Published online before print January 2, 2007, doi: 10.1161/01.HYP.0000254350.62876.b1
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(Hypertension. 2007;49:631.)
© 2007 American Heart Association, Inc.
Original Articles, Part 2 |
The –20 and –217 Promoter Variants Dominate Differential Angiotensinogen Haplotype Regulation in Angiotensinogen-Expressing Cells
From the Interdisciplinary Genetics Program (M.E.D., C.D.S.), Medical Scientist Training Program (M.E.D.), Department of Biostatistics (M.B.Z.), Department of Internal Medicine (K.R., C.D.S.), and Molecular Physiology and Biophysics (C.D.S.), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City.
Correspondence to Curt D. Sigmund, Departments of Internal Medicine and Physiology and Biophysics, 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242. E-mail curt-sigmund{at}uiowa.edu
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Abstract |
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A number of naturally occurring polymorphisms exist in the human angiotensinogen locus, some of which have been associated with essential hypertension, preeclampsia, and other medical disorders. However, to date there has been no comprehensive determination of the significance of specific haplotypes in relation to the regulation of angiotensinogen expression. We cloned the promoters extending from –1219 to +125 bp from 11 ethnically diverse individuals to acquire a representative cross-section of known haplotype diversity. Eight nonredundant haplotypes were identified, fused to luciferase, and studied for their effect on transcriptional regulation in human astrocyte, proximal tubule, and hepatocyte cell lines endogenously expressing angiotensinogen and in a mouse adipocyte cell line. The studies were carried out under baseline conditions, in the presence of the angiotensinogen enhancer, and in response to hormonal stimulation by dexamethasone, ß-estradiol, or testosterone. A statistical model was then constructed to assess the significance of individual polymorphisms. The polymorphisms with the greatest effect on transcription in these cell lines were located at –20 and –217. There were modest haplotype-specific effects of the angiotensinogen enhancer and no haplotype-specific effects of ß-estradiol, dexamethasone, or testosterone treatment. We conclude the following: (1) the –20 and –217 polymorphisms have the largest influence on angiotensinogen transcription, (2) other polymorphisms have a much smaller impact on angiotensinogen transcription, and (3) the transcriptional influence of the promoter polymorphisms may act cell specifically. Therefore, our data support a hypothesis that polymorphisms in the angiotensinogen promoter may act cell specifically to differentially regulate the level of angiotensinogen transcription in angiotensin-producing tissues.
Key Words: transcription • genetics • renin–angiotensin system • hypertension • transfection
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Introduction |
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The etiology and pathogenesis of high blood pressure are complicated by the varied molecular pathways, genetic polymorphisms, and environmental conditions that converge on blood pressure regulation. The genetic component of hypertension is strong, as shown by twin studies,1 and multiple genes from diverse molecular pathways have been linked to blood pressure. Among these pathways, the renin–angiotensin system is important because of its effects on salt and water homeostasis and autonomic control, as well as vascular structure and function. It is generally accepted that the rate-limiting step of the renin–angiotensin system is the enzymatic cleavage of angiotensinogen (AGT) by renin. Increases in plasma AGT or renin may translate into a physiological effect, because the level of plasma AGT is close to the Michaelis constant for the reaction.2 Transgenic models overexpressing AGT and renin exhibit increased blood pressure and manifest hypertensive sequelae.3,4 Gene dosing experiments in gene-targeted mice have suggested that each additional copy confers a modest elevation of plasma AGT and approximately an 8-mm Hg increase in blood pressure.5 In addition, certain polymorphisms in the AGT gene in humans correlate with modestly increased circulating AGT and with hypertension.6,7
Since the initial report in 1992 that the AGT locus is linked with hypertension in humans,8 hundreds of studies, using mainly the case–control design, have been reported implicating specific polymorphisms in the AGT locus with high blood pressure, its sequelae, and other cardiovascular and noncardiovascular end points (reviewed in Reference 9). Early studies focused primarily on 2 coding polymorphisms, T174M and M235T,8 but these were ultimately reported to not affect the kinetics of the enzymatic reaction with renin.6
