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Diabetes-Associated Sustained Activation of the Transcription Factor Nuclear Factor-B

¹ Department of Medicine I and Department of Neurology, University of Heidelberg, Heidelberg, Germany
² Department of Medicine IV, University Tübingen, Tübingen, Germany
³ Department of Anatomy, Department of Immunology, Department of Pathology and Institute of Food Chemistry, Technical University Dresden, Dresden, Germany
⁴ Department of Laboratory Animal Science, Institute of Pathophysiology, University Greifswald, Karlsburg, Germany
⁵ Columbia University, Department of Physiology, New York, New York
⁶ ASTA-Medica, Frankfurt am Main, Germany
⁷ Institute of Pathology, Case Western Reserve University, Cleveland, Ohio

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Activation of the transcription factor nuclear factor-B (NF-B) has been suggested to participate in chronic disorders, such as diabetes and its complications. In contrast to the short and transient activation of NF-B in vitro, we observed a long-lasting sustained activation of NF-B in the absence of decreased IB in mononuclear cells from patients with type 1 diabetes. This was associated with increased transcription of NF-Bp65. A comparable increase in NF-Bp65 antigen and mRNA was also observed in vascular endothelial cells of diabetic rats. As a mechanism, we propose that binding of ligands such as advanced glycosylation end products (AGEs), members of the S100 family, or amyloid-ß peptide (Aß) to the transmembrane receptor for AGE (RAGE) results in protein synthesis–dependent sustained activation of NF-B both in vitro and in vivo. Infusion of AGE-albumin into mice bearing a ß-globin reporter transgene under control of NF-B also resulted in prolonged expression of the reporter transgene. In vitro studies showed that RAGE-expressing cells induced sustained translocation of NF-B (p50/p65) from the cytoplasm into the nucleus for >1 week. Sustained NF-B activation by ligands of RAGE was mediated by initial degradation of IB proteins followed by new synthesis of NF-Bp65 mRNA and protein in the presence of newly synthesized IB and IBß. These data demonstrate that ligands of RAGE can induce sustained activation of NF-B as a result of increased levels of de novo synthesized NF-Bp65 overriding endogenous negative feedback mechanisms and thus might contribute to the persistent NF-B activation observed in hyperglycemia and possibly other chronic diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

The transcription factor nuclear factor-B (NF-B) has been proposed as a critical bridge between oxidant stress and gene expression (1,2,3,4,5,6,7,8). Exposure of cells to inflammatory, infectious, or other stressful stimuli results in rapid phosphorylation and degradation of IB and the subsequent release and translocation of NF-B into the nucleus (1,2,3,4,5,6,7,8,9,10,11). This mechanism ensures quick and finely tuned cellular responses in the absence of de novo protein synthesis. Because transcription of IB is positively autoregulated by NF-B (9,10,11), activation of NF-B is usually self-terminated within minutes to hours (1,2,3,4,5,6,7,8,9,10,11). Such a scenario lends itself to analysis by short-term in vitro studies in which stimulus-induced responses are transient and the system returns to the baseline state over hours. Consequently, induction of NF-B and enhanced transcription of its target genes in vitro have been studied mainly in the setting of acute cellular responses.

Reactive oxygen intermediates are generated by processes that occur over seconds. However, increasing evidence suggests a role for oxidative stress in chronic degenerative diseases such as atherosclerosis (1,6,12,13), diabetes (14,15,16), and Alzheimer’s disease (17,18,19). This indicates the relevance of signal transduction systems such as NF-B, which are capable of transforming the appearance and disappearance of short-lived oxygen free radicals into more sustained signals for cellular activation (8,16). Consistent with this concept, a recent study demonstrated that hyperglycemia-dependent overproduction of mitochondrial superoxide results in NF-B activation (16). NF-B, however, also acts as a protective factor against programmed cell death by inducing antiapoptotic genes such as A1, A20, XIAP, Bcl-2, and BcL-X_L and thus ensures cell survival in situations of perpetuated cellular activation (20,21,22,23). Therefore, more sustained protein synthesis–dependent pathways of NF-B activation might be present in addition to immediate, self-controlled protein synthesis–independent pathways of NF-B activation to rescue cells from cell death in the presence of a persistent stimulus.

Pathophysiologically relevant cellular perturbants associated with oxidant stress and chronic disorders are advanced glycosylation end products (AGEs) (14,16,24,25,26), S100 proteins (27), and amyloid-ß peptides (Aß) (28,29). AGEs are the result of nonenzymatic glycation and glycoxidation that accumulate during normal aging (14,24,25,26). Their deposition is enhanced with concomitant hyperglycemia, oxidant stress, carbonyl stress, and disorders associated with delayed macromolecular turnover, such as renal failure and amyloidosis (16,18,24,25,26,28,29,30,31,32,33,34,35,36,37,38,39,40). Carboxymethyllysine (CML), a rapidly formed glycoxidation product that increases in mononuclear blood cells of healthy volunteers undergoing hyperglycemic clamps already within 2 h (S.S., P.P.N., unpublished observation), is one of the earliest markers of AGE formation (36,37,38,39,40). Aß is the primary component of the extracellular senile plaques in Alzheimer’s disease (29) and has been strongly linked to the pathogenesis of this devastating neurodegenerative disease (18,29,30). AGEs, S100 proteins, and Aß are ligands of the receptor for AGEs (RAGE) (24,26,27,28,29,30,31,32,41,42,43,44,45,46,47,48,49). Due to their long-life nature, their ubiquitous presence in the plasma, vessel wall, and tissues, and their recognition by RAGE, whose expression levels seem to increase in the course of chronic disorders (24,29,31,32,41,42,43,44,45,46,47,48,49), we considered the hypothesis that ligand-receptor interaction may alter cellular homeostasis in a self-perpetuating manner.

