Published online before print February 16, 2006, doi: 10.1161/01.ATV.0000209998.73303.b5
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© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Molecular Dissection of Angiotensin II–Activated Human LOX-1 Promoter
From the Departments of Internal Medicine (J.C., Y.L., H.L., P.L.H., J.L.M.) and Physiology and Biophysics (J.C.), University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Ark.
Correspondence to J.L. Mehta, MD, PhD, Division of Cardiovascular Medicine, University of Arkansas for Medical Sciences, 4301 W Markham St, Slot 532, Little Rock, AR 72205-7199. E-mail MehtaJL{at}uams.edu
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Abstract |
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Objective— LOX-1, a receptor for oxidized low-density lipoprotein, plays a critical role in atherosclerosis. Its expression is upregulated by pro-atherogenic stimuli, such as angiotensin II (Ang II). In this study, we explored LOX-1 transcriptional promoter activation in response to Ang II in human coronary artery endothelial cells (HCAECs).
Methods and Results— We constructed full-length and deletion LOX-1 promoter mutants and examined their activation in response to Ang II in HCAECs. The Ang II (1 µmol/L for 24 hours) markedly induced LOX-1 promoter activity beyond the basal level, and a 116-bp fragment (between nt –2247 and –2131) was necessary for this induction. Within this 116-bp promoter fragment, there is a potential binding motif for transcription factor NF-
B. By EMSA, we observed the activation of NF-B by Ang II. The critical role of NF-B in Ang II–induced LOX-1 promoter activation was confirmed by mutagenesis assay, and further confirmed by blocking NF-B activation with the NF-B inhibitor caffeic acid phenethyl ester or NF-B p65 siRNA.
Conclusion— This study strongly suggests that Ang II, by activating NF-B, induces LOX-1 promoter activation.
In this study, we explored LOX-1 transcriptional promoter activation in response to angiotensin II (Ang II) in human coronary artery endothelial cells. This study strongly suggests that Ang II, by activating NF-B, induces LOX-1 promoter activation.
Key Words: LOX-1 • Ang II • oxLDL • transcription factor • NF-B • oxidative stress
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Introduction |
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Atherosclerosis and associated diseases are major causes of death in the Western world. High plasma level of low-density lipoprotein (LDL), especially in the form of oxidized LDL (oxLDL), is a major risk factor for atherogenesis.1 oxLDL plays a critical role in endothelial dysfunction, which appears at the early stages of atherogenesis.2 In vitro studies have demonstrated that during oxLDL–induced endothelial dysfunction, vascular endothelial cells internalize oxLDL through a receptor-mediated pathway. Although several scavenger receptors are involved in the internalization of oxLDL in macrophages, these receptors are absent or present only in a very small amount on endothelial cells.3 Recently, an endothelial oxLDL receptor, LOX-1, was identified on cultured bovine aortic endothelial cells4 and human coronary artery endothelial cells (HCAECs).5 Many of the pro-atherogenic effects of oxLDL in endothelial cells appear to be mediated through LOX-1.6
In vitro and in vivo studies have shown that LOX-1 expression is upregulated by oxLDL.7,8 Angiotensin II (Ang II), which participates in atherogenesis,9 also upregulates the expression of LOX-1.10 Singh and Mehta11 recently proposed an interaction between hyperlipidemia and renin-angiotensin system (RAS) activation in the development of atherosclerosis, during which LOX-1 plays a critical role.
The nucleotide sequence of human LOX-1 promoter has been identified.12 Although only a few details are known about the transcription factors that modulate LOX-1 gene expression, a number of potential cis-regulatory elements, such as nuclear factor-B (NF-B) and activator protein-1 (AP-1) binding motif, are found within the LOX-1 promoter sequence.12 Nonetheless, human LOX-1 transcriptional promoter has not been thoroughly examined on the molecular level.
In a recent study, we examined LOX-1 promoter activation in response to oxLDL in HCAECs, and found the transcription factor octamer-1(Oct-1) is essential for this process.13 To delineate whether Ang II activates LOX-1 promoter through this (Oct-1) or another pathway, we analyzed Ang II–mediated activation of human LOX-1 promoter in HCAECs.
