Published online before print May 1, 2006, doi: 10.1161/01.HYP.0000222892.37375.4d
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(Hypertension. 2006;47:1189.)
© 2006 American Heart Association, Inc.
Original Articles |
Hypoxia-Induced Endothelial NO Synthase Gene Transcriptional Activation Is Mediated Through the Tax-Responsive Element in Endothelial Cells
From the Department of Biomedical Sciences, National Institute of Health, 194 Tongilo, Eunpyeong-gu, Seoul 122-701, South Korea.
Correspondence to Inho Jo, Department of Biomedical Sciences, National Institute of Health, 194 Tongilo, Eunpyeong-gu, Seoul 122-701, Korea. E-mail inhojo{at}nih.go.kr
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Abstract |
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Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (–1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(–1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36±0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(–1600). However, progressive 5'-deletion from –1600 to –873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element–like site, located at –924 to –921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.
Key Words: nitric oxide synthase • nitric oxide • endothelium • gene regulation • vasodilation • signal transduction
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Introduction |
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In response to hypoxia, systemic arteries vasodilate, allowing more blood to be delivered to peripheral tissues. This vasodilation occurs within seconds after hypoxia and is maintained for several hours.1 The molecular basis of hypoxic vasodilation is still somewhat unknown, although potassium channel activation, decreased ATP levels, increased intracellular acidification, decreased release of vasoconstrictors, such as endothelin and thromboxane, and increased release of vasodilators, such as adenosine, prostacyclin, and NO, have all been suggested as key components.2,3
NO plays an important role in vasodilation, and in endothelial cells, NO is catalyzed from the amino acid L-arginine and molecular oxygen by endothelial NO synthase (eNOS). The activity4 and expression5–11 of eNOS have been found to increase in response to hypoxia, but conflicting studies have observed a decrease12,13 or lack of response.14 The discrepancies between these studies can potentially be explained by differences in either the duration of exposure to hypoxia or the endothelial cell type used.
Coulet et al11 reported recently that 2 contiguous hypoxia-responsive elements (HREs) located at –5375 to –5366 were identified in the eNOS gene promoter, and that transcription factor hypoxia-inducible factor (HIF)-2
preferentially bound to these elements under hypoxia, leading to the activation of eNOS gene transcription. However, several other studies using a construct of a shorter 5'-region flanking the eNOS gene (1600-bp), which is devoid of HRE, also clearly showed an increased transcriptional activity by hypoxia,9,11 implying that mechanism(s) other than the HIF/HRE-mediated signaling pathway may be involved. To date, there has been only 1 study showing that enhanced eNOS gene transcription was mediated by redox-sensitive activating protein 1 signaling pathways.9
In this study, we searched for other element(s) of the eNOS promoter that are responsible for hypoxia-induced transcriptional activation. We identified a Tax-responsive element (TRE) located from –924 to –921 within the eNOS promoter and showed that phosphorylated cAMP-responsive element (CRE) binding (CREB) protein (pCREB) preferentially binds to this response element under hypoxia and activates eNOS gene transcription.
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Materials and Methods |
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Materials
H-89 was purchased from Calbiochem. Minimum essential medium, Hanks’ balanced salt solution, newborn calf serum, penicillin–streptomycin, L-glutamine, trypsin-EDTA solution, and other plastic wares for cell culture were obtained from Gibco-BRL. Antibodies against eNOS and HIF-1
were purchased from Transduction Laboratories and BD Biosciences, respectively. Antibodies against pCREB (phosphorylated at Ser133) and CREB were purchased from Cell Signaling Technology, Inc.