RAGE is a member of the immunoglobulin superfamily of cell surface molecules (24,27,28,29,31,32,41,50) and is expressed by endothelial cells, monocytes/macrophages, neurons, and a range of other cells (50) whose dysfunction has been linked to chronic disorders such as diabetes, Alzheimer’s disease, inflammation, and atherosclerosis. Binding of ligands to RAGE triggers signal transduction mechanisms, including formation of lipid peroxides, activation of NF-B, and subsequent expression of genes regulated by NF-B (27,28,29,31,32,41,42,43,44,45,51,52,53).

There is increasing evidence that NF-B activation in vivo occurs in a more sustained manner. Constitutive NF-B activation has been observed in different malignant cells (54,55,56,57,58) as a result of a defect or dysregulation of IB and IBß or aberrant activation of IB-kinase (58). A persistent NF-B activation has also been suggested in atherosclerosis (13,59), Crohn’s disease (60,61,62), and Listeria monocytogenes infection (63). In vitro data have suggested that hypophosphorylated IBß (64,65,66,67) or de novo synthesis of c-rel can mediate longer-lasting NF-B activation (68,69).

However, experiments performed in nonimmune and nonmalignant cells were not directed toward understanding mechanisms underlying chronic NF-B activation in vivo, and the mechanism by which the normally short-acting response of NF-B activation is converted to a persistent cell perturbation is not understood. RAGE was recently shown to be involved in chronic disease, such as atherosclerosis, amyloidosis, diabetes, inflammation, tumor proliferation, and metastasis (27,28,29,31,43,44,45,46,47,48,49,53). Therefore, we studied whether ligand-stimulated RAGE activation can act like a master switch to turn short-lasting activation into a prolonged activation of NF-B.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents.
Reagents were obtained as follows: Dulbecco’s modified Eagle’s medium (DMEM; 4,500 mg/l glucose), RPMI 1648, HEPES buffer solution, L-glutamine, penicillin-streptomycin mixture, and phosphate-buffered saline (PBS; pH 7.4) were from Biowhittaker (Walkerville, MD). Fetal calf serum (FCS) was from Gibco/BRL (Dreieich, Germany). [-³²P]ATP (3,000 Ci/mmol at 10 mCi/ml), L-[³⁵S]methionine (>1,000 Ci/mmol at 10 mCi/ml), Hybond-N-nylonfilter, enhanced chemiluminescence (ECL)-nitrocellulose membranes, ECL detection reagents, and Hyperfilm X-ray films were obtained from Amersham (Freiburg, Germany). Aß peptides (1–40 and 25–35), S100B, phenylmethylsulfonyl fluoride (PMSF), actinomycin D, and cycloheximide were purchased from Sigma (Deisenhofen, Germany). Poly dI/dC and oligo(dt) was from Pharmacia (Freiburg, Germany). Anti-p65 (#sc-109), anti-IB (#sc-847), anti-IBß (#sc-945), anti–c-Jun KM-1 polyclonal antibodies, the respective second antibodies, and protein A/G PLUS-agarose were obtained from Santa Cruz (Heidelberg, Germany). Monoclonal anti-p65 antibodies, specific for active NF-B, were obtained from Boehringer Mannheim (Mannheim, Germany). Soluble RAGE (sRAGE) preparations used throughout this study have previously been described in detail (29,31,32,45,49,70). Polyclonal anti-RAGE antibodies, generated in goat with recombinant RAGE prepared in Escherichia coli as antigen, were a gift of Dr. M.A. Shearman (Merck, Sharpe & Dome, Essex, U.K.). Anti–heme oxygenase-1 (HO-1) antibodies were from Dr. M.A. Smith (Case Western Reserve University, Cleveland, OH). Vectastain ABC kits were purchased from Vector Laboratories (Burlingame, CA). The vector NF-B-Luc and primers for human HPRT were from Clontech (Heidelberg, Germany), and Effectene transfection reagent was obtained from Qiagen (Hilden, Germany).

Preparation of AGE-albumin, CML-albumin and Aß peptides.
AGE-albumin was prepared by incubating bovine or mouse serum albumin with 200 mmol/l glucose-6-phosphate at 37°C for 4 to 8 weeks in 100 mmol/l phosphate (pH 7.4) and 0.5 mmol/l sodium azide. At the end of incubation, AGE-albumin preparations were dialyzed against 5 l of 100 mmol/l phosphate and 10 mmol/l EDTA for 24 h and 0.9% NaCl for 12 h. Control albumin was also dialyzed against these buffers. Alternatively, glycated bovine albumin was purchased from Sigma. Nonglycated bovine albumin, heat-inactivated AGE-bovine albumin, and bovine albumin incubated with the synthetic substrate sorbitol served as negative controls. AGE-albumin preparations were characterized by chromatographic means (see below).

Quantification of furosine, pentosidine, and other amino acids after acid hydrolysis.
AGE-albumin samples were hydrolyzed in the presence of 6 N hydrochloric acid for 23 h at 110°C; the hydrolysate was dried in vacuo on a Speedvac concentrator (Savent, Farmingdale, NY) and dissolved in 0.2N sodium citrate buffer (pH 2.2). Analysis of amino acids, including CML, was performed on an Alpha Plus amino acid analyzer (LKB Biochrom, Cambridge, U.K.), using a stainless steel column (150 x 4 mm) filled with ion-exchange resin, DC4A-spec sodium form (Benson, Reno, NV). Composition of elution buffers, ninhydrin reagent, and running conditions are described elsewhere (71,72). Injection volume was 10–80 µl. After separation on the ion-exchange column, amino acids were initially detected using either a photodiode array detector PDA 996 (Waters, Eschborn, Germany; for furosine quantification, a detection wavelength of 280 nm was used) or a fluorescence detector SFM 25 (Kontron, Eching, Germany; for pentosidine quantification, measuring was achieved at excitation/emission wavelengths of 335/385 nm), connected between the outlet of the column and the ninhydrin reaction coil. After subsequent ninhydrin derivatization, amino acids were detected at 570 and 440 nm. Protein content of the plasma samples was calculated as the sum of the amino acids. From values of furosine, fructoselysine was calculated by multiplying the molar furosine values by 3.1, the known conversion factor for the degradation of fructoselysine during acid hydrolysis (73,74). For quantification of pentosidine, reference material was synthesized according to Henle et al. (75). Furosine was obtained from Neosystem (Strasbourg, France).