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Methods and Materials |
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LOX-1 Promoter-Luciferase Reporter Plasmid Construction
The promoterless luciferase reporter vector (pGL3 Basic) was obtained from Promega. Fragments of human LOX-1 promoter (both full-length and deletion mutant) were amplified by polymerase chain reaction (PCR), and cloned into pGL3 Basic vector. To confirm the role of NF-
B in Ang II–induced LOX-1 promoter activation, 3 double-strand DNA were synthesized by Invitrogen. They contained 1, 2, or 3 copies of NF-B binding motif, and were cloned into pGL3 Basic vector.
Promoter-Luciferase Constructs Nomenclature
For ease in understanding, full-length LOX-1 promoter-luciferase (from nt –2336 to +36) construct is referred to as "LOX-1 to 2336/+36." The name of the deletion mutant promoters reflects their structure. For example, a 5' deletion promoter construct, which is deleted from nt –2336 to –2000 and retains sequence from nt –1999 to +36, is termed "LOX-1 to 1999/+36." All 3' deletion and subsequent 5' or 3' deletion promoters retain the sequence from nt –35 to +36, which is termed "LOX-1 to 35/+36" and is regarded as the core promoter. A 3' deletion construct, which retains sequence from nt –2336 to –477 (plus nt –35 to +36), is termed "LOX-1 to 2336/–477."
DNA Sequencing
The nucleotide sequences of full-length and deletion mutant LOX-1 promoters amplified from PCR were verified by dideoxy chain-termination method with appropriate primers by the UAMS DNA sequencing laboratory. Nucleotide sequence of all PCR products was analyzed by comparing with human LOX-1 promoter sequence in the DataBase (NCBI Accession number AB021922). For all promoters, the consistency was >99.8%.
Cell Culture, Transient Transfection, and Ang II Treatment
HCAECs were cultured as described previously.5 On reaching 80% confluence, 1 µg LOX-1 promoter-luciferase reporter vector was transfected into HCAECs by FuGENE 6 transfection reagent (Roche). As an internal control for transfection efficiency, 1 µg Renilla luciferase vector (pRL-SV40, Promega) was cotransfected. Six hours after transfection, the transfection reagent was removed, and the transfected HCAECs were cultured in a complete culture medium for 36 hours. These HCAECs were then treated with 1 µmol/L Ang II for 24 hours. The concentration and incubation time of Ang II were based on a previous study.10
Dual Luciferase Assay
The luciferase expression/activity was measured by Dual-Luciferase Report Assay System (Promega), and luminescence was read by luminometer. This system allows the quantitation of activities of both Firefly luciferase (encoded by LOX-1 promoter-pGL3 plasmid construct) and Renilla luciferase (encoded by the pRL-SV40 plasmid).
Computer Analysis of LOX-1 Promoter
TRANSFAC database (MatInspector software) was used to search for the potential cis-regulatory elements within human LOX-1 promoter. The threshold was set at 0.88.
Electrophoretic Mobility Shift Assay (EMSA), Supershift, and Competition Assays
HCAEC nuclear extracts were prepared as previously described.13 The 116-bp LOX-1 promoter fragment (between nt –2247 and –2131), required for Ang II–induced LOX-1 promoter activation, was obtained by PCR. Complementary oligonucleotides containing putative NF-B binding site (5'-AGT TGA GGG GAC TTT CCC AGG C-3'), NF-1 binding site (5'-TTT TGG ATT GAA GCC AAT ATG ATA A-3'), Sp-1 binding site (5'-ATT CGA TCG GGG CGG GGC GAG C-3') or AP-2 binding site (5'-GAT CGA ACT GAC CGC CCG CGG CCC GT-3') were obtained from Invitrogen. All probes were end-labeled with [
-32P] ATP by T4 polynucleotide kinase, and unincorporated [-32P] ATP was removed. The radiolabeled probes were incubated with nuclear extracts for 30 minutes at room temperature. For competition assay, 100-fold excess of cold nonradiolabeled probes were incubated with nuclear extracts for 10 minutes before the addition of radiolabeled probes. As negative controls for competition assay, 100-fold excess of cold nonradiolabeled oligonucleotides containing mutant NF-B binding site (5'-AGT TGA GGC GAC TTT CCC AGG C-3') (Invitrogen) were used as competitors. For supershift assay, rabbit polyclonal antibody to NF-B p65 subunit (Santa Cruz) was used. The experimental protocol was identical to EMSA except that 1 µg, 2 µg, 3 µg, or 5 µg anti-NF-B p65 antibody was incubated with nuclear extracts on ice for one hour before the addition of radiolabeled probes. The specificity was verified by conducting a control assay with 5 µg normal rabbit IgG (Santa Cruz). The DNA–protein complexes were separated by electrophoresis in a 6% nondenaturing polyacrylamide gel using 0.25xTBE running buffer at 150 V for 4 hours.