Two regions of the AGT gene have been implicated as important regulators of AGT expression: the promoter and nearby elements located 
1.2 kb upstream of exon 1 and an enhancer found just after the second polyadenylation site in the 3' flanking region.10 Although the 3' flanking enhancer greatly increases expression of a minimal AGT promoter in HepG2 cells, its importance in vivo has been questioned.11 One of the original polymorphisms identified in AGT, M235T, was reported to exist in nearly complete linkage disequilibrium with a promoter polymorphism located 6 bp upstream of the transcription start site.6 Polymorphisms in the promoter region are of significance, because they may influence the strength of the AGT promoter and, consequently, the levels of AGT and angiotensin II. They are thought to garner significance by differentially binding transcription factors, but only 1 transcription factor has been identified to bind overlying –6 (in an allele-independent manner).12 Like –6, other polymorphisms have been identified in the AGT promoter region, and 2 of these located at –20 and –217 have been reported to exhibit differential transcription factor binding. The factors reported to bind the region surrounding –20 include upstream stimulatory factor (USF), estrogen receptor (ER) 
, and apolipoprotein A1 regulatory protein 1, whereas glucocorticoid receptor and CCAAT box/enhancer binding protein (C/EBP) have been reported to bind at –217.13–16
Individual polymorphisms do not exist in isolation. Instead, a series of polymorphisms in the AGT 5' flanking region are linked to form haplotypes. Differential transcriptional activity of the promoter results from the influences of each polymorphism and the interaction among polymorphisms making up the haplotype. Although several studies have reported haplotypes in the AGT 5' flanking region,17,18 there have been no studies that have systematically examined the transcriptional effects of combinations of polymorphisms in the AGT promoter that exist in naturally occurring haplotypes. Therefore, the first goal of this study was to evaluate the transcriptional activity of the AGT promoter in 8 naturally occurring haplotypes containing polymorphisms at –6, –20, –217, and at 11 other positions between –1219 and +125 bp.
Previous studies examining the regulation of the AGT promoter or the effects of its polymorphisms were largely performed in cell lines derived from the liver. Indeed, AGT is also expressed in kidney, adipose tissue, and brain, among other tissues. The importance of AGT synthesis in these tissues in blood pressure control has been documented in transgenic models specifically targeting either overexpression or ablation of the renin–angiotensin system genes.19–22 Therefore, the second goal of this study was to assess the transcriptional consequences of natural AGT promoter variation in 4 AGT-expressing cells lines derived from liver (HepG2), renal proximal tubule (HK-2), glial cells in the central nervous system (CCF), and adipose tissue (differentiated 3T3-L1). We did this to test the hypothesis that polymorphisms in the AGT promoter may have cell-specific influences on transcriptional activity.
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Methods |
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Cell Culture
Cell culture media and supplements were purchased from Gibco (Invitrogen) unless otherwise specified. HepG2 cells were cultured in DMEM containing 4.5 g/L of D-glucose, L-glutamine, 10% FBS, 1.0 mmol/L of sodium pyruvate, 0.1 mmol/L of nonessential amino acids, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. HK-2 cells were cultured in RPMI 1640 medium with 10% FBS, 2 mmol/L of L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. CCF cells were cultured in RPMI 1640 medium with 10% FBS, 10 mmol/L of HEPES, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. 3T3-L1 cells were cultured in DMEM containing 4.5 g/L of D-glucose and L-glutamine, with 10% FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. 3T3-L1 preadipocytes were differentiated as described previously23 and as detailed in the Supplemental Methods (available online at http://hyper.ahajournals.org). Oil Red O staining (Sigma) of 3T3-L1 cells was performed as described previously to confirm their differentiation into adipocytes (Supplemental Figure I, available online at http://hyper.ahajournals.org).23 All of the cells were split using brief treatment with 0.25% trypsin-EDTA at 37°C after rinsing with Dulbecco’s PBS. Hormones for stimulation studies included dexamethasone, testosterone, and ß-estradiol (Sigma). Stock solutions were made with 2% ethanol and 98% culture medium, which also served as vehicle.
