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J. Biol. Chem., Vol. 275, Issue 33, 25781-25790, August 18, 2000

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The Receptor for Advanced Glycation End Products Is Induced by the Glycation Products Themselves and Tumor Necrosis Factor- through Nuclear Factor-B, and by 17-Estradiol through Sp-1 in Human Vascular Endothelial Cells^*

Nobushige Tanaka^§, Hideto Yonekura, Sho-ichi Yamagishi, Hideki Fujimori, Yasuhiko Yamamoto, and Hiroshi Yamamoto^¶

From the  Department of Biochemistry and the ^§ Department of Ophthalmology, Kanazawa University School of Medicine, Kanazawa 920-8640, Japan

Received for publication, February 15, 2000, and in revised form, May 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The binding of advanced glycation end products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. Here we show that AGE, tumor necrosis factor- (TNF-), and 17-estradiol (E₂) up-regulated RAGE mRNA and protein levels in human microvascular endothelial cells and ECV304 cells, with the mRNA stability being essentially invariant. Transient transfection experiments with human RAGE promoter-luciferase chimeras revealed that the region from nucleotide number 751 to 629 and the region from 239 to 89 in the RAGE 5'-flanking sequence exhibited the AGE/TNF- and E₂ responsiveness, respectively. Site-directed mutation of an nuclear factor-B (NF-B) site at 671 or of Sp-1 sites at 189 and 172 residing in those regions resulted in an abrogation of the AGE/TNF-- or E₂-mediated transcriptional activation. Electrophoretic mobility shift assays revealed that ECV304 cell nuclear extracts contained factors which retarded the NF-B and Sp-1 elements, and that the DNA-protein complexes were supershifted by anti-p65/p50 NF-B and anti-Sp-1/estrogen receptor  antibodies, respectively. These results suggest that AGE, TNF-, and E₂ can activate the RAGE gene through NF-B and Sp-1, causing enhanced AGE-RAGE interactions, which would lead to an exacerbation of diabetic microvasculopathy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glucose and other reducing sugars can react nonenzymatically with the amino groups of proteins to form reversible Schiff bases and, then, Amadori compounds. These early glycation products undergo further complex reactions to become irreversibly cross-linked, heterogeneous fluorescent derivatives termed advanced glycation end products (AGE)¹ (1). AGE are known to accumulate in various tissues at an extremely accelerated rate under a diabetic state, and are implicated in the development of diabetic vascular complications, e.g. retinopathy and nephropathy (1). We have shown previously that AGE exert their effects on endothelial cells and pericytes, the constituents of microvessels, through interactions with a cell-surface receptor for AGE (RAGE); AGE stimulate the growth of microvascular endothelial cells through an induction of vascular endothelial growth factor (VEGF) leading to angiogenesis on the one hand (2), and inhibit prostacyclin production and stimulated plasminogen activator inhibitor-1 synthesis by the endothelial cells on the other (3). AGE exhibit a growth inhibitory action on pericytes (4), which would lead to pericyte loss, the earliest histological hallmark in diabetic retinopathy (5).

RAGE belongs to the immunoglobulin superfamily of cell surface molecules (6, 7). It is expressed in multiple tissues (8) and interacts with various ligands including AGE (9, 10). The engagement of RAGE by AGE has been reported to induce cellular oxidant stress, activating the transcription factor nuclear factor-B (NF-B) (11, 12), resulting in the perturbation of a variety of vascular homeostatic functions (9, 10). AGE-RAGE interaction thus has been thought to play a central role in the development of diabetic vasculopathy. To determine how the RAGE gene is regulated under a diabetic state is, therefore, important for clarifying the pathogenesis of diabetic complications as well as for understanding the physiological roles of RAGE.

It has been reported that AGE-rich blood vessels show enhanced RAGE immunoreactivity (13); this implies the possibility that AGE themselves may up-regulate the RAGE expression. Among cytokines, tumor necrosis factor- (TNF-) is thought to be involved in the development of diabetes (14). Evidence has accumulated that serum TNF- levels are increased in non-insulin-dependent diabetes mellitus (15-17) and that TNF- can activate the NF-B pathway (18-20). Recently, NF-B has been reported to play a role in the basal and lipopolysaccharide-induced expression of the RAGE gene (21). It has also been reported that diabetic vasculopathy is often aggravated during pregnancy, probably due to the increased level of serum estrogen (22-24). From these observations, the possibility that TNF- and estrogen worsen the diabetic complications through the induction of RAGE gene expression should also be considered.

In the present study, we thus examined the effects of AGE, TNF-, and 17-estradiol (E₂) on RAGE gene expression and found that the three agents are capable of up-regulating the RAGE mRNA and protein levels in human microvascular endothelial cells. On the other hand, non-glycated BSA, other cytokines, and an anti-estrogen did not affect the RAGE mRNA levels. mRNA stability and promoter assays demonstrated that the induction was at the transcriptional level, and that AGE and TNF- induced the RAGE gene through an activation of NF-B while E₂ induced the gene through Sp-1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Bovine serum albumin (BSA) (fraction V, fatty acid-free, endotoxin-free) was purchased from Roche Molecular Biochemicals (Mannheim, Germany). TNF- was purchased from Becton Dickinson Labware (Bedford, MA). Transforming growth factor-1 (TGF-1) and interferon- (IFN-) were from R&D Systems, Inc. (Minneapolis, MN). E₂ and 4-hydroxytamoxifen (4-OH tamoxifen) were from Sigma. [-³²P]ATP (6,000 Ci/mmol) was from NEN Life Science Products. Restriction enzymes and T₄ polynucleotide kinase were from Takara (Kyoto, Japan). Rabbit polyclonal antibodies raised against recombinant human RAGE extracellular domain (amino acids 24-321) (7) were kindly provided by the Institute of Biological Science, Mitsui Pharmaceutical Inc (Mobara, Japan).

