Published online before print May 30, 2008, doi: 10.1161/CIRCRESAHA.108.176990
© 2008 American Heart Association, Inc.
Molecular Medicine |
Aryl Hydrocarbon Receptor Is Activated by Glucose and Regulates the Thrombospondin-1 Gene Promoter in Endothelial Cells
From the Department of Molecular Cardiology, Cleveland Clinic, Ohio.
Correspondence to Olga I. Stenina, 9500 Euclid Ave, NB50-66, Cleveland, OH 44195. E-mail stenino{at}ccf.org
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Abstract |
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Hyperglycemia is an independent risk factor for development of diabetic vascular complications. The molecular mechanisms that are activated by glucose in vascular cells and could explain the development of vascular complications are still poorly understood. A putative binding site for the transcription factor aryl hydrocarbon receptor (AhR) was identified in the glucose-responsive fragment of the promoter of thrombospondin-1, a potent antiangiogenic and proatherogenic protein involved in development of diabetic vascular complications. AhR was expressed in aortic endothelial cells (ECs), activated, and bound to the promoter in response to high glucose stimulation of ECs. The constitutively active form of AhR induced activation of the thrombospondin-1 gene promoter. In response to high glucose stimulation, AhR was found in complex with Egr-1 and activator protein-2, which are 2 other nuclear transcription factors activated by glucose in ECs that have not been previously detected in complex with AhR. The activity of the DNA-binding complex was regulated by glucose through the activation of hexosamine pathway and intracellular glycosylation. This is the first report of activation of AhR (a receptor for xenobiotic compounds) by a physiological stimulus. This report links the activation of AhR to the pathological effects of hyperglycemia in the vasculature.
Key Words: aryl hydrocarbon receptor • glucose • thrombospondin-1 • endothelial cells
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Introduction |
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Hyperglycemia is an independent risk factor for vascular complications of diabetes.1–5 Endothelial dysfunction is the earliest sign of developing diabetic vascular complications (reviewed recently6–8). Hyperglycemia affects the expression of numerous endothelial proteins,9–11 including thrombospondin (TSP)-1,12 a potent antiangiogenic and proatherogenic protein implicated in the development of a variety of vascular diabetic complications.12–15 We report here that high glucose activates aryl hydrocarbon receptor (AhR) in endothelial cells (ECs), which activates transcription of the TSP-1 gene (THBS1).
AhR is a transcription factor (TF) known to be activated by aromatic hydrocarbons, eg, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) present in industrial waste, tobacco smoke, and byproducts of herbicides.16–18 Although the connection between AhR expression or activity and atherogenesis has not been explored directly, multiple epidemiological and animal studies have established the association between known AhR activators and heart disease.18–23 Recent reports demonstrated that AhR negatively affects angiogenesis in cancer and ischemia models,24–26 further implicating this TF in regulation of endothelial function.
The abnormalities observed in AhR knockout mice include cardiac hypertrophy,27,28 altered insulin regulation and responsiveness, altered glucose tolerance in pregnant females,29 and immune system impairment.30,31 Although AhR is clearly required for a variety of physiological processes,32–36 physiological activators of AhR are unknown, and only a few recent reports describe activation of AhR in response to pathological stimuli.24,37,38 The mechanism of AhR transcriptional activity and the target genes have not been comprehensively studied. There is no information on regulation of gene expression by AhR as a result of metabolic abnormalities.
Our results demonstrate that AhR is rapidly activated in ECs in response to high glucose. Active AhR associates with the thrombosponsin-1 gene (THBS1) promoter and activates it. AhR forms a complex with several other TFs activated by glucose: activator protein (AP)-2, Egr-1, upstream stimulatory factor (USF)-2, and Pax-5. This complex is different from the complex formed by AhR and AhR nuclear translocator (ARNT) (hypoxia-inducible factor [HIF]1β) in response to xenobiotics, and the activity of the complex is regulated by glycosylation.
This is the first report of AhR activation by high glucose that links AhR to the physiological regulation of gene expression by glucose and the pathological effects of hyperglycemia in the vasculature.
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Materials and Methods |
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Cell stimulation with high glucose has been described previously.12,15
Antibodies Used
Anti-AhR was from Novus Biologicals (Littleton, Colo) and Abcam (Cambridge, Mass); anti–Egr-1 was from Cell Signaling Technology (Danvers, Mass); anti–USF-1 and anti–USF-2 were from Santa Cruz Biotechnology Inc (Santa Cruz, Calif); RL2 was from Abcam; and anti–AP-2 was from AbD Serotec (Raleigh, NC).