Genomic DNA Extraction and Plasmid Construction
Genomic DNA was prepared from human blood leukocytes. Human blood leukocytes were obtained from Ansan Health Study,15 and all of the procedures for human research were approved by the Institutional Review Board of Ansan Health Study. Using genomic DNA as a template, a DNA fragment of the 5'-flanking region of the eNOS gene (–1600 to +22 nucleotides) was amplified by PCR and designated eNOS(–1600). The sense and antisense primers containing KpnI and XhoI sites, respectively, for PCR amplification are shown as follows: sense, 5'-TTGACCCTGAGGATGCCAGT-3'; antisense, 5'-TGCTGCCTGCTCGAGCAGAGC-3'. Furthermore, 5'- deletion DNA fragments of the eNOS promoter at –1400, –962, –873, and –428 were also amplified by PCR and designated eNOS(–1400), eNOS(–962), eNOS(–873), and eNOS(–428), respectively. Each sense primer is shown as follows: eNOS(–1400), sense 5'-ACTGGGAAGGCGAGGTGGTG-3'; eNOS(–962), sense 5'-AGTCTCTGAGGTACCGAAATC-3'; eNOS(–873), sense 5'-GAGTGCTGGGGTACCCCACAA-3'; and eNOS(–428), sense 5'-ATCCCCTGCTACAGAAACAA-3'. The same antisense sequences were used to construct all of the 5'-deletion DNA fragments. In a separate experiment, site-directed mutagenesis (A
G at –922) of eNOS(–962) was also carried out using a modified sense primer 5'-TCCGGTACCTCAGATGGCACAGAA-3' and designated m- eNOS(–962). The synthesized PCR products containing each of the 6 DNA fragment sequences were digested and fused into luciferase reporter gene plasmids, which were designated pGL2-eNOS(–1600), pGL2-eNOS(–1400), pGL2-eNOS(–962), pGL2-eNOS(–873), pGL2-eNOS(–428), and pGL2-m-eNOS(–962), respectively (Figures 3B, 4B, and 4
E).
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Cell Culture, Transfections, and Hypoxia Treatment
Bovine aortic endothelial cells (BAECs) were isolated and cultured as described.16 BAECs were transfected with (3 µg each) pGL2-eNOS(–1600), pGL2-eNOS(–1400), pGL2-eNOS(–962), pGL2-eNOS(–873), pGL2-eNOS(–428), and pGL2-m-eNOS(–962) using Lipofectin (Invitrogen Inc) according to the manufacturer’s instructions. In a separate experiment, BAECs were also transfected with 3 µg of dominant-negative inhibitor of CREB, termed A-CREB.17 BAECs or transfectants were then subjected to either hypoxia or normoxia for the indicated times, respectively. Protocols for cell culture, transfections, luciferase assays, and hypoxia treatments are described in detail in the online data supplement (available online at http://www.hypertensionaha.org).
Measurement of NO Release
Measurement of NO release is described in detail in the online data supplement.
Electrophoretic Mobility Shift Assay
The method for electrophoretic mobility shift assay (EMSA) is described in detail in the online data supplement. The sequences of the sense strands of double-stranded oligonucleotides used as probes and competitors in the EMSA were as follows, with the consensus motifs of CRE and TRE3 italicized: The CRE probe (sense strand) used was 5'-CGGGAAGCGTGCGTCACTGAATGACA-3' (Figure 3C). At this moment, it should be noted that the CRE sequence indicated in the eNOS gene is not a "classical" palindromic DNA sequence (TGACGTCA) of CRE, but it has been widely used as a putative CRE sequence in the previous studies.18,19 In a separate experiment, a positive control for CRE probe with a classical palindromic DNA sequence of CRE (italicized) used was 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3.20 The wild-type TRE3 probe used was 5'-CCAGCCCCTCAGATGACACAGAACTACAAAC-3' (Figure 4C), and mutant TRE3 (mTRE3, with mutation sequence in boldface) was 5'-CCAGCCCCTCAGATGGCACAGAACTACAAAC-3' (Figure 4C). For the mutation of TRE3 sequence, the site-specific mutation (AG) at –922 of the eNOS promoter was directed from the discovery of eNOS gene single-nucleotide polymorphic sites.21,22
Western Blot Analysis
Western blot analysis is described in detail in the online data supplement.
Real-Time RT-PCR
Measurement of the levels of eNOS mRNA is described in detail in the online data supplement.
Statistical Analysis
All of the data are presented as mean (SD) with n indicating the number of experiments. Statistical significance was determined by the Student t test for 2 points. Differences were deemed statistically significant at P values <0.05.
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Results |
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Hypoxia Increases eNOS Protein Level and NO Production
Hypoxia (1% O2 concentration), as evidenced by the increase in HIF-1
expression (Figure 1A), increased the NO production by BAECs in a time-dependent manner (Figure 1B). The maximal increase in NO levels (2.78±0.14-fold of the control) was observed after 24-hour treatment, and this increase subsequently fell over the next 48-hour period. Therefore, unless otherwise stated, all of the subsequent experiments were performed using this condition (1% O2 concentration for 24-hour treatment). Western blot analysis revealed that this increase was accompanied by an increase in eNOS protein expression (Figure 1B).