Cells-- Human skin microvascular endothelial cells (HMVEC) (Cascade Biologics, Inc., Portland, OR) were maintained in HuMedia-EB2 supplemented with 5% fetal bovine serum (FBS), gentamycin (50 µg/ml), amphotericin B (50 ng/ml), basic fibroblast growth factor (5 ng/ml), heparin (10 µg/ml), epidermal growth factor (10 ng/ml), and hydrocortisone (1 µg/ml) according to the supplier's instructions (Kurabo Corp., Osaka, Japan) in humidified incubators containing 5% CO₂. The human umbilical vein endothelial cell-derived cell line ECV304 (25) was kindly donated by Dr. Yoshio Sawasaki (National Defense Medical College, Tokorozawa, Japan), and was maintained in M199 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 µg/ml). All experiments using HMVEC and ECV304 cells were carried out in media lacking epidermal growth factor and hydrocortisone, and FBS, respectively. These media are referred to as the experimental media.

Preparation of AGE-BSA-- AGE-BSA was prepared by incubating BSA with 0.5 M glucose at 37 °C for 6 weeks under sterile conditions as described previously (2). After unincorporated sugars were removed by dialysis against phosphate-buffered saline (PBS), glucose-modified higher molecular weight materials were used as AGE-BSA. Control non-glycated BSA was incubated under the same conditions except for the absence of glucose. The concentration of AGE-BSA and control BSA were determined by the method of Bradford (26).

Measurement of RAGE mRNA by Quantitative Reverse Transcription-PCR (RT-PCR) Method-- Poly(A)⁺ RNAs were isolated from subconfluent cultures of HMVEC or ECV304 cells incubated under various conditions, using a Quick Prep Micro mRNA purification kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), and analyzed by RT-PCR with a GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA) as described previously (27). Oligodeoxyribonucleotide primers and probes for human RAGE, VEGF, and -actin mRNA were the same as described previously (4, 28). The amounts of poly(A)⁺ RNA templates and cycle numbers for amplification were chosen in quantitative ranges where reactions proceeded linearly as described previously (28, 29); 30 ng of templates and 30 cycles were chosen for amplifying human RAGE mRNA, 30 ng and 40 cycles for human VEGF mRNA, and 30 ng and 20 cycles for -actin mRNA. The fragments amplified with PCR were sequence-verified on both strands by the chain termination method (30). Five-µl aliquots of each RT-PCR reaction mixture were electrophoresed on a 2% agarose gel and transferred to a Hybond-N⁺ nylon membrane (Amersham Pharmacia Biotech), and the membrane was hybridized with the respective probes, which had been ³²P-end-labeled with polynucleotide kinase (27). Signal intensities of hybridized bands were measured by a BAS 1000 BioImage analyzer (Fuji Photo Film Co. Ltd., Hamamatsu, Japan).

Analysis of RAGE mRNA Stability-- HMVEC or ECV304 cells were treated with TNF-, E₂, or AGE-BSA for 4 h, and further cultured in the presence of 10 µg/ml of actinomycin D (Sigma) for various time periods. Total RNAs were isolated from the cultures with Isogen (Nippon Gene, Toyama, Japan) according to the method described by Chomczynski and Sacchi (31), and analyzed by the quantitative RT-PCR method described above. The amounts of total RNA templates and cycle numbers for amplification were chosen in quantitative ranges; 300 ng of templates and 35 cycles were chosen for amplifying human RAGE mRNA, and 300 ng and 20 cycles for -actin mRNA.

Western Blot Analysis-- Subconfluent cultures of HMVEC or ECV304 cells were incubated with TNF-, E₂, or AGE-BSA for 24 h. After the incubation, cells were washed with cold PBS, scraped off in cold PBS, and pelleted by centrifugation at 300 × g at 4 °C for 5 min. The cells were lysed immediately by sonication in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue) and boiled at 95 °C for 5 min. Twelve µg of the cell lysates were resolved by SDS-PAGE (12.5%), and then transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was treated with the anti-RAGE polyclonal antibodies, and the immunoreactive bands were visualized with an ECL detection system (Amarsham Pharmacia Biotech) as described previously (32). Signal intensities of the resultant bands were determined by densitometry using BIO-PROFIL 1-D (version 5.08) software (Vilber Lourmat Biotechnology, Marne La Vallée, France).

Construction of the RAGE Promoter-Luciferase Chimeras-- The chimeric genes for transfection experiments were constructed by ligating the 5'-flanking regions of differing lengths of the human RAGE gene upstream of the luciferase gene in a pGL3-basic vector (Promega Corp., Madison, WI) (see Fig. 5A). The genomic DNA fragments of the human RAGE gene were amplified by PCR using a cosmid named KS71 (33) as a template, which was kindly provided by Professor Toshimichi Ikemura (National Institute of Genetics, Shizuoka, Japan). The PCR primers employed in the amplification reactions are shown in Table I. Furthermore, we constructed two additional chimeric genes. The DNA fragments containing exons 1-11 plus introns 1-10 or the 3'-flanking region of the human RAGE gene were amplified by PCR using the cosmid KS71 and specific primers shown in Table I, and were ligated to downstream of the luciferase gene in the pGL3-Basic vector that had carried the longest fragment of the 5'-flanking region (see Fig. 5A). All the fragments obtained were sequence-verified.

