Identification of a Novel Polymorphism in the 3′UTR of the l-Arginine Transporter Gene SLC7A1
Contribution to Hypertension and Endothelial Dysfunction
Background— Endothelial dysfunction because of reduced nitric oxide bioavailability is a key feature of essential hypertension. We have found that normotensive siblings of subjects with essential hypertension have impaired endothelial function accompanied by altered arginine metabolism.
Methods and Results— We have identified a novel C/T polymorphism in the 3′UTR of the principal arginine transporter, solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 gene (SLC7A1). The minor T allele significantly attenuates reporter gene expression (P<0.01) and is impaired in its capacity to form DNA-protein complexes (P<0.05). In 278 hypertensive subjects the frequency of the T allele was 13.3% compared with 7.6% in 498 normotensive subjects (P<0.001). Moreover, the overall genotype distribution observed in hypertensives differed significantly from that in normotensives (P<0.001). To complement these studies, we generated an endothelial-specific transgenic mouse overexpressing l-arginine transporter SLC7A1. The Slc7A1 transgenic mice exhibited significantly enhanced responses to the endothelium-dependent vasodilator acetylcholine (−log EC50 for wild-type versus Slc7A1 transgenic: 6.87±0.10 versus 7.56±0.13; P<0.001). This was accompanied by elevated production of nitric oxide by isolated aortic endothelial cells.
Conclusions— The present study identifies a key, functionally active polymorphism in the 3′UTR of SLC7A1. As such, this polymorphism may account for the apparent link between altered endothelial function, l-arginine, and nitric oxide metabolism and predisposition to essential hypertension.
Received September 19, 2006; accepted December 18, 2006.
The vascular endothelium plays a crucial role in the regulation of vascular tone, the modulation of vascular architecture, and the control of cellular adhesion. Endothelial dysfunction has been widely associated with various cardiovascular risk factors and disease states including hypertension, diabetes, smoking, atherosclerosis, and heart failure.1–4 There is good evidence that the underlying degree of endothelial dysfunction confers incremental cardiovascular risk.5 Although in the context of diabetes and heart failure, for example, endothelial dysfunction is generally attributed to the underlying disease state per se, in hypertension and atherosclerosis it has been proposed that endothelial dysfunction may precede the onset of these conditions.
Clinical Perspective p 1274
Hypertension affects ≈25% of the population in westernized societies and is a major risk factor for cardiovascular disease. Despite the enormous prevalence of hypertension, little progress has been made in the identification of the key underlying mechanisms, probably as a result of complex gene-environment interactions.6
Several studies, including our own, have observed endothelial dysfunction in the normotensive siblings of hypertensive individuals.7,8 These data suggest a genetic contribution from the pathway responsible for nitric oxide (NO) bioavailability. In particular, we noticed that in normotensive siblings of hypertensive subjects, endothelial dysfunction is accompanied by reduced arginine transport.8 In the present study, we report the discovery of a likely molecular mechanism responsible for this finding. This involved our identification of a novel polymorphism in the 3′UTR region of the solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 gene (SLC7A1, previously “CAT-1”; chromosome 13q12-q14). The variant allele is associated with altered expression of SLC7A1, thereby explaining, at least in part, the pathophysiological observations.
DNA Samples and Genotyping
Twelve primer pairs corresponding to different intronic sites of human SLC7A1 gene (SLC7A1, GenBank accession numbers NM_003045 for mRNA, NT_009799 for genomic sequence) were designed and used to resequence all of its exons. DNA and demographic details were obtained from 498 normotensive subjects (systolic/diastolic pressure of 121±1/74±1 mm Hg) and 278 hypertensive subjects (systolic/diastolic pressure of 160±2/98±1 mm Hg) whose parents both had the same blood pressure status as the subjects. The respective human ethics committees at each institution approved the present study. A 20-sample pool was used initially as polymerase chain reaction (PCR) template to detect possible polymorphisms by means of direct DNA sequencing. Each of the individual samples was then reassessed to confirm the polymorphisms. During this process, 1 novel single-nucleotide polymorphism (SNP) was found in the 3′UTR with the use of primers pri047 (5′-AGCTGTCTGGAGGTGACCAG-3′) and pri048 (5′-GCCTGAGAGGGTTTGCTGT-3′) and PCR conditions of 94°C for 1 minute, followed by 35 cycles of 94°C, 60°C, and 72°C for 1 minute each, with an additional 8-minute extension at 72°C at the end of last cycle before 4°C for at least 2 minutes. PCR products were then gel-purified to remove excess primers and dNTPs before DNA sequencing performed at the Baker Heart Research Institute.