Site-Directed Mutagenesis
Site-directed substitution mutation of specific nucleotide within human LOX-1 promoter region was performed using In Vitro Site-Directed Mutagenesis System (Promega). We first cloned a LOX-1 promoter-luciferase plasmid construct (LOX-1 to 2247/–2131), which contains a potential NF-B binding site (5'-CAGGAGTT-3'). To make site-directed substitution mutations within this NF-B binding site, a 19-base mutagenic oligonucleotide 5'-GAG GAC ATT AGT trichloroacetic acid (TCA) AGA T-3' was synthesized. After mutagenesis, the original NF-B binding motif (5'-CAGGAGTT-3') was changed to mutant binding motif (5'-CATTAGTT-3'), and the mutation was verified by DNA sequencing.
Confirmation of the Role of NF-B in Ang II–Mediated LOX-1 Promoter Activation
To confirm the role of NF-B in Ang II–induced LOX-1 promoter activation, a separate batch of cells were transfected and pretreated with 10, 20, or 40 µg/mL NF-B inhibitor caffeic acid phenethyl ester (CAPE),14 followed by Ang II treatment. In another set of experiment, cells were cotransfected with NF-B p65 siRNA plasmid (3.1-H1 hygro-p65siRNA) or control plasmid (3.1-H1 hygro), which lacks p65 siRNA,15 followed by Ang II treatment.
Western Blot Analysis of the Expression of NF-B p65 Subunit in HCAECs
Whole cell protein extracts were prepared as described previously,7 and were separated by 10% SDS-PAGE followed by being transferred to nitrocellulose membranes. After incubation in blocking solution (5% nonfat milk, Sigma), membranes were incubated with primary antibodies overnight at 4°C. Antibodies used were 1:500 dilution rabbit polyclonal antibody to NF-B p65 (Santa Cruz), and 1:5000 dilution mouse monoclonal antibody to ß-actin (Sigma). Membranes were then washed with 1xTris-buffered saline Tween 20 solution and incubated with 1:5000 dilution secondary antibody at room temperature for 1 hour. The proteins were detected with the ECL system (Amersham Life Sciences).
Data Analysis
All data represented the mean of samples from at least 3 separately performed experiments. Data were presented as mean±SD and were analyzed with Student t test. A value of P<0.05 was considered significant.
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Deletion Analysis of Ang II–Induced Human LOX-1 Promoter Activation
Compared with the basal level, the full-length LOX-1 promoter (LOX-1 to 2336/+36) was significantly activated by Ang II, whereas all 5' deletion LOX-1 promoter fragments (LOX-1 to 1999/+36, LOX-1 to 1500/+36, LOX-1 to 996/+36, and LOX-1 to 498/+36) were not (Figure IA, available online at http://atvb.ahajournals.org). For 3' deletion LOX-1 promoters, all promoter fragments (LOX-1 to 2336/–477, LOX-1 to 2336/–1059, LOX-1 to 2336/–1502, and LOX-1 to 2336/–1990) were activated by Ang II treatment. However, the core promoter was not affected by Ang II (Figure IB).
From these experiments, we concluded that the LOX-1 promoter region, which responds to Ang II, lies between nt –2336 and –1990 and is a 346-bp fragment. Accordingly, this 346-bp promoter fragment (LOX-1 to 2336/–1990) and a series of 5' and 3' deletion promoter fragments were constructed. All these LOX-1 promoter constructs contained an extra nucleotide sequence from nt –35 to +36, serving as the core promoter. After Ang II treatment, only LOX-1 to 2336/–1990 and 5' deletion promoter LOX-1 to 2247/–1990 were found to be activated, whereas other 5' deletion promoters (LOX-1 to 2149/–1990 and LOX-1 to 2048/–1990) were not activated by Ang II (Figure IIA, available online at http://atvb.ahajournals.org). Next, we observed that 3' deletion promoter fragments (LOX-1 to 2336/–2033 and LOX-1 to 2336/–2131) were activated by Ang II, whereas LOX-1 to 2336/–2231 was not (Figure IIB). These data suggest that the Ang II–responsive cis-regulatory elements reside within the region between nt –2247 and –2131.