Western Analysis
Total protein extracts stored at –80°C were prepared by rinsing cells with PBS; lysing in 50 mmol/L Tris (pH 8), 10 mmol/L EDTA, 10 µg/mL leupeptin, and 1% Triton X-100; and then centrifuging. Western blotting was as described previously24 using purified human plasma AGT as the positive control (Biodesign International). T-52 rabbit anti-human AGT polyclonal primary antibody was used at 1:10 000 dilution (a gift from Duane Tewksbury, Marshfield Medical Research Foundation, Marshfield, WI).
Vectors, Transfections, and Transcriptional Reporter Assays
Vectors for transfection analyses included pGL3-Control, pGL3-Basic, pRL (Promega), and modified versions of the pGL3-Basic vector containing the haplotypes of the human AGT promoter with or without the human AGT enhancer. The human AGT promoter was amplified from 11 individuals of the Coriell Polymorphism Discovery Resource using the primers 5'-AGATCTCTCGAGACAA GTGATTTTTGAGGAGTCCCTATC-3' and 5'-AGATCTAAGCTT CTCCTCCCGGCCTTTTCCTCCTA-3' including sites for XhoI and HindIII. The Coriell Resource is composed of DNA samples from unrelated individuals designed to reflect the diversity in the human population representing the following ethnicities: European, African, Mexican, Native American, and Asian. Because the DNA samples were completely anonymous, the research was deemed exempt by the University of Iowa Institutional Review Board. Clones were sequenced and compared with the NCBI database, yielding 8 nonredundant haplotypes. The AGT enhancer was amplified from 1 individual of the Coriell panel and cloned upstream of the AGT promoter into each of the 8 nonredundant haplotype vectors. Primers used to amplify the AGT enhancer contained the sequences 5'-CGGGGTACCGTGCAAACGAAAGTGC-3' and 5'-GCCGAGCTCACTGGCTCAGACCTCC-3' with restriction sites for KpnI and SacI. All of the clones were confirmed by sequencing.
Transfection of HepG2, CCF, HK-2, and 3T3-L1 cells was optimized in our laboratory to use 35 µL of LipoTAXI (Stratagene) for HepG2 and HK-2 cells, 10 µL Lipofectamine 2000 (Invitrogen) for CCF cells, or 10 µL Lipofectamine 2000 in addition to 20 µL Plus reagent (Invitrogen) for differentiated 3T3-L1 cells. Quantifications of firefly luciferase and Renilla luciferase expression were performed according to the manufacturer’s protocol for the Dual-Luciferase Reporter Assay System (Promega) using a 96-well MicroLumatPlus luminometer (Berthold Technologies). Each measurement was performed in duplicate. Additional details of the transfection are provided in the Supplemental Methods.
Statistical Analysis
The statistical analyses were performed using SigmaStat (Systat Software, Inc) and SAS (version 9.1, SAS Institute Inc). The normality assumption of data distribution was checked using Shapiro–Wilk’s test, which found that baseline luciferase data from each cell line were not normally distributed. To normalize the data distribution, the log normal transformation was applied. All of the graphs plot error bars ± SE and were prepared using SigmaPlot (Systat Software, Inc). A P value 
0.05 was considered significant in all of the cases. The determination of which individual single nucleotide polymorphisms (SNPs) accounted for the greatest variation in AGT reporter expression under baseline cell culture conditions was made possible by developing a multiple linear regression model in which the polymorphisms were each assigned an independent variable within each cell type. A detailed explanation of the model is provided in the Supplemental Methods.