The novel SNP, involving a C/T substitution, was genotyped with the use of 2 allele-specific primers designed in such way that the only difference between them was the polymorphism site at the very last nucleotide of their 3′ ends: 5′-GCAAGTGACGCACAGCCC-3′ (pri049) and 5′-GCAAGTGACGCACAGCCT-3′ (pri050). Two parallel PCRs were performed for each DNA sample. These contained both pri047 and pri048, plus either pri049 or pri050, under the same conditions of PCR as described above. After PCR products were run on a 1.5% agarose gel, DNA genotypes were called directly without the need for further DNA sequencing.
Reporter Gene Assays
The reporter gene vectors pGL3-Basic and pGL3-TK (Promega, Madison, Wis) were used as background and positive control, respectively, for luciferase assays. Human DNA with homologous genotypes of the 3′UTR polymorphism (either CC or TT) was used as PCR template to generate allele-specific amplicons. Primer sets pri047-pri049 and pri047-pri050 were again used to generate 216-bp PCR products containing allele C and allele T, respectively. Other primers were also used to generate different sized allele-specific amplicons as well as “nonrelated” DNA from the last intron of SLC7A1. All DNA fragments were inserted downstream of luciferase gene at an XbaI site, immediately preceding the poly(A) sequence. pSV-β-Galactosidase control vector (Promega) was used as internal control to correct for transfection efficiency among samples.
Chinese hamster ovary cells were grown at 37°C under 5% CO2 in DMEM (GIBCO/BRL) supplemented with 2 mmol/L l-glutamine and 10% heat-inactivated FCS. At 50% to 70% confluence, cells were cotransfected with equimolar amounts of each reporter gene construct and 2 μg of internal control DNA (pSV-β-Galactosidase control vector) by electroporation in 0.45-cm cuvettes. The electroporation conditions were 500 μF, 270 V. After incubation for 24 and 48 hours after transfection, cells were washed twice with PBS, harvested in the reporter lysis buffer provided in the luciferase assay system (Promega), and centrifuged at 13 000 rpm for 1 minute at 4°C. The supernatant was assayed for both luciferase and β-galactosidase activities. β-Galactosidase activity was measured colorimetrically with the use of Emax precision microplate reader (Molecular Devices, Sunnyvale, Calif). Luciferase activity was normalized to β-galactosidase activity to correct for differences in transfection efficiency. All of the assays were performed in triplicate, and the means of relative luciferase activity were plotted as percentage with respect to pGL3-TK. At least 3 independent experiments were performed for each reporter gene construct.
Electrophoretic Mobility Shift Assay
Nuclear extracts from HeLa cells were purchased from Promega. Together with the primer pri047, either allele C-specific (pri049) or allele T-specific (pri050) primers were used to generate 216-bp 3′UTR DNA fragments as described above. After PCR, the 5′ phosphate groups of the DNA fragments were removed by alkaline phosphatase before end-labeling with T4 polynucleotide kinase with the use of [γ-32P]ATP. The 32P end-labeled probes were preincubated with or without unlabeled competitors for 10 minutes in the presence of 1 μL of HeLa nuclear extracts, then incubated at room temperature for 20 minutes in 15 μL of binding solution (25 mmol/L HEPES buffer, pH 8, 50 mmol/L KCl, 0.5 mmol/L MgCl2, 0.5 mmol/L dithiothreitol, 2 μg poly[dI-dC]-poly[dI-dC], 10% glycerol). Competition assays were performed with 100-fold molar excess of unlabeled DNA fragments from relevant unlabeled PCR products or double-stranded oligonucleotides containing AP1, AP2, CREB, nuclear factor-κB, OCT1, SP1, and TFIID binding recognition elements (Promega). The reaction mixture was then electrophoresed on a 4% polyacrylamide gel in ×0.5 TBE buffer. The gels were wrapped and exposed to Kodak BioMax MR films (Sigma-Aldrich, St Louis, Mo) for 12 to 16 hours. The autoradiography results were then quantified.