Pyrraline analysis after enzymatic hydrolysis.
Enzymatic hydrolysis was performed using four enzymes (pepsin, pronase, aminopeptidase, and prolidase) as described previously (75). For pyrraline detection, amino acid analysis was performed as described above with the combination of the PDA (set at a wavelength of 297 nm) and ninhydrin derivatization (75). Alternatively, pyrraline analysis was performed via isocratic ion-pair reversed-phase high-performance liquid chromatography (HPLC) with UV detection (76). Pyrraline reference material was synthesized according to Henle and Bachmann (77).

Preparation of CML-albumin and Aß peptides.
In vitro synthesis of CML-albumin was performed as described by Schleicher et al. (36). Assays for endotoxin showed AGE-albumin and CML preparations to contain virtually undetectable levels of lipopolysaccharide (LPS; <10 pg/ml at a protein concentration of 5 mg/ml according to the Limulus assay [Sigma]). Synthetic Aß peptides (1–40) were purchased from Sigma and made up freshly every time. To allow fibril formation, we kept preparations at room temperature (rt) for at least 4 h before use.

Isolation of CML-modified proteins from erythrocytes.
Erythrocytes were lysed in 0.9% NaCl, 1.5 mmol/l PMSF, 0.1 mmol/l leupeptin, 20 mg/ml soybean inhibitor, and 2 mmol/l benzamidine by three freeze-thaw cycles followed by pulsed ultrasonication for 3 min and a final freeze-thaw cycle. Insoluble material was removed by a 5-min centrifugation at 6,000g at 4°C, and the supernatant was used for extraction of CML-modified proteins as previously described (70). Ten micrograms of total erythrocyte lysate was loaded onto a 1-ml anti–CML-Sepharose column (36,70) and incubated for 2 h at rt (70). After extensive washing, CML-modified proteins were eluted with 1 mol/l glycine (pH 3.0), neutralized, and dialyzed in DMEM. Protein concentration was determined using the BCA assay system (Pierce, Rockford, IL).

Plasmids.
The SV-40 driven luciferase control plasmid pGL2-control, the promoterless plasmid pGL2-basic, and the ß-galactosidase (ß-Gal) control plasmid pSV-ß-Gal were obtained from Promega (Heidelberg, Germany). The plasmid NF-B-Luc, which contains four tandem copies of the NF-B consensus sequence fused to a TATA-like promoter region from the herpes simplex virus thymidine kinase promoter, was from Clontech (Heidelberg, Germany).

Transgenic mouse model.
Mice transgenic for an NF-B–driven ß-globin reporter gene (tg14) were provided by Dr. Thomas Wirth (Würzburg, Germany) and have been previously characterized in detail (78). Eight-week-old female mice were left untreated or treated once with 1,000 µg of mouse AGE-albumin (500 µg i.p. and 500 µg i.v.) at time point 0 and kept for an additional 6 days. Where indicated, mice received sRAGE (25 µg/mouse) or anti-RAGE antibodies (40 µg/mouse) as described (29,31,32). At the end of the experiments, mice were killed and organs were removed, immediately snap-frozen, and analyzed by reverse transcription–polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assays (EMSAs).

Diabetic rat model.
The kidneys of diabetic rats and of nondiabetic Sprague-Dawley rats were provided by Dr. I. Köting (Karlsburg, Germany). Diabetic BB/O(ttawa)K(arlsburg) rats were bred as previously described in detail (79), developed diabetes at the age of 104 days (±16 days), and were kept for 57 days (±9 days) before harvest. During this time, rats were treated with sustained-release insulin implants (Linplant; Mollegaard, Skensved, Denmark) with an insulin rate of 2 units/24 h. With this treatment, the mean plasma glucose amounted to 136 ± 24 mg/dl.

Patient characteristics.
Blood from six patients with newly manifested diabetes type 1 (two men, four women; age 35.1 ± 11.7 years; HbA_1c 12.4 ± 2.3%) was collected on the day the diagnosis was made. Four healthy volunteers (three men, one woman; age 31.5 ± 5.5 years) served as control subjects. From three of these patients, repetitive blood sampling was performed over 3 consecutive days. HbA_1c levels were routinely determined by HPLC (Diamat; Biorad, Munich, Germany; normal range 4.5–6.1%) in the central laboratory unit of the hospital. The study was approved by the ethical committee of the Department of Medicine, University of Tübingen, and informed consent had been obtained from all patients studied.

Preparation of peripheral blood mononuclear cells and immunocytological detection of activated NF-Bp65.
Immediately after venipuncture, peripheral blood mononuclear cells (PBMCs) were separated as previously described in detail (80,81,82). Isolated PBMC were washed three times in cold PBS (pH 7.4, 4°C), seeded on coverslips, and fixed in 4% paraformaldehyde according to standard methods. Staining for NF-Bp65 was performed as described below using a monoclonal antibody (Boehringer Mannheim), which exclusively recognizes the activated form of NF-B (13).