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Increase in eNOS Protein Level and NO Production by Hypoxia Is Mediated Through pCREB-Mediated Pathway
Hypoxia is reported to increase the phosphorylation of CREB.23 This result, considered in context with a previous finding that beraprost sodium (BPS), an orally active prostacyclin analogue, increased eNOS gene transcription by pCREB-mediated pathways,18 prompted us to test whether these hypoxia-stimulated increases in eNOS expression and NO production are also mediated by pCREB-CRE signaling pathways. As shown in Figure 2A, hypoxia increased the phosphorylation of CREB with no alteration of CREB expression. The maximal increase in CREB phosphorylation was observed after 24-hour treatment, showing a similar time-dependent pattern to that of eNOS expression. To determine whether the increase in CREB phosphorylation mediates hypoxia-induced increases in eNOS protein level and NO production, we first used H-89, a specific protein kinase A (PKA) inhibitor. PKA is well known to phosphorylate CREB in a variety of cells.24,25 As shown in Figure 2B, pretreatment with H-89 (10 µmol/L for 1-hour treatment) significantly decreased CREB phosphorylation by hypoxia. This drug, however, did not alter CREB expression. Furthermore, H-89 significantly attenuated hypoxia-induced increases in eNOS expression and NO production. To further clarify a role of pCREB in this mechanism, we next transfected A-CREB, a dominant-negative inhibitor of CREB, into BAECs. A-CREB, devoid of Ser133 phosphorylation site, is well known to inhibit CRE-mediated transcription by DNA binding of pCREB17 to the specific promoter sequence of several target genes. Recently, A-CREB was also reported to inhibit the phosphorylation of CREB, leading to a decrease in CRE-mediated steroidogenic acute regulatory protein gene transcription induced by insulin-like growth factor 1.26 Parallel to the results with H-89, we found that transfection of A-CREB also decreased CREB phosphorylation by hypoxia (Figure 2C). Furthermore, this transfection significantly decreased hypoxia-stimulated eNOS expression and NO production by hypoxia. Taken together, our data suggest that the stimulatory effect of hypoxia on eNOS expression and NO production in BAECs is mediated at least in part by CREB phosphorylation.
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CRE Within –1.6-kb eNOS Gene Promoter Is Not Involved in Transcriptional Activation of eNOS Gene by Hypoxia
To test whether hypoxia increased the eNOS expression via the interaction between pCREB and CRE within the eNOS promoter, we first examined the transcriptional activation of eNOS by hypoxia. In agreement with previous reports,8,9,18,27 we clearly found that hypoxia (for 24-hour treatment) increased the amount of eNOS mRNA (Figure 3A), suggesting that the stimulatory effect of hypoxia on eNOS gene expression occurs at least at the level of transcription. We next constructed pGL2-eNOS(–1600), as described in the Methods section, to explore the role of the interaction between pCREB and CRE in the transcriptional activation of the eNOS gene. As shown in Figure 3B, hypoxia clearly increased the promoter activity (2.36±0.18-fold of the control) in cells transfected with pGL2-eNOS(–1600). Based on a Genomatix database search (http://genomatix.de), we identified 1 putative CRE site located from –733 to –727 of the eNOS promoter (Figure 3B). However, using pGL2-eNOS(–873) containing this putative CRE site, no increase in promoter activity was observed in response to hypoxia, suggesting that this CRE site is not involved in hypoxia-stimulated eNOS transcription. To further corroborate this finding, we explored EMSA to find whether the pCREB induced by hypoxia bound to the CRE. As shown in Figure 3D, the supershift experiment using anti-pCREB antibody revealed that pCREB did not bind to the CRE site. This result is valid, because we found that pCREB clearly bound to the positive control. We also found that this binding was specific, because it was inhibited in a dose-dependent manner by a 10- and 50-molar excess of unlabeled CRE (from positive control or from eNOS gene) oligonucleotides. These data suggest that pCREB may not bind to the CRE in the eNOS promoter. A positive control contains a "classical" palindromic DNA sequence (TGACGTCA) of CRE, whereas CRE in the eNOS gene is very similar in sequence to the classical CRE with only 1 nucleotide (A) deleted (TGCGTCA).