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Results |
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Previous studies of the AGT promoter have focused primarily on the association of individual SNPs in cells derived mainly from the liver13,14 or in cells representative of other AGT-expressing tissues that do not express AGT themselves.25 Because these SNPs coexist in close proximity and in linkage disequilibrium with other SNPs, we used an experimental protocol to examine the transcriptional function of naturally occurring polymorphisms in an unbiased way. We cloned
1.3 kb of the AGT promoter from DNA of 11 ethnically diverse individuals obtained from the Coriell Polymorphism Discovery Resource. An analysis of 21 clones revealed 8 nonredundant haplotypes that varied at 14 different positions, including –6, –20, –217, and 11 others (Table 1). These 8 haplotypes seemed to achieve the following: (1) they accurately represent known haplotypes genotyped previously at either 5 or 8 of the 14 positions that we identified; (2) they were found in proportion to their previously reported frequency; and (3) they accounted for 93%, 88%, and 89% of known haplotype diversity in whites, Japanese, and Africans, respectively.17,18 Given this coverage, we did not continue to sequence other samples from the resource.
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We performed transfection experiments and measured luciferase activity in 3 human cell lines and 1 mouse cell line of diverse origin that represent physiologically important sites of AGT synthesis in vivo. These include HepG2 (derived from hepatocytes), HK-2 (derived from renal proximal tubule), CCF (derived from astrocytes of the brain), and differentiated mouse 3T3-L1 adipocytes. We confirmed that each cell line expresses endogenous AGT protein, although at substantially differing levels (Figure 1A and 1B). Importantly, the strength of hAGT promoter activity in transfected HepG2, CCF, and HK-2 cells varied in proportion to the level of endogenous AGT protein (Figure 1C).
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Transcriptional assays were first performed under unstimulated conditions (Figure 2). Baseline reporter expression varied from 2- to 5-fold between haplotypes, and the overall pattern of transcriptional activity was very similar among HepG2, HK-2, and differentiated 3T3-L1 cells. In these 3 cells lines, haplotypes 1 and 8 generally exhibited the highest activity, whereas haplotypes 2 to 4 generally exhibited the lowest. In contrast, the dominant expression pattern of haplotypes 1 and 8 observed in HepG2, HK-2, and 3T3-L1 cells was not evident in CCF cells where there was much less variation of transcriptional activity among haplotypes.
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To assess the importance of individual SNPs in each haplotype, we developed a linear regression model where each SNP (or combination of SNPs in some cases) was considered an independent variable. This model allowed us to determine the relative strength of an individual polymorphism while controlling for variation that occurred at other positions within a haplotype and that may have transcriptional effects on their own. The need for the model becomes evident when trying to assess the relative importance of the –20 and –217 polymorphisms. From the data presented in Figure 2 it is clearly evident that changing –20A to –20C increases transcriptional activity in 3 of the 4 cell lines (compare haplotypes 2, 3, or 4 that contain –20A with haplotypes 1 or 8 that contain –20C). On the contrary, the effect of –217A is difficult to directly assess, because it always covaries with –20A (see haplotypes 6 and 7 in Table 1), whereas –217G covaries with either –20C (haplotype 1) or –20A (haplotype 2). The importance of the –217 polymorphism is easily observed in HepG2 and HK-2 cells by comparing transcriptional activity of haplotypes 2, 3, or 4 (–217G and –20A) with haplotypes 6 or 7 (–217A and –20A), but its importance in CCF and 3T3-L1 cells is only uncovered after the model takes into account the effects of other linked polymorphisms (Table 2).
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The data in Table 2 show the following: (1) there is a dominant effect of the –20 and –217 polymorphisms in all 4 of the cell types; (2) –20 and –217 are equally important in HepG2, HK-2, and 3T3-L1 cells; (3) the relative importance of –217 is greater than –20 in CCF cells; (4) the polymorphisms at positions –812 and –792 generally have a weak negative influence on AGT transcription; (5) the influence of the –775 polymorphism is not significant in 2 cell lines, weakly stimulatory in CCF, and weakly inhibitory in 3T3-L1 cells; and (6) the combination of 2 (C2) set of SNPs at positions –6 and +67 lacks a significant effect in all of the cell lines. These data suggest some cell specificity by which polymorphisms in the AGT promoter exert their transcriptional effects. However, because of the structure of the 8 haplotypes, there was no clear method to examine the specific function of every polymorphism. For example, the combination of 3 (C3) set of SNPs at positions –1178, –1074, and –532 varied together as a group in haplotype 6, whereas the combination of 4 (C4) set of SNPs at positions –742, –604, –282, and –88 only varied as a group in haplotype 5. Therefore, these had to be modeled separately (Table 2). The observation that C3 seemed to have a large effect on transcriptional activity in all of the cell lines may have to be mitigated by the fact that haplotype 6 also carries with it –217A, which on its own has robust effects, especially in CCF cells.