Immunocytochemistry.
Immunocytochemistry was performed by indirect immunoperoxidase techniques using PBMC fixed onto poly-L-lysine–coated glass slides with 4% paraformaldehyde; slides were washed twice in PBS (pH 7.4) for 5 min. After the last wash, slides were treated with 0.3% hydrogen peroxide, dissolved in methanol, for 30 min at rt. Blocking was performed with 0.6% normal goat or horse serum for 30 min (rt) before the affinity-purified polyclonal rabbit antibody against IB (concentration: 0.01 µg/µl; 60 min; rt) or the monoclonal mouse antibody for activated NF-Bp65 (concentration: 0.01 µg/µl; 60 min; rt) were used as primary antibodies. Slides were washed 3 times for 10 min in PBS (pH 7.4), and detection of signals was performed using Vectastain ABC kits (Vector Laboratories) according to the manufacturer’s instructions, as previously described in detail (61,80,81). Peroxidase activity was visualized with 0.05% 3,3-diaminobenzidine-tetrahydrochloride (Vector Laboratories) before the slides were counterstained with Mayer’s hematoxylin (Vector Laboratories). Controls for immunospecificity were included in all experiments by omission of the primary antibody and its replacement by PBS and matching concentrations of normal rabbit serum (data not shown).

RT-PCR.
RT and PCR for ß-globin, NF-Bp65, hypoxanthine guanine phosphoribosyl transferase (HPRT), and ß-actin were performed as described by Lernbecher et al. (78). cDNA was reverse-transcribed from 2 µg of total RNA with oligo-dT primers (Pharmacia), AMV-reverse transcriptase, and Taq-polymerase (Promega). When RT-PCR was performed from PBMC, Taq-polymerase was replaced by PFU-polymerase (Stratagene, Heidelberg, Germany). Amplification was performed using the following primer pairs: ß-globin transgene forward 5'-TTTCTGATAGGCAGCCTGCACTGGT-3', ß-globin transgene reverse 5'-CATAGTTGTGTTCAGATCGATCTGG-3'; p65-forward-1 5'-GCTACAAGTGCGAGGGGC-3', p65-reverse-1 5'-GGGGTCTGCGTAGGGAGGG-3'; p65-reverse-2 5'-GGCCTGCCTGATGGGTCCC-3; actin-forward 5'-AGAGGTATCCTGACCCTGAAGTACC-3', actin-reverse 5'-CCACCAGACAACACTGTGTTGGCAT-3'; HPRT-forward-1 5'-GGCGTCGTGATTAGTGATGATGAACC-3', HPRT-reverse 5'-CTTGCGACCTTGACCATCTTTGGA-3' The following conditions were used: 1x [95°C, 360 s]; 40x [94°C, 40 s; 65°C, 60 s.; 72°C, 120 s]; 1x [72°C, 600 s] for ß-globin and actin, 1x [95°C, 360 s]; 35x [94°C, 40 s; 68°C, 60 s; 72°C, 120 s]; 1x [72°C, 600 s] for NF-Bp65, 1x [95°C, 360 s]; 1x [94°C, 60 s; 52°C, 120 s; 72°C, 120 s]; 1x [94°C, 60 s; 58°C, 60 s; 72°C, 120 s]; 32x [94°C, 40 s; 62°C, 60 s; 72°C, 60 s]; 1x [72°C, 600 s] for HPRT and separated onto 2% agarose gels. Verification of PCR products was performed by reamplification with different primers recognizing sequences internal to the amplification products and by Southern blot analysis. Reactions that lacked template RNA or AMV-reverse transcriptase served as internal controls.

EMSA.
Nuclear proteins were harvested as described elsewhere (51,52,61,80,81,82,83,84) and assayed for transcription factor binding activity using the NF-B consensus sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3'. Specificity of binding was ascertained by competition with a 160-fold molar excess of unlabeled consensus oligonucleotides and by supershift experiments (data not shown).

Immunoblot (Western blot) analysis.
Cytoplasmic and nuclear fractions were prepared as previously described in detail (51,52,61,84). Twenty micrograms of cytoplasmic extracts or 10 µg of nuclear extracts was separated onto 10–12% SDS-PAGE, followed by electroblotting to ECL-nitrocellulose membranes. Membranes were incubated with primary antibodies for NF-Bp65, IB, and IBß (1:100) for 60 min at rt. After washing (2 x 7 min in Tris-buffered saline [TBS], 0.05% Tween), the secondary antibody (horseradish peroxidase–coupled rabbit IgG, 1:2000) was added, and incubation was continued for 30 min at rt. Membranes were washed 3x 5 min as above followed by a last 5-min wash in TBS. Immunoreactive proteins were detected with the ECL-Western blot system and subsequent autoradiography for 2 min.

In vitro phosphorylation of IB.
For in vitro phosphorylation, 5 x 10⁶ bovine aortic endothelial cells (BAECs) were incubated in the presence of 500 nmol/l AGE-albumin for the times indicated in the figure legends. At the end of incubation, cells were washed 3 times in 0.9% NaCl, harvested by scraping, collected by a 5-min centrifugation at 500g, 4°C, and resuspended in 1 ml of 0.9% NaCl. Preparations were counted in a Neugebauer chamber and adjusted for the same number of cells, collected by centrifugation as above, and resuspended in 500 µl of 0.9% NaCl. Whole-cell lysate was obtained by mechanical disruption with an Ultrathurrax (Wheaton, Milleville, NJ), cleared by centrifugation at 10,000g at 4°C for 5 min, and immediately supplemented with 5 µl of 100 mmol/l PMSF (80). In vitro phosphorylation of a 70-kDa IB-GST fusion protein (Santa Cruz) was performed for 30 min at 37°C in 35 mmol/l Tris (pH 7.5), 15 mmol/l MgCl₂, 1 mmol/l MnCl₂, 2 µl of 100 mmol/l rATP, and 1 µl of -³²P-ATP (10 µCi) with 15 µl of the above whole-cell extract (85). The reaction was completed by the addition of 180 µl of IP buffer (20 mmol/l Tris [pH 7.5], 150 mmol/l NaCl, 5 mmol/l EDTA, 0.2% SDS, 1% Triton-X100, 0.1 mmol/l PMSF, 1 unit/ml aprotinin) supplemented with 1 mmol/l sodium orthovanadate (85). After addition of 2 µl of IB (FL) antibody and 10 µl of protein A/G Sepharose, incubation was continued for 16 h at 4°C. At the end of incubation, precipitates were collected by centrifugation at 10,000g for 10 min at 4°C; washed twice in IP buffer followed by a final wash in 150 mmol/l NaCl, 50 mmol/l Tris-HCl (pH 7.5), and 5 mmol/l EDTA; and then resuspended in 40 µl of denaturing buffer (125 mmol/l Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) (86). The immunoprecipitate was analyzed by SDS-PAGE and autoradiography (85,86).

