuclear extracts prepared from both human K562 cells and rat liver (Ammendola et al., 1994; Knoepfel et al., 1994). While it has also been reported that there was a decrease in the DNA-binding activity of Sp1 with H2O2 treatment in nuclear extracts prepared from rat liver or with purified Sp1 (Ammendola et al., 1994), previous studies have shown that H2O2 exposure can lead to the generation of the hydroxyl radical, via Fenton reaction (Wardman and Candeias, 1996). To examine the involvement of hydroxyl radical in decreasing eNOS promoter activity, we treated the transfected PAECs with hydroxyl radical scavenger, deferoxamine myslate, in the presence of H2O2. We did not observe any protective effect on the H2O2-mediated decrease in eNOS promoter activity. We also did not observe detectable levels of hydroxyl radical in OPAECs treated with H2O2. This suggests that the reduction of eNOS promoter by H2O2 is not mediated indirectly through hydroxyl radical generation but rather directly through H2O2 itself. Indeed we found that treatment with PEG-catalase blocked the H2O2-mediated decrease in eNOS promoter.
Previously, it has been reported that H2O2 cause increase in eNOS mRNA levels by increasing its stability (Drummond et al., 2000). This induction of eNOS through H2O2 has been shown to be Ca2+ dependent, and H2O2 induced autophosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased its activity (Cai et al., 2001). In our study, we also observed an increase in eNOS mRNA in OPAECs treated with H2O2. However, as we have previously shown that treatment of OPAECs with H2O2 leads to decreased eNOS protein levels after 24h (Wedgwood et al., 2005), this suggests that decreases in promoter activity outweighs the increase in mRNA stability. The main promoter construct used in our study (−650) represents the part of the full eNOS promoter, and we found that H2O2 caused only a modest (~25%) decrease in promoter activity through the inhibition of Sp1 transcription factor binding. Thus, it is interesting to speculate that there are other elements further upstream or downstream of eNOS promoter that may lead to more severe decreases in eNOS transcription in H2O2-exposed OPAECs and at least one of these regions is AP-1 as we have shown (Kumar et al., 2008), but further studies will be required to identify other potential sequences.
Overall, our data suggest a major role for the zinc finger transcription factor Sp1 in the downregulation of eNOS expression in OPAECs treated with H2O2. H2O2 treatment appears to cause the oxidation of cellular proteins, including Sp1, causing loss of the structural zinc atom thereby increasing labile cellular zinc. This results in decreased binding of Sp1 to its cis-element in the eNOS promoter. This is the first report suggesting that Sp1 plays a major role in regulating eNOS expression in response to H2O2. By characterizing the molecular mechanisms of redox regulation of eNOS expression in vivo, it may be possible to identify potential therapies to reduce or prevent endothelial dysfunction in diseases such as PPHN where decreased eNOS expression is associated with increased ROS production.
This research was supported in part by Grants HL60190 (to S.M.B.), HL67841 (to S.M.B.), HL72123 (to S.M.B.), and HL70061 (to S.M.B.), all from the National Institutes of Health, and by a Transatlantic Network Development Grant from the Fondation Leducq (to S.M.B.).
No competing financial interests exist.