Next, in an effort to examine whether certain polymorphisms act synergistically, statistical association was determined for those SNP pairs for which all 4 of the permutations were present within the 8 haplotypes (Table 3). For example, in 3T3-L1 cells, altering position –775 has relatively small effects irrespective of the allele at –20, whereas altering –20 from A to C had larger effects on transcription in the presence of –775C than –775T. Similarly, particularly strong effects of –20 were noted in HepG2, HK-2, and 3T3-L1 cells when –792G was present. Of course, one must recognize that whereas a number of statistically significant associations were found when examining –20 with –775, –20 with –792, and –217 with –792, this statistical model was unable to compensate for SNPs at the other positions that covaried within each 4-way comparison.
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The AGT enhancer was reported previously to markedly stimulate the transcriptional activity of a very short hAGT promoter.10,11 However, in the context of the 1.3-kb promoter used herein, the enhancer had a much smaller effect (Figure 3). There was virtually no effect of the enhancer in differentiated 3T3-L1 cells, and in CCF cells the effect was generally 1.2-fold, except for haplotype 3, where it increased to 1.7-fold. In HepG2 cells, enhancer activity averaged 1.4-fold but varied from 1.0- to 1.7-fold. Interestingly, haplotypes 2, 3, and 4, which exhibited the lowest baseline transcriptional activity, exhibited the largest stimulation by the enhancer (1.5-, 1.7-, 1.6-fold, respectively). On the contrary, haplotype 8, which exhibited the highest baseline activity, exhibited the smallest stimulation by the enhancer. The most interesting observations were made in HK-2 cells where enhancer activity varied considerably among haplotypes. Transcriptional activity decreased by 40% in haplotypes 1, 2, and 6; had no effect in haplotypes 4 and 7; and increased transcription by 50% in haplotypes 3 and 8. The increase in haplotype 3 was consistent between CCF and HK-2 cells, suggesting some haplotype-specific effect of the enhancer in certain AGT-expressing cell types.
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) with those containing the enhancer (
). Middle panel shows the relative strength of the enhancer. Right shows a matrix with multiple pairwise comparison analysis of the log normal-transformed data using the Tukey adjustment. •, P<0.05. Graphs depict mean±SEM. N=4 for HepG2, CCF, and differentiated 3T3–L1 cells and N=8 for HK-2 cells.
We also examined effects of certain steroid hormones (testosterone, estrogen, and dexamethasone) on haplotype-specific transcriptional activity. We tested testosterone because renal proximal tubule cells are a recognized androgen target cell, and androgens induce AGT expression in kidney.26 Dexamethasone has been reported to induce AGT expression in the liver, and the polymorphism at –20 may involve differential binding of the estrogen receptor.14,16 To ensure the presence of the correct steroid hormone receptor in each cell line, we first performed transfections in the presence or absence of hormone with reporter constructs containing the androgen response element, estrogen response element (ERE), or glucocorticoid response element. All 3 of the cell lines robustly responded to dexamethasone induction of the test plasmid (4.3-, 2.0-, and 18.2-fold in HepG2, HK-2, and CCF cells, respectively) without addition of glucocorticoid receptor expression vector. However, there was no testosterone- or estrogen-mediated response. We, therefore, cotransfected each haplotype vector with an expression vector encoding either the androgen receptor or ERß. Although an interaction between ER and the –20 polymorphism has been reported previously,16 we used ERß, because it recognizes the same ERE sequence, can transactivate ERE-containing constructs, can form homodimers or heterodimers with ER, and is sensitive to the same antagonists as ER.27,28 Indeed, cotransfection of ERß caused a 2.4-, 10.0-, and 4.2-fold increase for the ERE reporter plasmid in HepG2, HK-2, and CCF cells, respectively, whereas cotransfection with androgen receptor caused an 2- to 3-fold induction with the androgen response element reporter plasmid in the 3 cell lines. In HepG2 cells, dexamethasone and estrogen had minimal effects on AGT transcriptional activity, whereas testosterone exhibited a 1.0- to 1.4-fold effect that was not significantly different among haplotypes (Supplemental Figure II). Similar results were obtained in HK-2 cells (Supplemental Figure III). CCF cells exhibited the largest induction of the glucocorticoid response element and androgen response element test plasmids (18.2- and 2.3-fold, respectively), and their average induction of the hAGT promoter in response to dexamethasone, testosterone, and estrogen was 1.9-, 1.1-, and 1.6-fold, respectively. Despite this induction, we observed no haplotype-specific effects of the hormone treatments (Figure 4).