Intrinsic labeling and immunoprecipitation of NF-Bp65.
BAEC in 35-mm² dishes that had been confluent for 3 days were stimulated with AGE-albumin (500 nmol/l) for the times given in the figure legends. Four hours before harvest, the medium was changed to methionine-free medium for 1 h. Thereafter, the medium was changed again to DMEM supplemented with 7 µCi/ml L-[³⁵S]methionine. Where indicated, BAECs were cultivated in the presence of 0.1 µmol/l antisense-p65 oligonucleotides, added 24 h before harvest. At the end of incubation, BAECs were lysed in 400 µl of IP buffer (20 mmol/l Tris [pH 7.5], 150 mmol/l NaCl, 5 mmol/l EDTA, 0.2% SDS, 1% Triton-X100, 0.1 mmol/l PMSF, and 1 unit/ml aprotinin) for 20 min at 4°C, before lysates were cleared by centrifugation (10,000g, 10 min, 4°C) (86). The protein concentration in the supernatant was determined according to Bradford. Total protein lysate (200 µg) was precleared by adding 1 µg of an irrelevant monoclonal mouse antibody (c-jun KM; Santa Cruz) and 20 µl of protein A/G PLUS-agarose. The reaction was incubated at 4°C for 30 min and centrifuged for 5 min at 10,000g, 4°C. The supernatant was incubated with 2 µg of a polyclonal anti-p65 antibody (Santa Cruz) for 1 h at 4°C before 20 µl agarose conjugate was added and incubation was continued for 16 h at 4°C. At the end of incubation, precipitates were collected by centrifugation at 10,000g for 10 min at 4°C; washed three times in IP buffer, followed by a last wash in 150 mmol/l NaCl, 50 mmol/l Tris-HCl (pH 7.5), and 5 mmol/l EDTA, and then resuspended in 40 µl of denaturing buffer (125 mmol/l Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 10% ß-mercaptoethanol) (86). Four microliters were counted in a ß-counter to determine incorporation of L-[³⁵S]methionine into each sample. Determination was repeated twice. The remaining 32 µl was subjected to SDS-gel electrophoresis and subsequent autoradiography to confirm the incorporation data.

Tissue culture and transient transfection experiments.
BAEC were cultured as previously described (83,87). Bovine arterial smooth muscle cells (SMCs) prepared from bovine aorta were cultured in DMEM containing 10% FCS (88). hT neurons (Stratagene), derived from Ntera/D1(NT2) precursor cells (Stratagene), were cultured according to the manufacturer’s instructions. THP-1 monocytic cells were cultured in RPMI 1648 medium supplemented with 10% FCS. BAECs and SMCs were cultured to confluence and kept so for 3 days in the respective medium containing 10% FCS before the experiments were started. Experiments with hT-neurons were started 1 day after the cells had reached confluence. THP-1 cells were seeded 24 h before the test protein was added. All experiments were performed without serum deprivation before stimulation.

For transfection experiments, BAECs growing in the logarithmic phase were transfected with an NF-B–driven luciferase reporter gene (Clontech) using the Effectene Transfection Reagent (Qiagen) according to the manufacturer’s instructions by adding 0.4 µg of NF-B DNA for each well at a concentration of 0.2 µg/ml of culture medium. After applying the DNA to the cells, cells were cultivated for 1 h before stimulation by AGE-albumin (500 nmol/l) or Aß (1 µmol/l) was performed for 48 h. To correct for efficiency in transfection, we included 0.05 µg of pSV-ß-Gal plasmid/ml medium (83). The ratio of luciferase activity to ß-gal activity served to normalize luciferase activity (83). Each experiment was performed in triplicate.

Antisense-phosphorothioate oligodeoxynucleotides.
Antisense-RAGE-phosphorothioate oligodeoxynucleotides ([PS]ODNs) (5'-G-[PTO]-ACCACTGCCCCTGCTGC-3'); sense-RAGE-[PS]ODNs-(5'-G-[PTO]-CAGCAGGGGCAGTGGTC-3'), antisense-NF-Bp65-[PS]ODNs (5'-G-[PTO]-GGGAACAG-[PTO]-TTCGTCCATGG-[PTO]-C-3), scrambled-antisense-NF-Bp65-[PS]ODNs (5'-A-[PTO]-GTCTGGAG-[PTO]-CCATCGAGGTG-[PTO]-C-3), and sense-NF-Bp65-[PS]ODNs (5'-G-[PTO]-CCATGGACGAA-[PTO]-CTGTTCCC-[PTO]-C-3') derived from the published DNA sequences for bovine RAGE and human NF-Bp65 were synthesized and purified by HPLC (MWG-Biotech, Ebersberg, Germany). [PS]ODNs were chemically modified by introducing phosphorothioate linkages in which a nonbridging phosphate oxygen atom was substituted with a sulfur atom to protect oligonucleotides from serum- and nuclease-mediated degradation, as previously described in detail (52). Uptake of ODNs by the cells occurred passively without any further pretreatment of the cells (52).