Site-directed Mutagenesis-- Mutations were introduced into the RAGE promoter-luciferase chimeras using a GeneEditor^TM in vitro site-directed mutagenesis system (Promega) according to the manufacturer's protocol. The nucleotide sequences of the mutagenic oligodeoxyribonucleotide primers were 5'-ACTGTCAGAGTGGGGTTCCCCTCCCATTAAAG-3' (nucleotides 683 to 652), 5'-GTGACTGTACCCAGAAACTGGTAGTACCCAGG-3' (202 to 171), 5'-CTGGTAGTACCCAGGAATGGGGTGATAATTAT-3' (185 to 154), and 5'-ACTGTACCCAGAAACTGGTAGTACCCAGGAATGGGGTGATAAT-3' (199 to 157) for yielding pGL-5 NF-B2m, pGL-7 Im, pGL-7 IIm, or pGL-7 ImIIm, respectively (mutated sites are indicated by underlines) (see schematic representations in Fig. 6A). pGL-5 NF-B2m contained the region 751 to +43, but with the mutation in an NF-B site (671 to 663). pGL-7 Im and pGL-7 IIm contained the region 239 to +43, but with the mutations in one of two Sp-1 sites (189 to 181 and 172 to 166), respectively. pGL-7 ImIIm had the mutations in both Sp-1 sites. All the mutated constructs were sequence-verified.

Transfection Experiments and Luciferase Assay-- ECV304 cells or HMVEC (2 × 10⁵ cells) were plated into the wells of six-well tissue culture plates (Becton Dickinson Labware) 1 day before transfection. For ECV304 cells, the DNA/cationic lipid mixture for transfection was composed of test plasmid (2 µg), pRL-SV40 vector (0.5 µg) (Promega) that served as an internal control to normalize luciferase activities, TransFast^TM transfection reagent (7.5 µl) (Promega), and 1 ml of the experimental medium. Cells were exposed to the DNA/cationic lipid mixture for 2 h, then received 5 ml of the FBS-containing medium and were further incubated for 36 h at 37 °C. For transfection of HMVEC, test plasmid (2 µg) and pRL-CMV vector (1 µg) (Promega) were added to a tube containing 100 µl of Opti-MEM I (Life Technologies, Inc.). Lipofectin reagent (6 µl) (Life Technologies, Inc.) was added to another tube containing 100 µl of Opti-MEM I. The plasmid DNA and lipofectin reagent were then mixed together. After incubation at room temperature for 30 min, DNA-liposome complex was diluted with 800 µl of Opti-MEM I. Cells were exposed to the mixture for 8 h, then received 2 ml of the FBS-containing medium and were further incubated for 36 h at 37 °C. After the incubation, the cells were treated with TNF-, E₂, or AGE-BSA for 8 h in the experimental medium. Luciferase activities were measured using a Dual-Luciferase^TM reporter assay system (Promega) according to the manufacturer's protocol with a luminometer (Fluoroskan Ascent FL version 2.2.4, Labsystems, Helsinki, Finland).

Preparation of Nuclear Extracts from Cultured Cells-- Nuclear extracts were prepared essentially as described by Schreiber et al. (34). Briefly, ECV304 cells (2 × 10⁶) were plated onto 75-cm² tissue culture flasks (Becton Dickinson Labware) in the complete medium and left for 24 h at 37 °C. The cells were further incubated in the experimental medium at 37 °C for 12 h and then treated with TNF-, E₂, or AGE-BSA for 4 h. After the treatment, the cells were washed twice with ice-cold PBS, scraped off in PBS (10 ml), and pelleted by centrifugation at 3,000 rpm in a Beckman GH3.7 roter at 4 °C for 5 min. The pelleted cells were resuspended in 0.4 ml of ice-cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) and placed on ice for 15 min. After the addition of 25 µl of 10% Nonidet P-40, the suspension was vortexed for 15 s and centrifuged at 13,000 rpm in a Hitachi T15S roter at 4 °C for 30 min. The resultant nuclear pellets were washed with buffer A and resuspended in 0.1 ml of a solution containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride by constant agitation for 15 min at 4 °C. The nuclear lysates were centrifugated at 13,000 rpm at 4 °C for 10 min, and the supernatants were collected as nuclear extracts. The protein concentrations of the nuclear extracts were determined by the method of Bradford (26).

Electrophoretic Mobility Shift Assay and Supershift Assay-- The wild type and mutant double-stranded oligodeoxyribonucleotides encompassing the NF-B site (nucleotide numbers 671 to 663) or the two Sp-1 sites (189 to 181 and 172 to 166) were prepared. Their sequences were 5'-AGAGTGGGGAACCCCTCCCA-3' and 5'-AGAGTGGGGTTCCCCTCCCA-3' (677 to 658) as the wild and mutated NF-B elements, respectively, and 5'-CCCAGAGGCTGGTAGTACCCAGGGGTGGGGTGA-3' and 5'-CCCAGAAACTGGTAGTACCCAGGAATGGGGTGA-3' (193 to 161) as the wild and mutated Sp-1 elements, respectively (mutated sites are indicated by underlines). Twenty-five fmol of wild-type oligodeoxyribonucleotides, which had been ³²P-end-labeled with polynucleotide kinase (27), were incubated with 5 µg of nuclear extracts at room temperature for 30 min. Samples were then loaded onto 6% polyacrylamide gels and run in 0.2× Tris borate/EDTA electrophoresis buffer at 10 V/cm for 2-3 h. The gels were dried and autoradiographed at 80 °C overnight. For competition assay, nuclear extracts were first incubated with a 50-fold excess of unlabeled wild-type or mutant oligodeoxyribonucleotides at room temperature for 15 min, and then incubated with the labeled wild-type probe for 30 min under the same conditions as described above. For supershift assays, antibodies to NF-B p65, NF-B p50, Sp-1, estrogen receptor (ER) , or ER (Santa Cruz Biotechnology Inc., Santa Cruz, CA) were added to the nuclear extracts and incubated at 4 °C for 12 h. The antibody-treated nuclear extracts were subsequently incubated with the labeled oligodeoxyribonucleotides for 30 min under the same conditions as described above.