Generation and Genotyping of Transgenic Mice Overexpressing SLC7A1-GFP
To establish the functional impact of an alteration in endothelial SLC7A1 expression in the range of that predicted by the reporter studies, we established an endothelial-specific Slc7A1 overexpressing transgenic mouse. Plasmid pT2BLacZpA1L7 containing mouse TIE2 promoter and longer enhancer fragment was a gift from Dr Thomas N. Sato (University of Texas Southwestern Medical Center at Dallas).9 mCAT-1-GFP was cloned into EcoRI–BamHI sites, followed by the insertion of TIE2 promoter fragment into the HindIII site of pBluescript KS(−) (Invitrogen, Carlsbad, Calif), resulting in the generation of plasmid pBSTIE2CAT-1gfp. The TIE2 longer enhancer fragment (≈10.6 kb), released from the plasmid pT2BLacZpA1L7 by complete digestion with NotI and partial digestion with XbaI, was then inserted into pBSTIE2CAT-1gfp. All the cloning products, as well as the mCAT-1-GFP fusion, were confirmed by enzyme digestion and sequencing.
The resulting plasmid, containing Slc7a1-GFP driven by TIE2 promoter and enhancer, was digested by SalI to remove the vector backbone. The remaining gene expression cassette was then gel-purified, resuspended in injection buffer (10 mmol/L Tris-HCl, pH 7.4, 0.1 mmol/L EDTA), and passed through a 0.45-μmol/L filter (Millipore, Billerica, Mass) before microinjection into oocytes from C57B/L mice. To screen for positive transgenic mice, 4 primer sets, each with at least 1 primer binding to GFP sequences, were used to amplify DNA and mRNA from mouse samples. PCR using the 3 primer pairs, 5′-CTTTGCTCAGGGCGGACT-3′ (pri502) and 5′-CTGACAGCAACTTGGACCAG-3′ (pri523), 5′-GTCCTCCTTGAAGTCGATGC-3′ (pri524); 5′-TCGTGACCACCCTGACCTAC-3′ (pri525), 5′-GATGTTGTGGCGGATCTTG-3′ (pri530); and 5′-GAGCAAGACCAAGCTCTCATTT-3′ (pri531), was performed by denaturation at 94°C for 3 minutes, followed by 35 cycles of 94°C for 30 seconds, 65°C for 30 seconds and 72°C for 2 minutes, and a final extension step at 72°C for 10 minutes. PCR involving primers 5′- CTAGTGGATCCTTACTTGTACAGCTCGTCCATGCC-3′ (pri508) and 5′-AAGCTTGAATTCACAGCAGATTCGCTCAGCACAATG-3′ (pri509) was performed under similar conditions except that the annealing temperature was 60°C and the extension time at each cycle was 3.5 minutes. Transgenic-positive mice were maintained by backcrossing to wild-type C57B/L and subjected to further PCR screening of their offspring.
Isolation and Fluorescence-Activated Cell Sorting of Endothelial Cells From Mouse Aorta
Primary murine aortic endothelial cells were isolated from both wild-type and transgene-positive C57B/L mice (Baker Institute Animal Center, Melbourne, Australia) as described. Briefly, aortas were harvested, the adventitia was removed, and strips were placed lumen side down into Matrigel in culture medium ECCM (each 500 mL containing 200 mL of DMEM without FCS, 200 mL of Ham’s F12 media (Invitrogen), 100 mL of FCS, 15 mg of endothelial mitogen, 30 mg of heparin, and 1 mL of antibiotic/antimycotic). The isolated murine aortic endothelial cells s were further purified by fluorescence-activated cell sorting (FACS) (model FACSAria, Becton Dickinson, Franklin Lakes, NJ) by utilizing their property of uptake of acetylated low-density lipoprotein (Ac-LDL). Briefly, murine aortic endothelial cells were incubated with DMEM containing 2 μg/mL Dil-Ac-LDL for 4 to 16 hours, were washed 3 times with PBS, and then were resuspended in FACS sorting buffer (each 100 mL S-MEM-Ca2+–free medium containing 2 mL of 0.5 mol/L EDTA, 2 mL of antibiotic/antimycotic, 1 mL of FCS). A 418-nm laser was used for excitation and 550 nm for emission, “scatter gates” were set to minimize the contribution of cell pairs, and “fluorescence gates” were chosen to eliminate the more highly fluorescent macrophages. The sorted cells were termed SLC7A1–murine aortic endothelial cells. They were maintained for up to 8 passages and used for experiments from passages 3 to 6.