TRE Site Located From –924 to –921 of the eNOS Promoter Is Identified as a pCREB Binding Sequence Responsible for Hypoxia-Stimulated eNOS Gene Transcription
To map the region(s) of the eNOS promoter responsible for hypoxia-stimulated eNOS transcription, we further searched a Genomatix database. In addition to CRE, we found that there are 4 more putative sequences, so-called TREs, for CREB binding (Figure 4A). Progressive 5'-deletion from –1600 to –962 [pGL2-eNOS(–1400) and pGL2-eNOS(–962)] did not significantly alter the increased eNOS promoter activity by hypoxia (Figure 4B). However, further deletion from –962 to –873 [pGL2-eNOS(–873)] completely attenuated the increased promoter activity, whereas deleting down to –428 [pGL2-eNOS(–428)] did not reduce activity further (Figures 3B and 4
B). This result suggests an important role for the TRE3 site in hypoxia-stimulated eNOS gene transcription. To further corroborate these findings, nuclear extracts after hypoxia were incubated with wild-type (TRE3) and mutant double-stranded oligonucleotides (mTRE3) corresponding with the TRE3 region and analyzed by EMSA (Figure 4C). The sequence of mTRE3 was adopted from the well-known single-nucleotide polymorphic sequence identified by several genomic epidemiology studies.20,21 As shown in Figure 4D, incubation with TRE3 resulted in significantly retarded complexes (left); more prominent retarded complex was found when incubated with extracts from hypoxic cells. The supershift experiment using anti-pCREB antibody demonstrated that pCREB bound to the TRE3 probe. The binding of pCREB to the TRE3 was specific, because this binding was significantly inhibited in a dose-dependent manner by a 10- and 50-molar excess of unlabeled wild-type TRE3 oligonucleotides. In contrast, competition and supershift assays showed that pCREB did not bind to the mTRE3 probe (right), although incubation with mTRE3 resulted in a more prominent retarded complex compared with incubation with TRE3. These results defined the hypoxia-responsive sequence as TRE3 located at –924 to –921 of the eNOS promoter. The importance of TRE3 for the observed effects was further supported by a finding that the construct pGL2-m-eNOS(–962), which contains mTRE3 in the context of pGL2-eNOS(–962), was unresponsive to hypoxia (Figure 4E). Furthermore, cotransfection of A-CREB significantly inhibited the luciferase activity of pGL2-eNOS(–962) induced by hypoxia, whereas no alteration in luciferase activity of pGL2-m-eNOS(–962) was observed, suggesting that pCREB bound to the TRE3 sequence, leading to an increase in eNOS gene transcription.
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Discussion |
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Our study demonstrates that hypoxia increases NO production by activating pCREB-mediated eNOS gene transcription. Promoter analysis of the eNOS gene mapped the hypoxia-responsive sequence to TRE, located at –924 to –921. These data are further confirmed by EMSA, supershift assay, and site-specific mutation analysis. We believe that this is the first solid evidence that eNOS gene expression is induced in response to hypoxia through the interaction between pCREB and TRE.
Our current finding, that hypoxia increases pCREB-mediated eNOS gene transcription, deserves particular attention, because cAMP-mediated vasodilation has been generally considered to be eNOS independent.28–30 However, it has been reported recently that cAMP increased NO production via either phosphorylation of eNOS30,31 or expression of eNOS,18 suggesting a direct effect of cAMP on eNOS modulation. A particularly interesting finding from a previous study, which relates to our current study, was that BPS, an orally active prostacyclin analogue, increased the promoter activity of the eNOS gene through the interaction between pCREB and CREs.18 In this previous study, PKA was found to phosphorylate CREB, and PKA inhibitors completely blocked the effect of BPS on eNOS gene transcription and NO production. This PKA-mediated, pCREB-stimulated eNOS gene transcription was fairly reproduced by our current data using H-89 treatment and A-CREB transfection (Figure 2B and 2C). However, it should be noted that the phosphorylation of CREB was first detected at 6 hours after hypoxia, peaked at 24 hours, and gradually declined thereafter (Figure 2A), whereas BPS increased pCREB at a much earlier time point (30 minutes) and peaked at 1 hour.18 Furthermore, the authors of the previous report defined 2 contiguous CRE sequences at –733 and –603 as a pCREB binding site. Interestingly, however, this result was not reproduced by our current study, because we did not observe any of the interactions of pCREB with CREs that they had defined (Figure 3D). Instead, we found that pCREB induced by hypoxia treatment bound to the TRE site. At present, the reason for these incompatible results is not fully understood, although it may be attributable to different experimental conditions: hypoxia treatment versus BPS treatment.