EMSA Analysis of the Activation of Transcription Factor NF-B by Ang II
We searched the cis-regulatory element(s) within this 116-bp fragment in the database TRANSFAC, which revealed multiple putative binding sites for transcription factors, including NF-B, NF-1, Sp-1, and AP-2.
To determine the transcription factor binding specificity of this 116-bp LOX-1 promoter region, and to determine the specific transcription factor(s) responsible for Ang II–induced LOX-1 promoter activation, we incubated nuclear extracts from Ang II–treated and control HCAECs with radiolabeled 116-bp LOX-1 promoter fragment, and performed EMSA. There was no shifted band without Ang II treatment. However, a shifted band was observed after Ang II treatment, implying the binding of transcription factor(s) (Figure 1). We also incubated nuclear extracts with radiolabeled oligonucleotides containing putative NF-B, NF-1, Sp-1, or AP-2 binding motif. There was no shifted band for all probes without Ang II treatment. After Ang II treatment, a shifted band was observed with radiolabeled probes containing putative NF-B binding motif, whereas there was still no shifted band with radiolabeled probes containing NF-1, Sp-1, or AP-2 binding motif (Figure 1).
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The binding of NF-B to 116-bp LOX-1 promoter fragment was verified by EMSA-supershift assay using anti-NF-B p65 antibody. We found that treatment of nuclear extracts with anti-NF-B p65 antibody attenuated Ang II–induced shifted band. Note that 5 µg of the antibody significantly abolished the Ang II–induced shifted band. Importantly, rabbit nonspecific IgG, serving as a control, did not affect Ang II–induced shifted band (Figure 2).
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The binding specificity of NF-B to LOX-1 promoter was further verified by EMSA-competition assay. We observed that the Ang II–induced band shift with radiolabeled 116-bp LOX-1 promoter and probes containing NF-B binding motif both were blocked by the presence of nonradiolabeled competitors, whereas 100-fold excess of mutant nonradiolabeled probes, with a mutation within NF-B binding motif, had no effect (Figure 3). We also used 100-fold excess of nonradiolabeled 116 bp LOX-1 promoter fragment as competitors, and identified that the Ang II–induced band shift with radiolabeled 116 bp LOX-1 promoter fragment and probes containing NF-B binding site both were blocked by the competitors (Figure 3).
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Mutations Within the NF-B Binding Motif Knockout Ang II–Induced LOX-1 Promoter Activation
The role of NF-B in Ang II–induced activation of human LOX-1 promoter was also examined by mutational analysis. Two site-directed substitution mutations were made in the potential NF-B binding motif within the 116-bp LOX-1 promoter fragment, changing the original NF-B binding motif (5'-CAGGAGTT-3') to a mutant binding motif (5'-CATTAGTT-3') (Figure IIIA, available online at http://atvb.ahajournals.org). The mutant 116-bp LOX-1 promoter fragment lost its transcription factor binding in response to Ang II treatment (Figure IIIB). Further, the mutant 116-bp LOX-1 promoter was barely activated by Ang II, unlike the wild-type 116-bp LOX-1 promoter (Figure IIIC).
Inhibition of NF-B Attenuates Ang II–Induced LOX-1 Promoter Activation
To confirm the role of NF-B in Ang II–induced activation of human LOX-1 promoter, we constructed luciferase reporter vectors containing 1, 2, or 3 copies of putative NF-B binding motifs upstream of luciferase gene, and examined their activation in response to Ang II. Dual luciferase assay showed that Ang II treatment slightly activated reporter vectors containing 1 and 2 copies of NF-B binding motif, whereas the reporter vector containing 3 copies of NF-B binding motif were greatly activated by Ang II (Figure IV, available online at http://atvb.ahajournals.org).
To further verify the role of NF-B in this process, NF-B activation was inhibited by a chemical NF-B inhibitor CAPE, as well as by NF-B p65 siRNA. The inhibitory effects of CAPE and NF-B p65 siRNA were examined by EMSA. As shown in Figure 4, CAPE in a dose-dependent fashion attenuated Ang II–induced NF-B activation, especially in high concentrations (20 and 40 µg/mL). Further, Ang II–induced NF-B activation was attenuated by NF-B p65 siRNA. The effect of siRNA on NF-B expression was confirmed by Western blot analysis, which showed that the expression of NF-B p65 was reduced after siRNA plasmid transfection (Figure V, available online at http://atvb.ahajournals. org). Note that the control plasmid had no inhibitory effect. In addition, both CAPE (20 µg/mL) and NF-B p65 siRNA attenuated the 116-bp LOX-1 promoter activation in response to Ang II treatment (Figure 5).
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Discussion |
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Analysis of Ang II–Induced LOX-1 Promoter Activation
The Ang II is a major stimulus for LOX-1 gene expression and atherosclerosis.8–11 However, it is not clear whether the regulation of LOX-1 gene expression is transcriptional, translational or both. Further, it is not clear whether the upregulation of LOX-1 gene expression in response to Ang II is caused by the stabilization or by the increased transcription of LOX-1 mRNA. The present study provides definitive evidence that LOX-1 promoter is constitutively active, but at a relatively low level in the basal state, and that it is activated by Ang II.
Our data suggest that the promoter region in response to Ang II lies in the nucleotide sequence between nt –2247 and –2131, a 116-bp fragment. This 116-bp promoter fragment, on treatment with Ang II, had the same activity as the full-length LOX-1 promoter. However, it should be noted that the LOX-1 core promoter (LOX-1 to 35/+36), containing the TATA box as well as the transcription initiation site, is indispensable for Ang II–induced LOX-1 promoter activation, because all LOX-1 promoter constructs without the core promoter cannot be activated by Ang II (data not shown). We postulate that after Ang II treatment, there is a conformational change in the LOX-1 promoter, which is induced by transcription factor(s) or cofactor(s) binding, bringing the region between nt –2247 and –2131 to the proximity of the core promoter. This conformational change and the interaction among transcription factors lead to the activation of the LOX-1 promoter.
Previous studies demonstrated that both Ang II and oxLDL stimulate intracellular oxidative stress and induce the expression of LOX-1 in endothelial cells.16,17 Further, pretreatment of endothelial cells with anti-oxidants attenuates Ang II– and oxLDL–induced LOX-1 expression,16,17 implying a common oxidant signaling pathway. As such, it appears that Ang II may activate LOX-1 promoter through the same mechanism as oxLDL. In a recent study, we found that a distinct promoter region, between nt –1595 and –1494, is required for LOX-1 promoter activation in response to oxLDL.13 There could be 2 explanations for this observation. First, Ang II and oxLDL both induce intracellular oxidative stress, which then activates the same transcription factor. The binding motif for this specific transcription factor is present within the region between nt –2247 and –2131 (required for Ang II–induced promoter activation), and within the region between nt –1595 and –1494 (required for oxLDL-induced promoter activation). After Ang II or oxLDL treatment, the activation of this specific transcription factor causes LOX-1 promoter activation followed by gene expression. The other possibility is that Ang II and oxLDL activate different transcription factors. The binding motif for one exists in the region between nt –2247 and –2131, and that for the other exists in the region between nt –1595 and –1494. Accordingly, although Ang II and oxLDL initiate a common signal, different downstream steps are involved in Ang II–induced and oxLDL-induced LOX-1 promoter activation.
The Critical Role of NF-B in Ang II–Induced LOX-1 Promoter Activation
Computer analysis showed that within the 116-bp promoter region, which is required for Ang II–induced LOX-1 promoter activation, there are at least 4 potential transcription factor binding motifs, including the NF-B binding motif, the NF-1 binding motif, the Sp-1 binding motif, and the AP-2 binding motif. Although the presence of these transcription factor binding motifs within the LOX-1 promoter has been suggested by previous studies,12 it is merely based on computer analysis. Ang II has been shown to activate transcription factors including NF-B, leading to the expression of several genes in endothelial cells.18 However, there is no definitive evidence to support its role in Ang II– induced LOX-1 promoter activation and gene expression.
We examined the role of these transcription factors by multiple approaches, ie, EMSA, supershift, and competition assays, and found that only NF-B, but not NF-1, Sp-1, or AP-2, was activated by Ang II. The role of NF-B in Ang II–induced LOX-1 promoter activation was confirmed by mutagenesis assay. Our results are consistent with previous findings showing the activation of NF-B by Ang II in HCAECs.19 Although we did not observe a supershift band with the anti-NF-B p65 antibody, we found that the Ang II– induced band shift was attenuated by the antibody. The activation of NF-B p50 subunit in response to Ang II in endothelial cells is controversial. Some investigators20,21 have shown the activation of both p50 and p65 subunits by Ang II, whereas others16,22 showed the activation of only NF-B p65 by Ang II. We performed EMSA-supershift assay with antibody against NF-B p50 subunit, and found that Ang II– induced shifted band was somewhat attenuated by the NF-B p50 antibody (data not shown). As such, we believe that both p65 and p50 subunits of NF-B molecule are activated by Ang II in HCAECs. We confirmed the critical role of NF-B by treating HCAECs with CAPE, a chemical inhibitor of NF-B, and a specific p65 NF-B siRNA. Both CAPE, especially in high concentrations, and the siRNA significantly reduced Ang II–mediated NF-B activation and promoter activation. Because the transfection of cells with control plasmid, which lacks p65 NF-B siRNA sequence, had no such inhibitory effect, the role of NF-B, especially p65 subunit, in this process appears to be critical. In this study, we did not observe the complete blockade of NF-B activity by the siRNA; we postulate that it is caused by the relatively low transfection efficiency of endothelial cells, as we observed previously.13 This explanation is supported by the observation that NF-B p65 subunit is still expressed in p65 NF-B siRNA plasmid-transfected cells, though the expression is marked lower than that in cells without transfection. Accordingly, because not all cells are transfected with siRNA plasmid, it is not surprising that p65 NF-B siRNA cannot completely block Ang II–mediated NF-B activation or LOX-1 promoter activation in HCAECs.
It is well-known that Ang II activates NADH/NADPH oxidase, resulting in the production of superoxide anion.23 Superoxide anion may serve as a signaling molecule, mediating the activity of NF-B, which coordinates the upregulation of pro-inflammatory genes such as monocyte chemotactic protein-1.24 Actually, Ang II has pleiotropic effects at multiple points during NF-B activation. It not only induces the translocation of sequestered cytoplasmic NF-B complex via targeted proteolysis of IB but also facilitates the DNA binding of NF-B.25 Ang II has been reported to activate NF-B in several different cell types, such as vascular endothelial cells,17 smooth muscle cells,26 and cardiac myocytes.18 However, ours is the first study to our knowledge demonstrating the role of NF-B in Ang II–induced LOX-1 promoter activation in HCAECs.
The Ang II also activates other transcription factors in vascular endothelial cells, such as AP-117 and transducer and activator of transcription,27 but their binding motifs do not exist within the 116 bp LOX-1 promoter region. Moreover, because a mutation in NF-B binding motif within the 116-bp promoter region results in a total loss of transcription factor binding and Ang II–induced LOX-1 promoter activation, we conclude that NF-B is the major transcription factor for Ang II–induced LOX-1 promoter activation. However, other transcription factors or cofactors may also contribute to the activation of the LOX-1 promoter, which needs to be studied.
Summary
Here we describe that a 116-bp LOX-1 promoter fragment, between nt –2247 and –2131, which, along with the core promoter, is required for Ang II–induced LOX-1 promoter activation. Within this fragment, there is a NF-B binding motif. The activation of NF-B plays an important role in Ang II–induced LOX-1 promoter activation in HCAECs.
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Acknowledgments |
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This study was supported in part by funds from the Department of Veterans Affairs (J.L.M.) and the American Heart Association. We are grateful to Dr Steven Grant of the Medical College of Richmond, Va, for providing NF-
B p65 siRNA plasmid (3.1-H1 hygro-p65 siRNA) and control plasmid (3.1-H1 hygro). Received August 25, 2005; accepted February 1, 2006.
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