Endothelial Function in Mouse Aortic Rings
Aortic rings were prepared from wild-type and Slc7a1 transgenic mice (10 to 14 weeks old) as described previously. In brief, aortic ring segments (2 mm in length) were mounted into an isometric myograph (myograph model 610 mol/L, JP Trading, Copenhagen, Denmark). After a 30-minute equilibration period, each vessel was subjected to a passive length-tension stretch. This procedure enabled each vessel to be normalized to an internal circumference equivalent to 90% the transmural pressure of 100 mm Hg. Endothelial integrity was determined initially by the demonstration of at least 50% vasodilation to 1 μmol/L acetylcholine. Full concentration-response curves to acetylcholine (1 nmol/L to 100 μmol/L) were constructed with the use of vessels preconstricted with cirazoline at a concentration that achieved 70% of the contraction induced by KPSS (in mmo/L: KCl 124, KM2PO4 1.18, MgSO4 1.17, NaHCO3 25, CaCl2 2.5, EDTA 0.026, glucose 5.5 at pH 7.4).
Endothelial Arginine Transport and NO Production
To examine the effect of endothelial Slc7a1 transgene expression, we compared the cellular uptake of [3H]l-arginine in wild-type and Slc7a1 transgenic aortic endothelial cells, isolated as above. [3H]l-Arginine was measured as described previously.10 In conjunction, the influence of SLC7A1 overexpression on NO production was determined with the NO fluorochrome 4-amino-5-methylamino-2′,7′-dichlorofluorescein diacetate (DAF-FM, Molecular Probes, Eugene, Ore), as described previously.
Data are presented as mean±SEM. Between-group comparisons were performed with the use of unpaired Student t tests for normally distributed data or χ2 tests for categorical data.
The authors had full access to and take full responsibility for the integrity of the data. All authors read and agree to the manuscript as written.
To discover potentially relevant SNPs in the exons of SLC7A1, we sequenced all 12 exons using pooled and individual DNA samples after amplification of each using flanking PCR primers. This identified a novel SNP located at nucleotide 2178 in the SLC7A1 3′UTR, 10 nucleotides (nt) downstream of the stop codon (nm_003045). Details of this SNP, subsequently referred to as ss52051869, have been placed online (www.ncbi.nlm.nih.gov/projects/SNP/snp_ss.cgi?ss =52051869).
To evaluate whether ss52051869 differentially affects SLC7A1 expression, we tested the effect of 3′UTR segments of contrasting genotype (CC versus TT) on luciferase reporter expression. Irrespective of genotype, the 3′UTR segment reduced luciferase expression. Notably, however, cells transfected with C allele construct (pGL3-TK-216-bp-Allele-C) had significantly higher luciferase activity than cells transfected with T allele construct (pGL3-TK-216-bp-Allele-T) (Figure 1). Constructs made with “nonrelated” DNA of similar size showed no alteration in reporter expression (data not shown). Furthermore, no inhibitory effects were seen when smaller DNA fragments were used, for example, a 114-bp DNA insert containing the polymorphism site (representing nt 2161 to 2174 of nm_003045), in the reporter gene assay (data not shown).
We performed gel shift assays to investigate whether differences in allelic expression between C and T allele variants could be attributed to the differential binding of nuclear proteins. In these assays, 2 PCR amplicons corresponding to the sequence from nt 2161 to 2376 in the 3′UTR were 32P-labeled and allowed to interact with HeLa nuclear extracts. Both probes formed DNA-protein complexes and showed a similar pattern of migration on the gel. The allele C fragment, however, demonstrated much stronger binding than allele T (P<0.05) (Figure 2). To determine the binding specificity of these DNA-protein complexes, competition experiments were performed with 100-fold excess of unlabeled probes before the addition of nuclear extracts. DNA-protein complex formation was completely abrogated by unlabeled probe (Figure 2). To determine the identity of the nuclear proteins that bound, we performed additional competition electrophoretic mobility shift assays using the consensus oligonucleotides containing AP1, AP2, CREB, nuclear factor-κB, OCT1, SP1, and TFIID binding elements. In particular, the preincubation of consensus oligonucleotides containing SP1, AP1, AP2, CREB, and TFIID binding elements resulted in abolition of the DNA-protein complexes, consistent with them being specific competitors (data not shown).