Statistical Analysis-- Paired t tests and one-way analysis of variance (ANOVA) with Tukey's range tests were used to test for significant differences between groups. All experiments were carried out at least three times.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TNF-, E₂, and AGE-BSA Increased the RAGE mRNA and Protein Levels in HMVEC and ECV304 Cells-- To examine the effects of TNF-, E₂, and AGE-BSA on the RAGE mRNA level in HMVEC, poly(A)⁺ RNAs were isolated from cells that had been exposed to various concentrations of these agents for 4 h, and analyzed by the quantitative RT-PCR method. As shown in Fig. 1A, TNF-, E₂, and AGE-BSA increased the RAGE mRNA levels in dose-dependent manners. The extents of induction and the peak concentrations were about 3-fold at 100 ng/ml TNF-, 10 nM E₂, and 50 µg/ml AGE-BSA, respectively, when normalized by -actin mRNA-derived signals used as an internal control. Next, we examined the time course of the RAGE mRNA induction. HMVEC were treated for various time periods with TNF-, E_2, and AGE-BSA at their most effective doses. As shown in Fig. 1B, the mRNA levels began to increase at 2 h and reached a maximum at ~8 h after the addition of either of the three agents. On the other hand, exposure of HMVEC to TGF-1 (10 ng/ml), IFN- (165 ng/ml), and non-glycated BSA (50 µg/ml) for 4 h did not affect the RAGE mRNA levels (Fig. 1C). 4-OH tamoxifen (10 nM), an anti-estrogen, abolished the E₂-induced RAGE mRNA induction (Fig. 1C).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effects of TNF-, E2, and AGE-BSA on RAGE mRNA levels in HMVEC. A, HMVEC were incubated for 4 h with the indicated concentrations of TNF-, E2, or AGE-BSA. Poly(A)+ RNAs were then isolated and analyzed by RT-PCR as described under "Experimental Procedures." B, HMVEC were treated with TNF- (100 ng/ml), E2 (10 nM), or AGE-BSA (50 µg/ml) for the indicated time periods, and assayed for RAGE mRNA levels. C, HMVEC were treated with TNF- (100 ng/ml = 5 × 103 units/ml), TGF-1 (10 ng/ml), IFN- (165 ng/ml = 5 × 103 units/ml), E2 (10 nM), 4-OH tamoxifen (10 nM), E2 plus 4-OH tamoxifen, AGE-BSA (50 µg/ml), or non-glycated BSA (50 µg/ml) for 4 h, and assayed for RAGE mRNA levels. The TGF-1 and IFN- concentrations employed were those that caused near maximal biological effects (57, 58). Graphs indicate the quantification of the RAGE mRNA levels. Intensities of the RAGE mRNA signals were normalized with those of -actin mRNA-derived signals, and are related to the value of the control without additives. Columns and bars indicate means and standard deviations, respectively, in three independent experiments. *, p < 0.05; **, p < 0.01 (versus control).

We next determined the RAGE mRNA levels in ECV304 cells, an immortalized cell line derived from human umbilical vein endothelial cells (25). This cell line exhibited the same responsiveness to AGE-BSA with respect to VEGF induction (Fig. 2A) as did HMVEC (2), the most effective dosage of AGE-BSA being 50 µg/ml in both cultures. The extent of induction was about 4-fold. When exposed to TNF- (100 ng/ml), E₂ (10 nM), or AGE-BSA (50 µg/ml) for 4 h, the RAGE mRNA levels in the ECV304 cells were also increased about 2-fold compared with those in the control unexposed cells (Fig. 2B).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   AGE-induction of VEGF mRNA and effects of TNF-, E2, and AGE-BSA on RAGE mRNA levels in ECV304 cells. A, ECV304 cells were treated with the indicated concentrations of AGE-BSA for 4 h, and then assayed for the VEGF mRNA levels by RT-PCR. Closed and open columns indicate signals derived from VEGF121 and VEGF165, respectively. Graph indicates the quantification of the VEGF mRNA levels. Intensities of the VEGF mRNA signals were normalized with those of -actin, and are related to the value of the control. B, ECV304 cells were treated with TNF- (100 ng/ml), E2 (10 nM), or AGE-BSA (50 µg/ml) for 4 h, and assayed for the RAGE mRNA levels. Graph indicates the quantification of the RAGE mRNA levels. Intensities of the RAGE mRNA signals were normalized with those of -actin mRNA-derived signals, and are related to the value of the control. Columns and bars indicate means and standard deviations, respectively, in three independent experiments. *, p < 0.05; **, p < 0.01 (versus control).

We next examined whether the increase in RAGE mRNA was actually followed by an increase in RAGE proteins in HMVEC and ECV304 cells. The cells were treated with TNF-, E₂, or AGE-BSA for 24 h, and subjected to Western blot analysis with anti-RAGE polyclonal antibodies. As shown in Fig. 3, one major immunoreactive band was marked at 46 kDa in either HMVEC (Fig. 3A) or ECV304 cells (Fig. 3B), being consistent with our previous report (3), and its intensity was increased by the treatment with the three agents. The RAGE protein levels in HMVEC and ECV304 cells treated with TNF-, E₂, or AGE-BSA were about 2-fold higher than the basal levels. The results indicated that ECV304 cells retained the ability to respond to those agents as did primary cultured endothelial cells, and subsequently we mainly used this cell line to examine the mechanisms of RAGE gene induction.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   RAGE protein levels. HMVEC (A) and ECV304 cells (B) were incubated in the presence of TNF- (100 ng/ml), E2 (10 nM), or AGE-BSA (50 µg/ml) for 24 h, and then lysed in SDS-PAGE sample buffer. Twelve µg of protein per lane were run on 12.5% SDS-polyacrylamide gel under reducing conditions, and subjected to immunoblot analysis using anti-RAGE polyclonal antibody as described under "Experimental Procedures." rRAGE, 50 ng of recombinant human RAGE extracellular domain expressed in Escherichia coli. Control, untreated cells. Graphs depict quantitative representations of RAGE protein levels in HMVEC (A) and ECV304 cells (B). Data were related to the value of the respective controls. Columns and bars indicate means and standard deviations, respectively, in three independent experiments. *, p < 0.05; **, p < 0.01 (versus control).