Statistical analysis.
All values are given as mean, with the bars showing standard deviations. The means of groups were compared by analysis of variance using the Student’s t test to correct for multiple comparisons. P < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Sustained NF-B activation in vivo.
Hyperglycemia promotes leukocyte adhesion to the endothelium at least in part through NF-B–dependent upregulation of adhesion molecules (1,6,89,90). This implicates a hyperglycemia-associated continuous activation of NF-B in both leukocytes and endothelial cells. Consistently, the presence of activated NF-Bp65 in PBMCs isolated from diabetic patients has been reported to depend on the state of glycemic control (80). When PBMCs were isolated from newly manifested diabetic patients with poor glycemic control (HbA_1c >13), almost all PBMCs were recognized by a monoclonal antibody that exclusively binds to activated NF-Bp65 (Fig. 1A, left) (13). Activated NF-Bp65 was localized within the cells and more pronounced in the nuclear region (Fig. 1A, left, insert), thereby indicating that most of the activated NF-Bp65 was present in the nucleus. In contrast, minor staining in much fewer cells, indicating only a little activated NF-Bp65, was observed in healthy control subjects (Fig. 1A, right). In these cells, staining for activated NF-Bp65 was located outside the nucleus in the cytoplasm, close to the outer cell membrane (Fig. 1A, right, insert). Repetitive sampling of PBMC from three patients with poor metabolic control also showed activated NF-Bp65 in almost all cells over 3 days (Fig. 1C). In contrast, no difference was found when PBMCs of the above diabetic patients and healthy control subjects were analyzed for full-length IB (Figs. 1B and C). An average of 86% PBMCs derived from diabetic patients were positive for IB, similar to the 87% found in healthy control subjects (Fig. 1C). Consequently, the sustained NF-Bp65 activation observed in PBMCs of diabetic patients (Figs. 1A and C) cannot be explained by a loss of IB. Therefore, pathways of sustained NF-B activation must be present in vivo, overriding the IB-dependent autoregulatory loop, and allow activation of NF-Bp65 in the presence of substantial amounts of IB. This hypothesis is consistent with an upregulation of NF-Bp65 mRNA in PBMCs of diabetic patients: When total RNA was isolated from blood samples at the same time PBMCs were prepared and subjected to RT-PCR for NF-Bp65, NF-Bp65 mRNA was markedly increased in PBMC of patients with diabetes compared with PBMCs of healthy control subjects (Fig. 1D). RT-PCR for HPRT served as an internal control and confirmed comparable RNA input in each reaction (Fig. 1D).

View larger version (64K): [in this window] [in a new window]   FIG. 1. Immunocytochemistry of NF-Bp65 and IB in circulating cells of patients with diabetes and in healthy control subjects. A: Immunocytochemistry for activated NF-Bp65 in PBMCs of a patient with newly manifested type 1 diabetes (left) and in a healthy control subject (right) (magnification: x40). Positivity for NF-Bp65 is indicated by the dark brown color. Higher magnification (x200) of single cells demonstrated nuclear localization of NF-Bp65 in PBMCs derived from diabetic patients (left insert, arrow), whereas staining for NF-Bp65 in control PBMCs is restricted to cytoplasmic areas close to the cell wall (right insert, arrow). B: Immunocytochemistry for full-length IB in PBMCs of a patient with newly manifested type 1 diabetes (left) and a healthy control subject (right). Positivity for IB is indicated by the dark brown color. C: Compared with healthy control subjects (n = 6), staining for activated NF-Bp65 was significantly increased in PBMCs of patients with diabetes (n = 6; P = 0.0006 at day 1, diabetes versus control). In contrast, there was no significant difference in IB expression (P = 0.846 at day 1, diabetes versus control). NF-Bp65 and IB levels did not change within 3 days. D: PBMCs were isolated from four healthy volunteers and four patients with diabetes; total RNA was prepared, and 1 µg of each preparation was analyzed by RT-PCR with primers specific for NF-Bp65 (top) and HPRT (bottom), respectively. Representative RT-PCR results from two diabetic patients (lanes 1 and 2) and two control subjects (lanes 3 and 4) are shown at the left. The signal intensity of the NF-Bp65 representing band and the HPRT band was quantified by densitometry, and the ratio of NF-Bp65 to HPRT was calculated for each patient (n = 4) and each control subject (n = 4). The results are summarized at the right side.

View larger version (95K): [in this window] [in a new window]   FIG. 2. Immunocytochemistry of NF-Bp65 in resting cells. A: Nine diabetic kidneys and five control kidneys were examined by immunocytochemistry with a rabbit-derived polyclonal antibody for NF-Bp65, which recognizes both the activated and the inactive protein. Selected representative stainings are shown. Blood vessel endothelia of diabetic kidneys demonstrated pronounced immunoreactivity for NF-Bp65 (left), whereas blood vessel endothelia of control kidneys showed no NF-Bp65 staining (right). Positivity for NF-Bp65 is indicated by the brown color. B: Immunocytochemistry of the above diabetic kidneys using the monoclonal antibody for activated NF-Bp65, demonstrating nuclear translocation of activated NF-Bp65 in the majority of endothelial cells (arrowheads). Positivity for NF-Bp65 is indicated by the dark brown color.

RAGE-mediated sustained activation of NF-B.

View larger version (65K): [in this window] [in a new window]   FIG. 3. Ligands of RAGE induce sustained NF-B DNA binding activity. A–D: BAECs were left untreated (0 h) or were stimulated with AGE-albumin (500 nmol/l; A) or Aß peptide (1 µmol/l; 1–40; B), unmodified control-albumin (500 nmol/l; C), or scrambled Aß peptide (1 µmol/l; 40–1; D) for 12 h to 6 days. Nuclear extracts were prepared as described (see RESEARCH DESIGN AND METHODS) and assayed for NF-B binding activity, monitored in EMSA. Radioactive labeled oligonucleotides, spanning the consensus NF-B recognition motif, were incubated with equal protein amounts of nuclear extracts, and complexes were separated onto nondenaturing 5% PAA gels. For confirming NF-B binding, binding observed after 72 h was competed with a 160-fold molar excess of cold consensus NF-B oligonucleotides (cons.). The position of NF-B is indicated by an arrow. E–G: Nuclear extract from hNT-neurons (E), SMC (F), or THP-1 cells (G), harvested before (0 h) or 6 days after AGE-albumin stimulation (500 nmol/l) (6 days) were subjected to NF-B–specific EMSA. NF-B binding was confirmed by competing the shift observed after 6 days with a 160-fold molar excess of cold consensus NF-B oligonucleotides (cons.). The position of NF-B is indicated by an arrow.