Given the role of the 3′UTR in the regulation of mRNA expression, we determined whether there was a difference in ss52051869 allele frequency between hypertensive and normotensive subjects. In hypertensive subjects the frequency of the T allele was 13.3% compared with 7.6% in the normotensive subjects (P<0.001). Moreover, the genotype distribution observed in hypertensives differed significantly from that in normotensives, as shown in the Table. With regard to the TT genotype specifically, this was observed in 1 normotensive subject (0.2%) and 6 hypertensive subjects (2.2%). All genotype frequencies accorded with Hardy-Weinberg equilibrium.
To evaluate the in vivo effect of altered SLC7A1 expression, we established endothelial-specific overexpression in a transgenic mouse. Full kinetic analysis of arginine transport in aortic endothelial cells from wild-type and Slc7A1 transgenic mice showed a significant increase in Vmax (2149±24 versus 1513±39 pmol/mg per minute, respectively; P=0.045), consistent with increased SLC7A1 expression (Figure 3A). Of note, the magnitude of the difference in arginine transport capacity in endothelial cells from wild-type and transgenic mice was in the range of that which might be expected for the effect of the major and minor SNP alleles on the basis of the reporter studies. In conjunction with the elevation of arginine transport, aortic endothelial cells from Slc7A1 transgenic mice also demonstrated a highly significant increase in NO production (Figure 3B).
Having demonstrated the cellular effect of modest SLC7A1 overexpression, we next investigated the effect on vascular pharmacology in isolated aortic rings. Vascular rings obtained from Slc7A1 transgenic mice showed significantly greater sensitivity (EC50) to the endothelium-dependent vasodilator acetylcholine (Figure 4), whereas responses to the endothelium-independent vasodilator sodium nitroprusside were not altered (data not shown).
Intracellular l-arginine is derived predominantly from the extracellular milieu and transported principally via the type 1 cationic amino acid transporter SLC7A1.7 Clinical and experimental paradigms involving an extracellular deficiency of l-arginine or its transport have been shown to be associated with reduced endothelial function and NO production.11–13 Furthermore, the administration of l-arginine to hypertensive animals and humans has been shown to reduce blood pressure and to restore endothelial function in both hypertensive subjects and those with a genetic predisposition toward hypertension.8,14 The present study has found that a functional variant of the l-arginine transporter gene SLC7A1 is increased in frequency in subjects with essential hypertension and that, in experimental models, altered expression of SLC7A1 results in physiologically relevant changes in NO production and endothelial function.
Although hypertension demonstrates significant familial aggregation, genomewide linkage studies have not provided consistent results, and in general the strength of various associations between putative loci of interest and blood pressure has been modest.15 At the same time, strong evidence supports the notion of environmental inputs into the subsequent development of hypertension, including overweight and elevated salt intake.16 In this context, acute intervention studies indicate that l-arginine may influence blood pressure.17 More generally, recent dietary intervention studies raise the possibility that combination diets rich in vegetables, nuts, and grains could exert influences on blood pressure by mechanisms beyond the effects of sodium reduction alone. In particular, the Dietary Approaches to Stop Hypertension (DASH) diet was shown to exert antihypertensive effects independent of sodium.18 As such, it has been shown that nuts alone improve endothelial function, and this may be accounted for by their significant l-arginine content.19
To establish the functional importance of relatively modest changes in SLC7A1 expression, we established an endothelial-specific transgenic mouse. Endothelial cells obtained from transgenic mice displayed a marked increase in NO under basal conditions, and isolated aortic rings from these mice showed evidence of increased endothelial function. It is acknowledged that in the generation of the Slc7A1 transgenic mice we did not specifically demonstrate the effect that might be observed in the context of restoration of l-arginine transport in a clinical paradigm or by a “knock-in” mouse model of reduced l-arginine transport. Nevertheless, these experimental findings are directionally consistent with the expected expects of increased substrate availability for NO synthesis. In conjunction with these data, we showed recently that another well-recognized cardiovascular risk factor, cigarette smoking, significantly reduced l-arginine transport and NO production in endothelial cells.10 Taken together, the present study and our previous work strongly support the notion that genetic or environmental alterations in l-arginine transport have the capacity to directly influence NO production and thereby vascular tone.
SLC7A1 is a high-affinity, low-capacity cationic amino acid transporter that facilitates uptake of arginine and lysine in mammalian cells. SLC7A1 is expressed almost ubiquitously, with the exception of adult liver, but its expression level varies considerably in different tissues and cell types.20 SLC7A1 expression can be modulated by a variety of stimuli including cell proliferation, growth factors, cytokines, certain hormones, microRNA, nutrients, and cellular stress, including amino acid deprivation.20 Interestingly, the 3′UTR of human SLC7A1 mRNA contains several potential target sites for miR-122, a liver-specific microRNA. Indeed, it was shown recently that activity of the endogenous SLC7A1 mRNA was translationally repressed by miR-122, and such repression could be reversed by the binding of HuR, an AU-rich element binding protein, to the 3′UTR of SLC7A1 mRNA.21 Our findings on the SNP in the 3′UTR of SLC7A1 mRNA are therefore consistent with the important regulatory role that the 3′UTR plays in controlling gene expression. In the present study, however, we did not directly correlate the genotype with SLC7A1 mRNA or protein expression because of the inability to obtain relevant vascular tissue or cells for such investigations.
In conclusion, we have identified a functionally relevant SNP in the 3′UTR region of SLC7A1, the principal l-arginine transporter in humans. In the context of hypertension, this finding provides the basis for an interaction between a genetically programmed influence on vascular endothelial function and blood pressure with environmental factors, including diet and traditional cardiovascular risk factors.
Sources of Funding
The present study was supported by grants from the National Health and Medical Research Council of Australia and the Atherosclerosis Research Trust (UK).
Kaye DM, Ahlers BA, Autelitano DJ, Chin-Dusting JP. In vivo and in vitro evidence for impaired arginine transport in human heart failure. Circulation. 2000; 102: 2707–2712.
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Zhang WZ, Venardos K, Chin-Dusting J, Kaye DM. Adverse effects of cigarette smoke on no bioavailability: role of arginine metabolism and oxidative stress. Hypertension. 2006; 48: 278–285.
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Taddei S, Virdis A, Mattei P, Ghiadoni L, Sudano I, Salvetti A. Defective L-arginine–nitric oxide pathway in offspring of essential hypertensive patients. Circulation. 1996; 94: 1298–1303.
Wu X, Kan D, Province M, Quertermous T, Rao D, Chang C, Mosley T, Curb D, Boerwinkle E, Cooper R. An updated meta-analysis of genome scans for hypertension and blood pressure in the NHLBI Family Blood Pressure Program (FBPP). Am J Hypertens. 2006; 19: 122–127.
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Ros E, Nunez I, Perez-Heras A, Serra M, Gilabert R, Casals E, Deulofeu R. A walnut diet improves endothelial function in hypercholesterolemic subjects. Circulation. 2004; 109: 1609–1614.
Hypertension currently affects ≈25% of the population in westernized societies. Despite the high prevalence of hypertension and the long history of hypertension research, the pathogenesis of essential hypertension remains controversial. Most current theories suggest that essential hypertension results from a complex set of gene-environment interactions. One hallmark of hypertension is the presence of abnormal endothelial function. Interestingly, this phenomenon is commonly also observed in nonhypertensive siblings of individuals with hypertension. In the present study, we report, in hypertensive subjects, the increased presence of a novel polymorphism in a key gene responsible for the delivery of arginine into cells. We demonstrate that its presence can alter endothelial function and nitric oxide production, thereby indicating its potential role in the pathogenesis of hypertension. Identification of this gene polymorphism in hypertensives may in the future assist in the selection of certain antihypertensive interventions.