Effects of TNF-, E₂, and AGE-BSA on RAGE mRNA Stability in HMVEC and ECV304 Cells-- We next determined the RAGE mRNA stability in HMVEC and ECV304 cells exposed or not exposed to the three agents to determine which step of gene expression accounted for the increase in RAGE mRNA levels. The cells were incubated in the presence or absence of TNF-, E₂, or AGE-BSA for 4 h, then incubated with actinomycin D for various time periods, and underwent quantitative RT-PCR analyses. As shown in Fig. 4 (A and B), the half-lives of RAGE mRNA in TNF--, E₂-, and AGE-BSA-treated or untreated HMVEC and ECV304 cells were calculated from the RAGE and -actin mRNA-derived signals to be between 2.1 and 2.8 h, and there was no statistically significant difference among them. The results suggested that the TNF--, E₂-, and AGE-BSA-induced increase in RAGE mRNA was achieved at the transcriptional level.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   RAGE mRNA stability. HMVEC (A) and ECV304 cells (B) were treated with TNF- (100 ng/ml), E2 (10 nM), or AGE-BSA (50 µg/ml) for 4 h, and further cultured in the presence of 10 µg/ml actinomycin D for the indicated time periods. RAGE mRNA levels were determined by RT-PCR with total RNAs extracted from the cells. Control, cultures without the additives but with actinomycin D. Graphs depict quantitative representations of the RAGE mRNA levels in HMVEC (A) and ECV304 cells (B). Data were normalized by -actin mRNA-derived signals. Essentially the same results were obtained in three independent experiments.

Identification of the cis-Elements Responsive to TNF-, E₂, and AGE-BSA in the RAGE Promoter-- To confirm whether TNF-, E₂, and AGE-BSA did induce the RAGE gene transcription and, if so, to delimit the regions involved in such transcriptional activations, a series of chimeric 5'-deletion promoter-luciferase reporter constructs were prepared. Schematic representations of the constructs are shown in Fig. 5A. ECV304 cells were transiently transfected with the constructs, and the effects of TNF-, E₂, and AGE-BSA on the luciferase activity in the transfected cells were determined. pGL-1 carried the longest 5'-flanking region of the RAGE gene (1689 base pairs upstream of the transcription start site; Ref. 14), and when the pGL-1-transfected cells were exposed to TNF- (Fig. 5B), E₂ (Fig. 5C), or AGE-BSA (Fig. 5D), the promoter activities (closed columns) increased significantly (1.5- to ~ 2-fold) compared with those in unexposed cells (open columns). The same concentration of non-glycated BSA did not induce the luciferase activity in the transfected cells (data not shown). Deletion of the 5'-flanking region of the RAGE gene to 751 (pGL-2 to -5) did not affect the TNF-- and AGE-induced luciferase expression, but deletion to 629 (pGL-6) abolished the induction (Fig. 5, B and D). In contrast, the construct with a deletion to 239 (pGL-7) still retained the E₂-induced luciferase expression but deletion to 89 (pGL-8) abolished the induction (Fig. 5C). The 751 to 629 region contained an NF-B site (671 to 663), and the 239 to 89 region contained two Sp-1 sites (189 to 181 and 172 to 166). The results thus suggested that the NF-B site and the Sp-1 sites might be required for the TNF-/AGE- and E₂-induced activation of the RAGE gene promoter, respectively.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Construction of RAGE promoter luciferase-chimeras and identification of TNF-, E2, or AGE-BSA-responsive regions. A, schematic representation of the 5'-deleted RAGE promoter-luciferase reporter fusion gene constructs. Transcription start site (14) is designated as +1. Numbers in parentheses indicate nucleotide positions 5' to the transcription start site. B-D, relative luciferase activities in ECV304 transfectants treated with TNF- (100 ng/ml) (B), E2 (10 nM) (C), and AGE-BSA (50 µg/ml) (D). Open and closed columns indicate the mean values of untreated and treated cells, respectively. Data were normalized by pRL-SV40-derived luciferase activities used as an internal control, and related to the value of untreated pGL-1-transfected cells. Bars, standard deviations of nine independent experiments. E, relative luciferase activities in ECV304 cells transiently transfected with pGL-1, pGL-e1-11, or pGL-3'fl in the absence (open columns) or presence of TNF- (100 ng/ml) (closed columns), E2 (10 nM) (lattice columns), or AGE-BSA (50 µg/ml) (dotted columns). Data were normalized by the activities derived from the internal control. Values are shown as the mean ± S.D. of four independent experiments. Statistical analysis was performed using ANOVA. B, pGL-1, -2, -3, -4, and -5 ± TNF-, p < 0.01; pGL-6, -7, -8, and pGL3-basic ± TNF-, not significant. C, pGL-1, -2, -3, -4, -5, -6, -7 ± E2, p < 0.01; pGL-8 and pGL3-basic ± E2, not significant. D, pGL-1, -2, -3, -4, and -5 ± AGE-BSA, p < 0.01; pGL-6, -7, -8, and pGL3-basic ± AGE-BSA, not significant. E, there were no significant differences in relative luciferase activities among cells transfected with pGL-1, pGL-e1-11, and pGL-3'fl, which received or did not receive TNF-, E2, or AGE-BSA, while p < 0.01 was noted between control versus each treatment within each construct.