Promoter Reporter Constructs
The fragments –280/+66 pTHBS1 and –265/+66 pTHBS1 (
AhR) were generated by PCR.
Mutants: (1) 5'-AGCCCGCGAGGCGA-3'; (2) 5'-AGCCCGGCTGGCGA-3'; (3) 5'-AGCCCGGCAGGCGA-3'; wild type: 5'-AGCCCGCGTGGCGCA-3'. Core sequence of the AhR binding element is underlined, and nucleotide substitutions are in bold,
Analysis of the Binding Sites for TFs in the THBS1 Promoter Region Responsive to Glucose
The sequence of pTHBS1 was analyzed using MatInspector 7.4.3 (Genomatix, http://www.genomatix.de).39
Plasmids for the Expression of AhR
The constitutively active form of AhR was prepared by constructing the AhR deletion mutant as described previously for murine AhR.40
Analysis of activation of TFs in glucose-stimulated human aortic ECs (HAECs) was performed using TranSignal Combo Protein/DNA array (Panomics).
Immunofluorescence
Anti-AhR antibody (Novus Biologicals) and goat antimouse Alexa Fluor–labeled secondary antibody (Invitrogen) were used to stain sections of rat aorta.12
Treatment of ECs with glycosylation inhibitors and metabolites of hexosamine pathway was done as described earlier.15
Statistical Analysis
All of the described experiments were performed more than 3 times, and the data are presented as mean values±SEM. Probability values were determined by t test using Microsoft Excel, and probability values of <0.05 were considered statistically significant.
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Results |
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The Minimal Fragment of Human THBS1 Gene Responsive to High Glucose in ECs
We have reported recently that the expression of TSP-1 is increased in response to high glucose (10 to 30 mmol/L) in all the major vascular cell types,12 and the increase in TSP-1 mRNA level is transcriptionally regulated.15 The increase in mRNA levels could be detected as early as 1 hour after the start of stimulation in cultured ECs and could still be detected at 72 hours in all vascular cell types.12 We have analyzed the activity of TSP-1 promoter deletion constructs to identify the promoter elements responsible for this regulation in ECs. The –280/+66 pTHBS1 fragment was activated in response to stimulation of human umbilical vein ECs (HUVECs) by 30 mmol/L glucose (indicated by a 6-fold increase in activity of luciferase), and this activation was abolished by deletion of 15 base pairs in –265/+66 pTHBS1 (
AhR) (Figure 1A), suggesting that a putative binding site for the TF AhR predicted in this 15-bp region may control the response to high glucose. The response to glucose was inhibited in –380/+66 and longer promoter fragments, suggesting a presence of an inhibitory element in the promoter between –280 and –380, which is active in ECs, but not in vascular smooth muscle cells15 or mesangial cells.41
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We analyzed the –280/+66 fragment of THBS1 using MatInspector (Genomatix) to identify putative binding sites for TFs. This analysis identified several putative binding sites (Figure 1B), including a binding site for AhR in the fragment responsible for glucose stimulation (–272; see Figure 1B). The putative binding site for AhR overlapped with the predicted binding site for USF (–274), and this sequence was also recognized by the program as a carbohydrate response element.
Identification of TFs Activated by High Glucose in ECs
We focused on TFs that are rapidly activated in response to high glucose and are still active at 24 hours.
To identify the TFs rapidly activated in response to acute treatment with high glucose (1-hour stimulation with 30 mmol/L glucose), we used 4 separate isolates of HAECs. A targeted proteomic approach was used to identify the activated TFs. TranSignal Protein/DNA array (Panomics Inc, Fremont, Calif) identifies only active TFs in nuclear extracts. Among the TFs activated in response to glucose treatment were six TFs whose putative binding sites have been predicted in the –280/+66 fragment of the THBS1 promoter: AhR, AP-2, Egr-1, USF, nuclear factor (NF)-
B, and Pax-5 (Table). We have confirmed the activation of Egr-1 and AP-2 using alternative methods of detection: Northern blotting and electromobility shift assay (EMSA) (see the online data supplement, available at http://circres.ahajournals.org). The values in the Table were calculated based on increase in 4 independent experiments. We have chosen to analyze only the values for TFs activated greater than 2.5-fold in at least 2 of 4 experiments. Activation of 5 of 6 TFs was statistically significant, but the values for Pax-5 did not reach statistical significance. Figure 2A demonstrates representative array results for the five TFs significantly stimulated in response to high glucose in HAECs. USF, NF-B, and Egr-1 are known to be activated in response to high glucose or in diabetics.42,43 However, to our knowledge, this is the first report of activation of AhR and AP-2 by glucose.