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Discussion |
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The current study examines for the first time the transcriptional activity of 8 naturally occurring haplotypes of the AGT promoter in 4 cells lines, each derived from a physiologically important AGT-expressing tissue. Our study was designed to accomplish 2 broad goals: to examine the transcriptional effects of AGT polymorphisms in the context of naturally occurring haplotypes and to examine the differential cell-specific effect, if any, of genetic variation in the AGT promoter. Our data suggest that genetic variation at positions –217 and –20 have the greatest effect in all of the cell lines, although there was some cell specificity in terms of the relative strength of each variant. For example, whereas –20 and –217 were equally important in HepG2, HK-2, and 3T3-L1 cells, the –217 variant seems to be more important in CCF cells. Variants at positions –812, –792, and, in particular, –775 have variable effects among the 4 cell lines. This suggests the possibility that the transcriptional effects of variants in the AGT promoter may manifest differently regarding the level of AGT mRNA in diverse tissues, each of which has an important individual role in blood pressure regulation.
The finding that the –20 SNP and the –217 SNP of AGT are the most important individual SNPs in the promoter region analyzed is in keeping with the fact that these are the only 2 SNPs in AGT known to differentially bind transcription factors. The –20C allele correlates with increased baseline AGT expression and exhibits a prevalence of 15% among the Center Etude Polymorphism Humain (CEPH) panel with a range of 7% to 27% among different ethnicities.29 The –20C allele was reported to differentially regulate AGT expression in HepG2 cells, presumably causing augmented transcription when bound by ubiquitously expressed USF transcription factors.16 By contrast, –20A may form an ERE that correlates with enhanced promoter activity in the presence of large amounts of exogenous ER. The presence of the –20A ERE may help explain the observation that pregnant women homozygous for –20A have higher plasma AGT than those homozygous for –20C.30 However, our results indicate that ß-estradiol treatment, even in the presence of ERß, has no significant effects on haplotype-dependent AGT transcriptional activity in any cell type tested. Indeed, we cannot rule out that the haplotype-independent effects of ß-estradiol observed in CCF cells may rely on ER binding to another ERE. The molecular mechanisms differentially regulating AGT promoter activity by –20 are further complicated by the report that the –20A allele also binds the transcription factor apolipoprotein A1 regulatory protein 1, which attenuates the transcriptional effects of ER.15
Despite this complexity, understanding how genetic variation at the –20 position affects AGT synthesis may be of particular significance. Because hypertension is prevalent among diabetic and obese patients, it is intriguing to consider that the levels of USF and its concomitant transcriptional activation of certain USF-target genes increase as glucose levels increase in cell culture and in mice. Increasing glucose concentration in mesangial cells resulted in 5-fold increases in USF1 and USF2 protein levels.31 In addition, USF1 and USF2 knockout mice exhibited an attenuated transcriptional activation of USF1/2 target genes after 24-hour fasting followed by 18 hours of high-carbohydrate diet refeeding.32–34 USF expression also increased in the kidneys of mice subjected to fasting–refeeding and in rats with streptozotocin-induced diabetes.31 USF1 is associated with familial combined hyperlipidemia, and approximately two thirds of such individuals fit the criteria for the metabolic syndrome.35 Interestingly, AGT was 1 of only 3 genes found to be differentially regulated in fat biopsies of patients from dyslipidemic families by a polymorphism in USF1 that may impact protein function.36 Therefore, a combination of environmental conditions, the genotype of AGT, and the genotype of other genes, such as USF1, may all factor into the susceptibility of an individual to cardiovascular disease.