We also tested additional constructs that carried genomic fragments containing exons 1-11 and introns 1-10 or the 3'-flanking region (pGL-e1-11 and pGL-3'fl, respectively) in addition to the pGL-1 5'-promoter region. As shown in Fig. 5E, when the pGL-e1-11- or pGL-3'fl-tranfected cells were stimulated by TNF- (closed columns), E₂ (lattice columns), or AGE-BSA (dotted columns), the extents of luciferase induction were almost indistinguishable from those in the pGL-1-transfected cells. The results indicated that there were neither stimulatory nor silencing elements in the exon/intron or 3'-flanking region of the RAGE gene that could affect its responsiveness to TNF-, E₂, or AGE-BSA.

To determine the role of the NF-B site at 671 to 663 in the TNF- and AGE activation of the RAGE promoter, site-directed mutagenesis was performed at that site (pGL-5 NF-B2m) (Fig. 6A). When luciferase activities were assayed in cells transfected with the mutant, the inducibility by both TNF- and AGE-BSA was found to be totally abolished (Fig. 6B). Similarly, we also determined the role of the two Sp-1 sites at 189 to 181 and 172 to 166 in the E₂-dependent transcriptional activation. We altered each of the two Sp-1 sites (pGL-7 Im and pGL-7 IIm) or both sites (pGL-7 ImIIm) (Fig. 6A), and the E₂ effect on luciferase activities in the mutant transfectants was tested (Fig. 6C). The E₂ inducibility of luciferase activities was decreased in pGL-7 Im- and pGL-7 IIm-transfected cells and totally abolished in pGL-7 ImIIm-transfected cells. The results indicated that the NF-B binding site and the two Sp-1 binding sites were essential for the TNF-/AGE- and E₂-induced activation of the RAGE gene, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of the mutated NF-B or Sp-1 sites in the inducibility of the RAGE promoter. A, schematic representation of mutated RAGE promoter-luciferase chimeras. B, relative luciferase activities in ECV304 cells transiently transfected with pGL-5 or pGL-5 NF-B2m in the absence (open columns) or presence of TNF- (100 ng/ml) or AGE-BSA (50 µg/ml) (closed or dotted columns, respectively). Data were normalized by the activities of the internal control. Values are shown as the mean ± S.D. of nine independent experiments. C, luciferase activities in ECV304 cells transfected with pGL-7, pGL-7 Im, pGL-7 IIm, or pGL-7 ImIIm in the absence (open columns) or presence of E2 (10 nM) (closed columns). Data were normalized by the internal control luciferase activities. Values are shown as the mean ± S.D. of nine independent experiments. Statistical analysis was performed using ANOVA. B, pGL-5; control versus TNF- or AGE-BSA, p < 0.01; pGL-5 NF-B2m, control versus TNF-, AGE-BSA, not significant. C, pGL-7 and pGL-7 Im; control versus E2, p < 0.01; pGL-7 IIm and pGL-7 ImIIm; control versus E2, not significant.

TNF-/AGE-BSA and E₂ Induced the Nuclear Protein-DNA Complex Formation on the NF-B and Sp-1 Sites-- To further characterize the roles of the NF-B and Sp-1 sites in the regulation of RAGE gene expression, we conducted electrophoretic mobility shift assay using synthetic oligodeoxyribonucleotides corresponding to the putative NF-B binding site and to the two Sp-1 binding sites, and nuclear extracts (5 µg) from ECV304 cells. As shown in Fig. 7, the extracts from cells treated with TNF- (panel A, lane 2, arrow) and AGE-BSA (panel B, lane 2, arrow) exhibited increased binding activities to the NF-B site compared with those from untreated cells (panels A and B, lane 1). In E₂-treated cells, the binding activities to the Sp-1 sites also increased compared with the basal conditions (Fig. 7C). In each case, the specificity of the gel-retarded band was demonstrated by competition with a 50-fold excess of unlabeled wild-type or mutant oligodeoxyribonucleotides, namely preincubation with wild-type probes resulted in decreased binding to the NF-B site (Fig. 7, A and B, lane 3) and the Sp-1 sites (Fig. 7C, lane 3). In contrast, preincubation with mutant probes had little effect on the binding to the NF-B and Sp-1 sites (Fig. 7, A-C, lane 4).

View larger version (49K): [in this window] [in a new window]   Fig. 7.   Electrophoretic mobility shift (A-C) and supershift (D-F) assays of the NF-B and Sp-1 binding sites in the RAGE promoter region. A-C, 5 µg of nuclear extracts from ECV304 cells treated for 4 h with TNF- (100 ng/ml) (A), AGE-BSA (50 µg/ml) (B), or E2 (10 nM) (C) were incubated with 25 fmol of the respective 32P-end-labeled double-stranded oligodeoxyribonucleotides for 30 min as described under "Experimental Procedures." For the competition assay, a 50-fold excess of unlabeled wild-type or mutant oligodeoxyribonucleotides was added to the extracts and preincubated for 15 min prior to the incubation with the 32P-end-labeled probe. The formation of the protein/DNA complexes was analyzed by electrophoresis on a 6% polyacrylamide gel. Closed arrows indicate NF-B/DNA and Sp-1/DNA complexes. Open and closed arrowheads indicate nonspecific binding to the probe and free probe, respectively. Essentially the same results were obtained in four independent experiments. D-F, for the supershift assay, the indicated antibodies were added to the extracts and preincubated at 4 °C for 12 h prior to incubation with the 32P-end-labeled probe. p65, anti-human p65 antibody; p50, anti-human p50 antibody; Sp-1, anti-human Sp-1 antibody; ER, anti-human estrogen receptor ; ER, anti-human estrogen receptor . The final concentration of each antibody employed was 0.5 µg/ml. Closed arrows indicate supershifted NF-B/DNA and Sp-1/ERa/DNA complexes. Closed arrowheads on the left indicate original NF-B/DNA and Sp-1/ER/DNA complexes. Open arrowheads, nonspecific binding. Essentially the same results were obtained in three independent experiments.