Although the involvement of TRE, the human T-cell leukemia virus type 1 (HTLV-1), a CRE-like site, in the regulation of hypoxia-stimulated eNOS gene transcription is of great interest, a detailed underlying mechanism is not clearly understood at the present time. It was reported previously that Tax, a unique oncoprotein encoded by a viral genome, interacts with HTLV-1 long terminal repeat indirectly via binding to CREB, which binds to TRE.32,33 Furthermore, Tax was known to stabilize CREB/DNA complexes and to enhance the binding affinity of CREB to TRE sites.32,34 Biochemical experiments have demonstrated that, in the presence of Tax, CREB binds to the TRE in a phosphorylation-independent manner,35,36 thereby serving as a cofactor for Tax activation of HTLV-1 transcription. However, in the absence of Tax, as shown in uninfected mammalian cells, CREB binds to the CRE sites only after its phosphorylation via the cAMP-dependent PKA signaling pathway. In this regard, it was expected that pCREB should bind to the CRE within the eNOS promoter. Unexpectedly, however, our data showed that pCREB induced by hypoxia bound to the TRE but not CRE (Figures 3D and 4D). These data strongly suggest that Tax may not be involved in our current system. Although CREB was reported to bind equally to the TRE at normoxia and hypoxia in virus-infected cells,37 to our knowledge, our study is the first to show that pCREB binds to the TRE in uninfected mammalian cells. Nonetheless, it is worthwhile to test whether there are other unknown Tax-like protein(s) in relation to TRE-mediated eNOS gene regulation in BAECs; such a test, however, is beyond the scope of our study.
The finding in our study of a specific TRE site responsible for hypoxia-stimulated eNOS gene transcription had not been observed by 2 recent reports. At present, the reasons for these incompatible results have yet to be clarified. Authors of 2 previous reports stated in their reports that they constructed and used an eNOS promoter sequence that was obtained either from GeneBank Accession No. AC09246611 or from 2 previously published reports.19,38 Interestingly, however, it was found that the eNOS promoter sequence presented from GeneBank or previously published reports was a mutant eNOS gene sequence containing a well-known single nucleotide polymorphism (SNP) site (AG mutation at –922). In this regard, it should be noted that this SNP loses the TRE consensus motif. We further tested our result by EMSA and found that pCREB did not bind to the mutant TRE3 site (Figure 4D), suggesting that the consensus motif (TGAC) of the TRE3 region in the eNOS gene promoter might play an important role in interacting with pCREB. From this result, our data demonstrate, for the first time, that an AG SNP site at –922 of the eNOS gene may be another important functional locus for regulating eNOS gene expression, in addition to a TC mutation at –786. Several studies have found previously that the repressed transcription of the eNOS gene is linked to a TC SNP at –786, which results in an increased risk of cardiovascular disease. Thus, a TC mutation at –786 has already been recognized as the most important functional locus for eNOS gene regulation,15,21,22 although this SNP was reported to be in linkage disequilibrium with 2 other SNPs (AG SNP at –922 and TA SNP at –1468).15,21 Further study will be needed to clarify this issue.
Perspectives
Our results shed light on the molecular mechanisms underlying hypoxia-stimulated eNOS gene transcription, which implicates pCREB-TRE mediation. This study extends our understanding of the mechanisms involved in NO regulation by other cardiovascular risk factor(s). It was reported previously that cigarette smoking, known as an important risk factor in cardiovascular disease, decreased eNOS gene expression in endothelial cells and decreased the amount of lung cAMP,39 an upstream molecule of PKA/pCREB-mediated signaling pathway. The identification of TRE site in the eNOS gene, thus, may hold a promise in unraveling cigarette smoking–induced vascular dysfunction. Finally, our results underscore the potential of the TRE site as a novel therapeutic target for the design of drugs aimed at improving endothelial function in NO dysregulation–related cardiovascular disease, such as hypertension.
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Acknowledgments |
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This work was supported in part by the Korea National Institute of Health intramural research grant (348-6111-213-000-207) awarded to I.J. and by the Biomedical Brain Research Center grant from the Ministry of Health and Welfare (0405-NS01-0704-0001) awarded to S.J.
Received February 15, 2006; first decision March 3, 2006; accepted April 7, 2006.
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