Like the –20 polymorphism, a variant at position –217 was reported previously to affect baseline activity of the AGT promoter and to be associated with hypertension.13 The –217A allele has a prevalence of 6% in the CEPH panel and from 4% to 42% in different ethnicities.29 The frequency of –217A is elevated in black hypertensive patients, and the A at that position increases binding of the C/EBP transcription factor, as well as the glucocorticoid receptor14 (although no glucocorticoid receptor–specific effect was noted in our study). C/EBP is involved in the acute-phase response (AGT is an acute-phase reactant), proper liver and white fat development, and glucose homeostasis.37–39 C/EBP can also activate USF in humans and thereby stimulate an increase in C/EBP expression.40 This suggests that C/EBP may modulate AGT expression during the inflammatory response and via interactive effects between the 2 transcription factors that seem to be responsible for high-level expression from –20C and –217A in the AGT promoter.
Our results may offer an explanation for the discrepancies of previous association studies that focus mostly on the –6 SNP. It is conceivable that the –6A allele is in linkage disequilibrium with the –20C or –217A alleles more frequently in some ethnicities than in others, which could account for its inconsistent association with elevated AGT expression and high blood pressure.6,7 The –6G allele, on the other hand, was only found in 1 of our haplotypes, albeit the most common one, that was not associated with high-level AGT expression in any cell line.
The effect of other AGT promoter SNPs may play a significant role in AGT transcriptional regulation, especially when one considers that only modest alterations in AGT expression can mediate detectable differences in blood pressure.5 There are few data available regarding the SNPs at –1178 and –1074, but there is evidence in a French population that –532T strongly correlates with plasma AGT levels and affects a putative AP-2 binding site.41 The –812A allele correlated with approximately a 30% to 60% reduction of reporter expression in all but HepG2 cells, and the –792A allele correlated with a 15% to 60% reduction in all of the cell lines. The fact that the directionality is the same among cell types lends support to the validity of the underlying statistical analysis and suggests that a similar mechanism may be operant in each cell type. The fact that the –775T allele is not significant in 2 cell lines and has small effects of opposite magnitude in 2 others casts into question its overall importance. Although we obtained some evidence suggesting interactive effects between SNPs, in particular –775 and –792 with –20 and –792 with –217, this analysis was limited to those haplotypes exhibiting each possible combination of the 2 SNPs analyzed.
Perspectives
The AGT gene is functionally expressed in several tissues and cells, and our reporter expression data in cultured hepatocyte, astrocyte, proximal tubule, and adipocyte lines suggest that the majority of the differential haplotype-dependent transcriptional regulation occurs because of the –20 and –217 SNPs. However, we also obtained evidence suggesting that there are some cell-specific differences in haplotype-dependent transcription. This suggests the possibility that, in vivo, specific haplotypes may control the level of AGT mRNA, AGT protein, and, ultimately, the level of angiotensin II generated locally by a tissue. This information, combined with a growing wealth of data supporting the importance of tissue renin–angiotensin system in blood pressure regulation, suggests that measurements of circulating AGT may not be a sufficient predictor of the contribution of AGT polymorphisms to blood pressure and may account for the huge disparity among reports on the association of AGT with hypertension. That there may be tissue-specific influences of AGT polymorphisms adds a level of complexity that must be considered in interpreting and designing genetic studies aimed at understanding the role of AGT in hypertension and other cardiovascular diseases.
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Acknowledgments |
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Sources of Funding
We gratefully acknowledge the generous research support of the Roy J. Carver Trust. This work was also supported by grants from the National Institutes of Health (HL48058, HL61446, and HL55006).
Disclosures
None.
Received October 3, 2006; first decision October 30, 2006; accepted November 21, 2006.
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