View larger version (63K): [in this window] [in a new window]   FIG. 4. AGE-mediated induction of NF-B DNA binding activity in vitro is dependent on RAGE. A: BAECs were left untreated (lane 1) or were incubated for 6 days with AGE-albumin (500 nmol/l) (lane 2) or with AGE-albumin (500 nmol/l) in the presence of either a 10-fold molar excess of sRAGE (lane 3) or 0.1 µmol/l antisense RAGE oligonucleotides (lane 4) before NF-B binding activity was monitored in EMSA. For confirming NF-B binding, binding observed at day 6 was competed with a 160-fold molar excess of unlabeled NF-B consensus oligonucleotides (lane 5). The result shown is representative of three independent experiments. B: BAECs were incubated for 5 days with S100B (400 nmol/l) in the absence (lane 1) or presence (lane 2) of a threefold molar excess of sRAGE, and NF-B binding activity was monitored in EMSA as above. C: BAECs were left untreated (lane 1) or incubated for 5 days with 400 nmol/l highly modified CML-albumin (lane 2), CML-modified proteins isolated from erythrocytes of a patient with diabetes and poor glycemic control (HbA1c 14.3%; lane 4), minimally modified CML-albumin (lane 6), or CML-modified hippuric acid (lane 8) in the absence (lanes 1, 2, 4, 6, 8) or presence (lanes 3, 5, 7, 9) of a threefold molar excess of sRAGE. NF-B binding activity was monitored in EMSA. The NF-B signal intensity was quantified by densitometry and is given below in the autoradiogram.

The observations in cell culture led us to predict that AGE/RAGE-dependent NF-B activation would also occur in vivo. Mice transgenic for an NF-B–controlled ß-globin transgene (78) were used. Under physiological conditions, constitutive expression of the transgene is restricted to lymphoid tissues, in which its expression is driven by constitutively present NF-B(p50/c-Rel) heterodimers (78). Activation of the p50/p65 heterodimer, however, can confer inducible transgene activation in all cell types (78). These mice received a single administration of 1,000 µg of AGE-albumin (500 µg i.v., 500 µg i.p.) or 1,000 µg of control albumin (500 µg i.v., 500 µg i.p.). Tissue was harvested 6 days later. The AGE concentration used was in the range of AGE serum levels determined in diabetic patients with end-stage renal disease (94,95,96,97,98). As a result of degradation and urinary AGE clearance (25,96,97,98), AGE concentrations present in these mice after 6 days probably gives rise to mouse serum levels even below AGE serum levels found in diabetic patients with good glycemic control (40,94,95,96). Total RNA was isolated from kidneys and subjected to RT-PCR for the ß-globin reporter gene (Fig. 5A). RNA isolated from the spleen served as control and demonstrated constitutive ß-globin transcription in all animals (data not shown). Despite the presence of endogenous AGE-degrading systems (25,97,98), an AGE-dependent induction of the NF-B–driven ß-globin transcription was still observed after 6 days (Fig. 5A, lane 2) and paralleled by an increase in NF-Bp65 mRNA (data not shown). No signal could be seen in mice that were infused with control albumin (Fig. 5A, lane 1) or in untreated control animals (data not shown). The strong ß-globin transgene signal in mice that were treated with AGE-albumin (Fig. 5A, lane 2) was reduced when mice were treated with anti-RAGE IgGs (40 µg/mouse i.v; Fig. 5A, lane 3) or an excess of sRAGE (25 µg/mouse i.v.; Fig. 5A, lane 4) at days 0 and 3 after stimulation. RT-PCR for actin served as an internal control and confirmed comparable RNA input in each reaction (Fig. 5A, bottom). In-parallel–performed EMSA with whole-cell extracts from these kidneys demonstrated prominent NF-B binding activity only in mice that had received AGE-albumin (Fig. 5B, lane 2). NF-B binding activity was significantly reduced in the kidney nuclear extracts of mice that had received anti-RAGE IgGs (40 µg/mouse i.v.; Fig. 5B, lane 3) or sRAGE (25 µg/mouse i.v.) (Fig. 5B, lane 4). Inhibition of ß-globin expression (Fig. 5A) and NF-B binding activity (Fig. 5B) by RAGE antibodies, however, was lower than the inhibition observed in the presence of sRAGE. Because sRAGE competes for AGE binding with all cellular AGE receptors, whereas blocking RAGE antibodies specifically inhibit ligation to RAGE, this implicates that other AGE receptors (25,43,99) or not-yet-identified RAGE-like receptors might also contribute to AGE-mediated sustained NF-B, though to a much lesser extent than RAGE. RAGE antibodies inhibit >80% of the AGE-dependent NF-B activation, thereby indicating that RAGE is the predominant receptor in mediating sustained NF-B activation.