Next, we examined by supershift assays which members of the NF-B family were responsible for the stimulation of the RAGE gene expression by TNF- and AGE-BSA. When the nuclear extracts from the cells exposed to TNF- (Fig. 7D) or AGE-BSA (Fig. 7E) were incubated with either anti-p65 or anti-p50 antibody, a more slowly migrating band (Fig. 7, D and E, arrow) newly appeared with a concomitant decrease in the original complex (Fig. 7, D and E, closed arrowhead). We also examined which factors were involved in the E₂ induction of the RAGE gene. When the nuclear extracts from the cells treated with E₂ (Fig. 7F) were incubated with either anti-Sp-1 or anti-ER antibody, the original band DNA-protein complex was supershifted upward (Fig. 7F, lanes 2 and 3), whereas anti-ER antibody gave no effect (Fig. 7F, lane 4). The results indicated that the DNA-binding complex for the NF-B site was composed mainly of p50 and p65 members of the NF-B family, and that the DNA-binding complex for the Sp-1 sites was composed mainly of Sp-1 and ER.

The NF-B and Sp-1 Sites Also Mediated the TNF--/AGE-BSA- and E₂-induced RAGE Gene Expression in HMVEC-- To confirm whether the NF-B site and Sp-1 sites are also involved in the TNF--/AGE-BSA- and E₂-induced up-regulation of RAGE gene in normal diploid microvascular endothelial cells, we performed luciferase assays in HMVEC using pGL-5 and its mutants for the TNF-/AGE-BSA-induced transcriptional activation, and pGL-7 and its mutants for the E₂-induced activation. As shown in Fig. 8A, TNF- and AGE-BSA induced luciferase expression ~3-fold in pGL-5-transfected cells, the extent of induction being comparable with those of the RAGE mRNA induction in HMVEC (Fig. 1). The inducibility by both TNF- and AGE-BSA was found to be totally abolished in pGL-5 NF-B2m- or pGL-6-transfected cells (Fig. 8A). E₂ induced luciferase expression about 2.6-fold in pGL-7-transfected cells (Fig. 8B). This extent of induction was also comparable with that of the RAGE mRNA induction by E₂ in HMVEC (Fig. 1). The E₂ inducibility of luciferase activities was totally abolished in pGL-7 ImIIm- or pGL-8-transfected cells (Fig. 8B).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Transcriptional activation in HMVEC. A, relative luciferase activities in HMVEC transfected with pGL-5, pGL-5 NF-B2m, or pGL-6 in the absence (open columns) or presence of TNF- (100 ng/ml) (closed columns) or AGE-BSA (50 µg/ml) (dotted columns). Data were normalized by the activities of pRL-CMV-derived luciferase used as an internal control. Values are shown as the mean ± S.D. of nine independent experiments. B, luciferase activities in HMVEC transfected with pGL-7, pGL-7 ImIIm, or pGL-8 in the absence (open columns) or presence of E2 (10 nM) (closed columns). Data were normalized by the activities of the internal control. Values are shown as the mean ± S.D. of nine independent experiments. Statistical analysis was performed using ANOVA. A, pGL-5; control versus TNF- or AGE-BSA, p < 0.01; pGL-5 NF-B2m; control versus TNF-, AGE-BSA, not significant; pGL-6; control versus TNF-, AGE-BSA, not significant. B, pGL-7; control versus E2, p < 0.01; pGL-7 ImIIm; control versus E2, not significant; pGL-8; control versus E2, not significant.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We (2-4, 35) and others (36-39) have shown previously that interactions between AGE and RAGE cause phenotypic changes in microvascular endothelial cells and pericytes that are characteristic of diabetic vasculopathy. In this paper, we have demonstrated for the first time that AGE, the RAGE ligand itself, TNF-, and E₂ specifically up-regulate RAGE mRNA and protein levels in human vascular endothelial cells, and that this process is mediated by two distinct nuclear complexes, namely p65/p50 NF-B and Sp-1/ER.

It has been reported that AGE-rich vasculature exhibits enhanced RAGE immunoreactivity in the sites of diabetic microvascular injury (13). This finding suggested that AGE themselves could activate the RAGE gene expression, and that increased RAGE might further transduce AGE signals within microvascular cells. The present study has clearly shown that AGE-BSA did, but non-glycated BSA did not, up-regulate the RAGE gene expression and that this was achieved at the level of transcription, because RAGE mRNA half-lives were unchanged by the treatment with AGE (Fig. 4), and because AGE elicited the expression of the reporter gene linked to the 5' promoter region of the RAGE gene (Fig. 5). The experiments with the RAGE promoter-reporter gene constructs demonstrated the presence of an AGE-responsive element in the 751 to 629 region of the human RAGE gene, and that an NF-B site residing at 671 conferred the responsiveness to AGE on the RAGE gene (Figs. 5, 6, and 8). Further, the AGE-dependent formation of the NF-B element-p65/p50 complex was demonstrated (Fig. 7). These results are considered to be consistent with the recent observations by others (11, 12) that AGE engagement of RAGE induces cellular oxidant stress, thereby activating the transcription factor NF-B. The fact that AGE themselves induce the RAGE gene appears to be important when considering the mechanisms of development of diabetic vascular complications. Such a positive feedback loop in the diabetic state may exacerbate diabetic vasculopathy, exemplified by retinopathy and nephropathy.