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Confirmation of Activation of AhR in Electromobility Shift Assay
Nuclear extracts from HAECs and HUVECs were used in an EMSA with a consensus AhR probe to confirm the activation of AhR by high glucose (Figure 2B). Two DNA-binding protein complexes in both EC types were activated in response to 30 mmol/L glucose (complex 1 and complex 2; Figure 2B). When anti-AhR antibodies were used, they either prevented binding of both complexes to the probe or resulted in a supershift in EMSA. Unrelated antibody (AbX) was used as a control. When the probe corresponding to the sequence of the THBS1 promoter containing the predicted binding site for AhR was used, activation and binding of AhR was also detected, and the binding was prevented by anti-AhR antibody (Figure 2C).
AhR Is Expressed in ECs
AhR is constitutively present in the cytosol and is translocated to the nucleus on activation by its ligand.44–46 We have detected AhR in both cultured HAECs and luminal ECs of the rat aorta (Figure 3A and 3B).
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AhR Associates With TSP-1 Promoter in Glucose-Stimulated HAECs
Chromatin immunoprecipitation from HAECs stimulated with 30 mmol/L glucose for 1 hour was performed with anti-AhR antibody, followed by the PCR amplification of the THBS1 promoter region. It revealed that AhR associates with TSP-1 promoter only on glucose treatment, whereas the general nuclear factor TFII is associated with the promoter in both control and glucose-treated ECs (Figure 4A).
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Constitutively Active AhR Activates the THBS1 Promoter
When the constitutively active form of AhR was cotransfected with the luciferase reporter/–280/+66 pTHBS1 promoter construct in HEK293 cells, the activity of luciferase was increased more than 4-fold as compared with cells transfected with the control plasmid (Figure 4B). The full-length AhR (inactive form) failed to induce any stimulation of the promoter (Figure 4C).
To confirm that the promoter activity in response to active AhR is dependent on the binding site for this TF (–272), we generated mutant –280/+66 pTHBS1 promoter constructs with 1, 2, and 3 nucleotide substitutions in the core of the AhR binding site as described in Materials and Methods (Mut1, -2, and -3). The activity of these mutants remained at the basal level when cotransfected with active AhR (Figure 4C). When HUVECs transiently transfected with these three mutants were stimulated with high glucose, none of the mutant –280/+66 pTHBS1 was activated in response to glucose (Figure 4D), clearly confirming that the activation of –280/+66 pTHBS1 promoter by glucose depends on the AhR-binding sequence.
Complexes Formed by AhR in High Glucose–Stimulated ECs
Several TFs are known to form complexes with active AhR. The most common is HIF1β (ARNT), which associates with AhR in response to xenobiotic activators (reviewed elsewhere47); other proteins, including TFs, have been identified (reviewed elsewhere45,48). We have used a TF/TF interaction array (Panomics) that detects only active TFs in complex with the TF of interest and is based on the immunoprecipitation. The experiments revealed that AhR can be precipitated in complex with several TFs activated in response to glucose stimulation and having predicted binding sites in close proximity to the AhR binding site in the THBS1 promoter. These are AP-2, Egr-1, USF, and Pax-5 (Figure 5A), suggesting that these TFs may form a complex on the promoter of THBS1 where the corresponding binding sites are in close proximity to each other in the region –280/+66. Neither of these proteins was precipitated with nonimmune mouse IgG (data not shown).
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To confirm these TFs form a complex in response to high glucose, we used corresponding antibodies in the binding reaction with consensus AhR probe (Figure 5B and 5C) and TSP-1 probe (Figure 5D). Formation of the specific complexes was prevented by anti-AhR antibody, whereas an unrelated antibody did not affect the binding (Figure 5B). To characterize the complex of TFs, the following antibodies were used in combination with the AhR consensus probe in EMSA: anti-AhR (Figure 2B and 2C and Figure 5B through 5D), anti–Egr-1 (Figure 5B through 5D), anti–AP-2 (Figure 5C and 5D), anti–USF-1, and anti–USF-2 (Figure 5C and 5D). With the exception of USF-1, all of these TFs were found in complex with AhR. Anti-AhR, anti–Egr-1, anti–AP-2, and anti–USF-2 antibodies either prevented the binding of the complex to the labeled probe or supershifted the complex band in EMSA. Anti–Egr-1 antibody prevented the formation of lower complex (complex 2) but did not affect the binding of complex 1 to the AhR probe, suggesting that Egr-1 is present in association with AhR in complex 2, but complex 1 is formed by AhR and other TFs (Figure 5B). When TSP-1 probe was used, anti–Egr-1 antibody inhibited the formation of the complex. USF-1 antibody supershifted USF-1 protein in EMSA in an unrelated experiment (data not shown). Antibody against HIF1β, the most common partner of AhR, did not affect either of the 2 complexes (data not shown), suggesting that HIF1β is not present in the complex.
Activation of DNA-Binding Complex Is Regulated by Glycosylation
We have recently reported that in vascular smooth muscle cells, intracellular glycosylation is responsible for activation of THBS1 transcription.15 We used RL2 antibody (anti–O-linked glucosamine N-acetyl [anti–O-GlcNAc]), which recognizes glycosylated intracellular proteins, in EMSA to detect glycosylation of a protein(s) in complexes formed on the AhR consensus or TSP-1 probes (Figure 5C and 5D). To confirm that glycosylation regulates the formation of the complex or its DNA-binding activity, we treated ECs with: (1) 6-diaz-O-5-oxonorleucine (DON), which is an inhibitor of glutamine:fructose 6-phosphate amidotransferase (GFAT), an enzyme controlling the hexosamine pathway of glucose metabolism leading to formation of metabolites for glycosylation; (2) glucosamine, a glycosylation residue precursor and a glucose metabolite entering the hexosamine pathway downstream of GFAT; (3) amino-N-phenylcarbamate (PUGNAc), known to increase O-linked protein glycosylation by effectively inhibiting β-N-acetyl-glucosaminidase (O-GlcNAcase), an enzyme responsible for cleavage of O-GlcNAc residues from intracellular proteins.15 The inhibitor of GFAT prevented activation of the AhR complex (Figure 6), and both glucosamine and PUGNAc resulted in activation of the complex without stimulation with high glucose, confirming that the activation depends on an intracellular glycosylation event. As expected, DON did not prevent complex formation in response to treatment of ECs with glucosamine, which acts downstream of GFAT, confirming that the effect of DON was specific. PUGNAc did not induce the formation of the complex in the DON-treated cells, and this is consistent with our previous observations15: PUGNAc is an inhibitor of deglycosylation, and a certain level of glycosylation is required to see its effect. However, in the DON-treated cells, glycosylation is inhibited.
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Discussion |
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Despite numerous studies on activation of AhR by xenobiotics,48,49 its physiological activators, ligands, and mechanism of activation remain unknown. Multiple indications of its role in normal physiology and pathology exist, including the AhR knockout mouse that develops metabolic abnormalities and cardiac hypertrophy.28–30 Recent publications have reported a new role for AhR in response to UV exposure,37 modified low density lipoproteins,38 and hypoxia,24 further establishing its role in pathological changes.
Our results clearly demonstrate that glucose activates AhR in ECs and that AhR controls the expression of TSP-1, a potent antiangiogenic and proatherogenic protein. EC function determines physiological and pathological angiogenesis and initiation of atherosclerotic lesions in the large blood vessels. Therefore, activation of AhR by high glucose in diabetics or during postprandial elevation of glucose levels may initiate a series of pathological events leading to endothelial dysfunction and resulting in vascular disease. The epidemiological association between heart disease and industrial wastes and cigarette smoke, which both contain activators of AhR, also indicates a possible role for AhR in the development of atherosclerotic changes caused by environmental factors.
The role of AhR in the activation of the THBS1 promoter was initially established by promoter analysis. The promoter region –280/+66, responsible for the increased transcription of THBS1, was identified, and the AhR/USF binding site was predicted in this region, also recognized by MatInspector as a carbohydrate response element. The regulation of the THBS1 promoter by high glucose in ECs appears to be different from the regulation in mesangial cells41 or vascular smooth muscle cells,27 where a longer promoter fragment is required for the response. The activation of AhR in ECs was detected by a targeted proteomic approach and confirmed using EMSA. Furthermore, active AhR bound to the promoter of the endogenous THBS1 gene in response to glucose. Overexpression of the constitutively active form of AhR but not the full-length AhR, together with the THBS1 promoter/reporter construct, further confirmed the activation of the promoter by AhR. The role of the AhR-binding sequence (–272) was confirmed using the mutant constructs with substitutions in the core sequence of the AhR-binding site: the mutant promoter fragments could not be activated in response to high glucose.
Gene transcription in response to extracellular and intracellular stimuli depends both on the promoter structure and on the signal- and cell type–specific patterns of activation of transcriptional activators, coactivators, and suppressors. TFs and coactivators form signal- and cell type–specific multiprotein complexes on the promoters. We have identified 5 proteins in complex with AhR in glucose-stimulated ECs: AP-2, Egr-1, USF-2, and Pax-5. Interestingly, but not surprisingly, the putative binding sites for all 4 TFs were predicted in the glucose-responsive fragment of the THBS1 promoter in close proximity to the AhR binding site (MatInspector). Of the multiple TFs with predicted binding sites in this region, only 5 were consistently activated (at least 2.5-fold) in response to high glucose in HAECs (AhR, AP-2, Egr-1, USF, NF-B), and 4 of them represented the proteins found in complex with AhR in both coprecipitation and supershift experiments with the corresponding antibodies. Although the probe-based analyses did not allow to distinguish between USF-1 and USF-2, which both can bind to the same DNA sequence and form homo- and heterodimers, further analysis suggested that USF-2 is present in the complex, but not USF-1. An anti–USF-1 antibody that we used to supershift USF-1 complex in an unrelated study with a different cell type (data not shown) failed to change the binding of the AhR complex to both AhR concensus and AhR THBS1 probe.
Although the activation of Egr-1 and NF-B in ECs and the activation of USF in other cell types and tissues in response to hyperglycemia or in diabetics has been reported previously,42,50–53 this is the first report of activation of AhR and AP-2 by high glucose. An antibody against HIF1β did not affect AhR complexes in EMSA, and this TF was not activated by high glucose in ECs, suggesting that AhR-dependent transcriptional mechanisms activated in response to glucose differ from the well-described mechanisms activated by xenobiotics.
Our data clearly demonstrate that activation of the transcriptional complex depends on glycosylation of at least 1 of the proteins: RL2 antibody recognizes proteins modified by O-linked N-acetylglucosamine, and this antibody supershifted the complex. Furthermore, inhibitors of the hexosamine pathway prevent formation of intermediates for intracellular glycosylation, and these inhibitors also prevent the activation of the DNA-binding protein complex, whereas the inhibitors of deglycosylation and downstream intermediates of the pathway caused formation of the complex. Further confirmation that AhR can be directly glycosylated and the identification of glycosylation sites are clearly mandated by these observations.
The activation of specificity protein (SP)-1 in response to stimulation with high glucose was previously reported in both ECs54 and vascular smooth muscle cells.55 SP1 undergoes posttranslational modification by O-linked N-acetylglucosamine, which prevents its degradation in vascular smooth muscle cells.55 Our array experiment did not detect any activation of SP1 in ECs or in vascular smooth muscle cells (data not shown), and our attempts to detect activated SP1 in nuclear extracts using a consensus probe were unsuccessful (data not shown). We believe that SP1 may be activated at the later time points but not at the earlier time points analyzed in our studies. THBS1 transcription is activated rapidly in response to high glucose,15 and this rapid activation suggests that only TFs activated before and at the onset of THBS1 activation are involved in the regulation of the promoter in response to acute glucose stimulation.
The results of this work document a novel physiological and pathological role for AhR in the response of vascular ECs to hyperglycemia. Despite the rapidly accumulating evidence that ECs respond to glucose by activation of a variety of genes, the transcriptional mechanisms of this regulation have not been well explored, with the exception of a few reports identifying the specific transcriptional factors mediating a change in gene expression in response to glucose.42,56–58
This transcriptional mechanism provides a novel and unexpected link between hyperglycemia and the expression of TSP-1, a potent antiangiogenic and proatherogenic protein involved in the development of multiple diabetic vascular complications.
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Acknowledgments |
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We thank Dr Paul Bornstein (University of Washington) for cDNA of THBS1 promoter, Dr DiCorleto and Lori Mavrakis (Cleveland Clinic) for HUVECs, Dr Priya Raman (Cleveland Clinic) for help with preparation of the biotinylated TSP-1 probe and advice on the design of TSP-1 promoter mutants, Yana Pleshivoy and Christy Harry (Cleveland Clinic) for technical help in preparation of the constructs and plasmids, and Tim Burke (Cleveland Clinic) for help with manuscript preparation.
Sources of Funding
This work was supported by NIH grants K01 DK62128, P50 HL077107, and R01 DK067532, American Heart Association Grant 0565284B, and funds from the Lerner Research Institute (Cleveland Clinic Foundation) (to O.I.S.).
Disclosures
None.
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Footnotes |
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Original received October 12, 2007; resubmission received April 3, 2008; revised resubmission received May 8, 2008; accepted May 16, 2008.
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