View larger version (39K): [in this window] [in a new window]   FIG. 5. AGE-mediated induction of NF-B DNA binding activity in vivo is dependent on RAGE. A: Mice transgenic for an NF-B–controlled ß-globin reporter gene (78) were infused once with 1,000 µg of AGE-albumin (500 µg i.v.; 500 µg i.p.) or control albumin (500 µg i.v.; 500 µg i.p.). At time points 0 and 3 days, mice were treated with RAGE IgGs (40 µg/mouse i.v.) or sRAGE (25 µg/mouse i.v.). After 6 days, mice were killed and total RNA from the kidney was prepared and 2 µg were analyzed by RT-PCR for ß-globin transgene and ß-actin expression (ß-globin transgene [top, 440 bp] and ß-actin [bottom, 720 bp]). B: Nuclear extracts were prepared from the other kidney of the above ß-globin transgenic mice. Ten micrograms of each nuclear extract were analyzed for NF-B (p50/p65) binding activity in EMSA using a 32P-radiolabeled oligonucleotide with the high-affinity NF-B(p50/p65) consensus sequence. The inducible NF-B complex is indicated by an arrow. For confirming NF-B–binding, kidney nuclear extracts from AGE-treated transgenic mice was competed with a 160-fold molar excess of unlabeled NF-B consensus oligonucleotides (lane 5).

AGE- and Aß-induced sustained NF-B activation mediating sustained gene expression.

View larger version (37K): [in this window] [in a new window]   FIG. 6. Sustained activation of NF-B by AGE-albumin and Aß induces NF-B–dependent gene expression. A: BAECs were transiently transfected with the plasmid NF-B-Luc, which contains four tandem copies of the NF-B consensus sequence fused to a TATA-like promoter region from the herpes simplex virus thymidine kinase promoter using the Effectene transfection reagent. One hour after application of DNA, cells were stimulated with control albumin (500 nmol/l), AGE-albumin (500 nmol/l), scrambled Aß (1 µmol/l), or Aß (1 µmol/l) for either 6 or 48 h. After harvest, luciferase activity was determined in the cell lysates and normalized for transfection efficiency by the amount of ß-gal activity expressed by the co-transfected control plasmid pSV-ß-Gal (83). Corrected values were expressed as relative luciferase (LUC) units and are given as percentage of luciferase expression determined in BAECs, stimulated with control albumin for 6 h. Three independent experiments were performed six times with identical results. The results are given ± SD. Statistical analysis was performed using a two-tailed Student’s t test. B: Immunoblot analysis of HO-1 in cytoplasmic extracts from BAECs that were untreated (0 h) or treated with AGE-albumin (500 nmol/l) for the times indicated demonstrated time-dependent induction of HO-1. HO-1 (molecular weight 39 kDa) is indicated by an arrow. The result shown is representative of three independent experiments. The HO-1 signal intensity was quantified by densitometry and is given below in the autoradiogram. Similar data were obtained after stimulation with 1 µmol/l Aß (data not shown).

AGE and Aß induce increased nuclear translocation of NF-Bp65.

View larger version (25K): [in this window] [in a new window]   FIG. 7. Relative contribution of NF-Bp50 and NF-Bp65 to the binding complexes formed during AGE-albumin–mediated sustained NF-B binding activity monitored by supershifting experiments. Characterization of the NF-B subunits, contributing to the observed shift formed at the NF-B consensus sequence, was performed by adding 2.5 µg of an irrelevant IgG or of anti–NF-Bp50 and anti–NF-Bp65 antibodies into the binding reactions with nuclear extracts of unstimulated BAECs or of BAECs stimulated with AGE-albumin for 24 h to 6 days. Signals obtained were quantified by densitometry. A: Signal intensity determined for the NF-B binding complex in the presence of a nonspecific IgG was taken as 100% at each time point investigated and compared with signal intensity for NF-B binding determined in the presence of anti-p50 or anti-p65 antibodies. Because of supershifted complexes and blockade of NF-B binding, the NF-B binding complex was significantly reduced in the presence of anti-p65 and anti-p50 antibodies. The extent of reduction served as a measure to calculate the relative contribution of NF-Bp65 and NF-Bp50 to each NF-B binding complex. Two different experiments were evaluated with identical results, and the data of one representative experiment are shown. Similar data were obtained after stimulation with 1 µmol/l Aß (data not shown). B: Ratio formed by the amount of NF-Bp65 divided by the amount of NF-Bp50 contributing to the NF-B binding activity observed over time.

View larger version (42K): [in this window] [in a new window]   FIG. 8. AGE-albumin induces sustained translocation of NF-Bp65 at time points at which IB proteins already reappeared in the cytoplasm. A and B: Immunoblot analysis of nuclear (A) and cytoplasmic (B) extracts of BAECs, stimulated with AGE-albumin for the times indicated, demonstrated AGE-albumin–dependent prolonged translocation of NF-Bp65 antigen into the nucleus (A). The NF-Bp65–specific complex is indicated by an arrow. The NF-B signal intensity was quantified by densitometry and is given on the right side of each autoradiogram. The result shown is representative of six independent experiments. Similar data were obtained after stimulation with 1 µmol/l Aß (data not shown). C: Immunoblot analysis of IB in cytoplasmic extracts from BAECs that were untreated (0 h) or treated with AGE-albumin (500 nmol/l) for the times indicated demonstrated a decrease in cytoplasmic IB antigen between 12 and 48 h. The IB-specific complex is indicated by an arrow. The IB signal intensity was quantified by densitometry and is given on the right side of the autoradiogram. The result shown is representative of four independent experiments. Similar data were obtained after stimulation with 1 µmol/l Aß (data not shown). D: Immunoblot analysis of IBß in cytoplasmic extracts from BAECs that were untreated (0 h) or treated with AGE-albumin (500 nmol/l) for the times indicated demonstrated a decrease of cytoplasmic IBß antigen between 12 and 48 h. The IBß-specific complex is indicated by an arrow. The IBß signal intensity was quantified by densitometry and is given on the right side of the autoradiogram. The result shown is representative of four independent experiments. Similar data were obtained after stimulation with 1 µmol/l Aß (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 9. AGE-albumin induces an IB phosphorylating kinase activity. BAECs were left untreated (0 h) or were st