We also demonstrated that TNF- is another inducer of the RAGE gene (Figs. 1-3). TNF- is known to be overexpressed in adipose tissue under obese and diabetic states (15-17) and to cause insulin resistance, the central and early component of non-insulin-dependent diabetes mellitus (18). TNF- affects not only insulin sensitivity by suppressing insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (40) but also cell survival by NF-B activation (20-22). We thus propose that an increased TNF- level in non-insulin-dependent diabetes mellitus patients may worsen diabetic vasculopathy via RAGE gene induction. The TNF--induced stimulation of human RAGE gene was found to be also transcriptional and to be achieved by the same NF-B element and binding complex (Figs. 5-8) as was the AGE stimulation. Recently, Li et al. (14) reported that the RAGE gene was activated by lipopolysaccharide via NF-B sites at 671 and 467 in bovine aortic endothelial cells and rat vascular smooth muscle cells, and that both sites were required for full activation by lipopolysaccharide. Our results indicated that only one NF-B site at 671 was required for RAGE gene induction by AGE-BSA and TNF- in human microvascular endothelial cells (Figs. 5, 6, and 8). The discrepancy may be due to differences in the species or ligand specificity of the gene activation. The results suggest that the factors that have the ability to activate the NF-B pathway can induce RAGE gene expression and have the potential to aggravate the AGE-mediated diabetic complications.

Pregnancy is known to worsen diabetic retinopathy (22-24). Schocket et al. (41) showed that a decrease in the retinal volumetric blood flow in diabetic patients during pregnancy might exacerbate retinal ischemia and aggravate retinopathy. Suzuma et al. (42) demonstrated that E₂ at the concentration often observed during pregnancy (~10 nM) stimulates VEGF-dependent angiogenesis through the up-regulation of VEGF receptor-2 expression. However, the mechanisms underlying the adverse effects of E₂ on diabetic complications are not yet fully understood. In this study, we demonstrated that E₂ is an alternative inducer of the RAGE gene in human endothelial cells, enhancing its transcription at the concentration of 10 nM (Figs. 1-3). An anti-estrogen, 4-OH tamoxifen, totally abolished the E₂-induced RAGE mRNA induction in HMVEC, while itself caused no change in the RAGE mRNA level (Fig. 1C). This was regarded as an indication that E₂ may act on RAGE gene through an estrogen receptor. The RAGE promoter does not contain any classical estrogen-responsive element (43) but contains several GC-rich boxes, which can bind to the transcription factor Sp-1 (14, 33, 44). Clearly, E₂ utilizes a device that is different from those employed by AGE and TNF- in the induction of RAGE gene. Two Sp-1 binding sites at 189 and 172 and an Sp-1/ER complex were involved in the E₂ activation, and full E₂ responsiveness required both cis-acting elements (Figs. 5-8). Recent studies from other laboratories have shown that an interaction of Sp-1/ER complex with GC-rich motifs in the promoter region is required for the transcriptional activation of several E₂-responsive genes (45-49). The results obtained in this study suggest that the E₂ induction of RAGE may partly underlie the exacerbation of diabetic retinopathy during pregnancy.

There are many genes that are regulated by the combination of NF-B and Sp-1, and in some cases a direct interaction between NF-B protein and Sp-1 protein has been demonstrated (50, 51). In the induction of the RAGE gene, however, such a direct interaction between the two factors would seem unlikely to occur because the Sp-1-mediated E₂ responsiveness was still retained in the constructs with the deletion of the more than 500-nucleotide region encompassing the NF-B element (Fig. 5).

Cytokines that did not affect the endothelial cell expression of RAGE gene included TGF-1 and IFN- (Fig. 1C). This indicated that Smad (52) or Janus kinase-signal transducers and activators of transcription (53) systems would not be involved in the regulation of RAGE gene.

In summary, we found that AGE and TNF- enhanced RAGE expression through activation of the p65/p50 complex of NF-B, and that E₂ also activated RAGE expression through activation of the Sp-1/ER complex. Although the extent was rather modest, the three agents consistently increased the RAGE mRNA and protein levels in vascular endothelial cells. Chronic exposure to AGE, TNF-, and/or E₂ and sustained enhancement of RAGE expression may cause a further accumulation of AGE in the vasculature, resulting in an exacerbation of AGE-RAGE-mediated vascular dysfunctions. Such mechanisms of RAGE gene activation probably have evolved to regulate functions of this multiligand receptor in various biologic processes, such as neurite formation of cortical neurons mediated by amphotericin engagement (54) and proinflammatory responses mediated by a recently discovered endogenous ligand, EN-RAGE (55). Obviously, diabetes abuses the molecular devices for the RAGE gene regulation, resulting in the formation of a vicious circle which may eventually lead to the development and progression of diabetic vasculopathy. The enhanced interaction between AGE and RAGE may further increase VEGF expression in endothelial cells and/or retinal pigment epithelial cells (2, 56), resulting in angiogenesis, and also inhibit pericyte growth, leading to pericyte loss (4). Although more studies are needed to better clarify the significance of the RAGE gene activation by AGE, TNF-, and E₂ as well as immediate post-RAGE signaling events that lead to NF-B and Sp-1 activations, inhibition of the RAGE gene activation may become a promising target for the prevention and treatment of diabetic vascular complications.

	ACKNOWLEDGEMENTS

We thank Shin-ichi Matsudaira, Reiko Kitamura, and Tomoko Yachi for assistance, and Brent Bell for reading the manuscript.

	FOOTNOTES

^* This work was supported by Grant 97L00805 from the Research for the Future Program of the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. Tel.: 81-76-265-2180; Fax: 81-76-234-4226; E-mail: yamamoto@med.kanazawa-u.ac.jp.

Published, JBC Papers in Press, May 26, 2000, DOI 10.1074/jbc.M001235200

	ABBREVIATIONS

The abbreviations used are: AGE, advanced glycation end products; RAGE, receptor for AGE; VEGF, vascular endothelial growth factor; NF-B, nuclear factor-B; TNF-, tumor necrosis factor-; E₂, 17-estradiol; BSA, bovine serum albumin; TGF-1, transforming growth factor-1; IFN-, interferon-; HMVEC, human skin microvascular endothelial cells; FBS, fetal bovine serum; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; ER, estrogen receptor; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES