Effects of Novel Polymorphisms in the RAGE Gene on Transcriptional Regulation and Their Association with Diabetic Retinopathy Barry I

Effects of Novel Polymorphisms in the RAGE Gene on Transcriptional Regulation and Their Association With Diabetic Retinopathy Barry I. Hudson, Max H. Stickland, T. Simon Futers, and Peter J. Grant Interactions between advanced glycation end products AGEs produce a plethora of effects through a number of (AGEs) and the receptor for AGE (RAGE) are impli- mechanisms. Firstly, AGEs form on the extracellular ma- cated in the vascular complications in diabetes. We have trix, altering vascular structure and trapping circulatory identified eight novel polymorphisms, of which the proteins, leading to a narrowing of the lumen (1,2). Sec- ,؊1420 (GGT)n, ؊1393 G/T, ؊1390 G/T, and ؊1202 G/A ondly, AGEs form on intracellular proteins and DNA were in the overlapping PBX2 3؅ untranslated region resulting in cellular changes. Thirdly, the principle means ؊ (UTR), and the 429 T/C (66.5% TT, 33.5% TC/CC), of derangement of AGEs is by specific AGE-binding recep- ؊407 to –345 deletion (99% I, 1% I/D, 0% D), ؊374 T/A TT, 33.6% TA/AA), and ؉20 T/A were in the tors, which include the AGE-receptor complex (3), the 66.4%) RAGE promoter. To evaluate the effects on transcrip- macrophage scavenger receptors (4), and the receptor for tional activity, we measured chloramphenicol acetyl AGE (RAGE) (5). The receptor most supported by studies transferase (CAT) reporter gene expression, driven by is RAGE, a member of the immunoglobulin superfamily. variants of the –738 to ؉49 RAGE gene fragment con- The gene for RAGE is found on chromosome 6p21.3 in the taining the four polymorphisms identified close to the MHC locus and is composed of a 1.7-kb 5Ј flanking region transcriptional start site. The –429 C, ؊374 A, and and 11 exons (6). RAGE is normally expressed at low 63-bp deletion alleles resulted in a mean increase of levels by the endothelium, smooth muscle, mesangial, and CAT expression of twofold (P 0.0001), threefold (P < < monocytes (7), but in both animal models and human 0.001), and fourfold (P < 0.05), respectively, with the –374 T and A alleles yielding highly differential binding diabetic subjects, high levels of RAGE expression have of nuclear protein extract from both monocyte- and been identified in the retina, mesangial, and aortic vessels, hepatocyte-derived cell lines. The prevalence of the concomitant with AGE accumulation (8,9). functional polymorphisms were investigated in subjects In normal homeostasis, it appears that RAGE functions with type 2 diabetes (106 with and 109 without retinop- to bind and internalize low levels of AGE for degradation; athy), with the –429 C allele showing an increase in the however, in diabetes, because of the sustained interaction retinopathy group (P < 0.05). These data suggest that of higher levels of AGEs, this appears to lead to receptor- the polymorphisms involved in differences in RAGE gene regulation may influence the pathogenesis of dia- mediated activation and secretion of various cytokines betic vascular complications. Diabetes 50:1505–1511, (10). These induce a cascade of protein expression (10), 2001 among which increased tissue factor (11) and fibrinolytic inhibitor plasminogen-activator inhibitor-1 (12) expression promote a procoagulant state. Further endothelial dys- function is brought about by the recruitment of monocytes he major complications associated with diabetes via a RAGE-dependent mechanism to these sites of AGE are diseases of the macro- and microvasculature, accumulation (13). From in vitro studies, it appears that a which have been postulated to be related to the feedback loop of increasing expression of RAGE, via Tformation of advanced glycation end products nuclear factor–␬␤, results from AGE-RAGE binding to (AGEs). AGEs result from the nonenzymatic glycation of enhance these effects (10). proteins and lipids (1), which form during aging and at an Inherited differences in key transcription binding sites accelerated rate in diabetes as a result of hyperglycemia. involved in RAGE gene regulation could alter this pathway of events by either increasing or decreasing RAGE expression. To investigate for the presence of novel polymor- From the Academic Unit of Molecular Vascular Medicine, University of Leeds, Ј Leeds General Infirmary, Leeds, U.K. phisms in the RAGE 5 flanking region, we screened the Address correspondence and reprint requests to Dr Barry I. Hudson, entire 5Ј gene regulatory region of RAGE for novel poly- Academic Unit of Molecular Vascular Medicine, Research School of Medicine, morphisms using a combination of single-strand con- G Floor, Martin Wing, Leeds General Infirmary, Leeds, LS1 3EX, UK. E-mail: [email protected]. formation polymorphism (SSCP) and denaturing high- Received for publication 30 May 2000 and accepted in revised form 2 March performance liquid chromatography (DHPLC). Because of 2001. AGE, advanced glycation end products; CAT, chloramphenicol acetyl the dense nature of genes within the MHC locus, part of transferase; DHPLC, denaturing high-performance liquid chromatography; the 5Ј flanking region of RAGE overlaps with the 3Ј EMSA, electrophoretic mobility shift assays; PCR, polymerase chain reaction; untranslated region (UTR) of the PBX2 gene (6), further RAGE, receptor for AGE; RFLP, restriction fragment–length polymorphism; SSCP, single-strand conformation polymorphism; TEAA, tri-ethyl ammonium complicating the correct identification of polymorphisms acetate; UTR, untranslated region. due to the presence of a pseudogene copy of PBX2 on DIABETES, VOL. 50, JUNE 2001 1505 FUNCTIONAL RAGE PROMOTER POLYMORPHISMS chromosome 3. Correctly identified polymorphisms were High Fidelity PCR system (Roche) using 2.6 U Expand enzyme mix, 200 ␮mol/l 2ϩ functionally characterized by transient transfection assays dNTPs, and 1.5 mmol/l Mg in High Fidelity Expand buffer. Amplification consisted of 94°C for 2 min, 10 cycles of 94°C for 15 s, 58°C for 30 s, 72°C for using RAGE promoter regions to drive the chlorampheni- 2 min, 20 cycles of 94°C for 15 s, 58°C for 30 s, 72°C for 2 min increasing 5 s col acetyl transferase (CAT) reporter gene, and nuclear a cycle, and a final extension at 72°C for 5 min. PCR fragments were gel protein interactions were characterized by electrophoretic purified using a NucleoSpin (Macherey Nagel) kit, and nested PCR was mobility shift assays (EMSA). Functionally active poly- performed to amplify each specific region. PCR products were purified using morphisms were assessed for their prevalence in a control QuickStep PCR purification columns (EDGE Biosystems), and cycle sequenc- population and in subjects with type 2 diabetes with and ing reactions were set-up with BigDye Ready Reaction mix (Applied Biosys- tems) according to manufacturers instructions. Purification was carried out without retinopathy. using AGTC Centriflex gel filtration columns (EDGE Biosystems) before automated sequencing on an ABI310 (Applied Biosystems). RESEARCH DESIGN AND METHODS Because of PCR slippage occurring in promoter regions I and J, the RPALL fragment was cloned into a pCR2.1 TOPO vector (Invitrogen), and six clones Initial polymerase chain reaction (PCR) optimizations were performed using were picked for each sample and mini-prep purified using Wizard Plus SV DNA from anonymous blood donors that was previously obtained from the Miniprep DNA purification system (Promega). Sequencing reactions were British Blood Transfusion Service Center, Leeds, U.K. Subsequent mutation setup using 200 ng of plasmid template with dRhodamine Ready Reaction mix detection was carried out with DNA from 80 random blood donors. Genotype (Applied Biosystems). A wild type RAGE 5Ј flanking sequence was then frequencies were investigated in subjects with type 2 diabetes with (n ϭ 106) and without (n ϭ 109) retinopathy who were previously recruited from the generated from all sequencing results and deposited with Genbank under outpatient clinic at the General Infirmary, Leeds. Type 2 diabetes was Accession No. AF208289. ϩ diagnosed according to World Health Organization criteria. Retinopathy was Reporter gene constructs. PCR primers to amplify from –738 to 49 Ј defined by opthalmoscopic examination performed by an opthamologist with relative to the transcription start site (RAGECATF 5 CAGCCCTGAACTAGC- Ј Ј Ј special interest in diabetic retinal disease. Subjects were classified as previ- TACCAT 3 RAGECATR 5 AGCACCCAGGCTCCAACT 3 ) were designed ously described (14). Each subject gave informed consent, and the study was because this construct gave the highest basal expression (16). PCR fragments approved by the United Leeds Teaching Hospitals (National Health Service) (RAGECAT) were amplified using Expand High Fidelity PCR system as before Trust Research Ethics Committee. Human genomic DNA was obtained from with a extension of 10 min to A-tail the PCR product to enable cloning into the 10 ml of whole blood in EDTA using the Nucleon extraction kit (Nucleon pCR2.1 TOPO vector (Invitrogen). Five RAGECAT constructs were generated: Biosciences). a wild type for all polymorphisms,aCat–429, an A at –374, a 63-bp deletion PCR. All available genomic sequences for human RAGE 5Ј flanking region from –407 to –345, andaTatϩ20, as described by Hudson et al. (17). Five were obtained from the internet site www3.ncbi.nlm.nih.gov/. RAGE se- clones for each genotype were mini-prep purified as before and sequenced quences U89336, AF001095, and D28769 were aligned using ClustalW at the using M13 primers (M13F 5Ј GTAAAACGACGGCCAG 3Ј M13R 5Ј CAGGAAA- Internet site www2.ebi.ac.uk/clustalw/ to determine discrepancies among CAGCTATGAC

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