Diabetes-associated sustained activation of the transcription factor nuclear factor-κB (original) (raw)

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-␤ peptide (A␤) 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 ␤-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 IB␤. 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. Diabetes . M.S. and G. P. serve as consultants/collaborators for Panacea Pharmaceuticals, Prion Development Laboratories, and Voyager Laboratories. A.-M.S. and D.M.S. are consultants for and have received a research grant from TransTech Pharma. P.P.N. has received a research grant from ASTA-Medica. -buffered saline; TNF-␣, tumor necrosis factor-␣. 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 A␤ 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; Ͻ10 pg/ml at a protein concentration of 5 mg/ml according to the Limulus assay [Sigma]). Synthetic A␤ 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 ␤-galactosidase (␤-Gal) control plasmid pSV-␤-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 ␤-globin reporter gene (tg14) were provided by Dr. Thomas Wirth (Wü rzburg, Germany) and have been previously characterized in detail (78). Eight-week-old female mice were left untreated or treated once with 1,000 g of mouse AGE-albumin (500 g i.p. and 500 g i.v.) at time point 0 and kept for an additional 6 days. Where indicated, mice received sRAGE (25 g/mouse) or anti-RAGE antibodies (40 g/mouse) as described . At the end of the experiments, mice were killed and organs were removed, immediately snapfrozen, 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. Kö ting (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 (Ϯ16 days), and were kept for 57 days (Ϯ9 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 Ϯ 24 mg/dl. Patient characteristics. Blood from six patients with newly manifested diabetes type 1 (two men, four women; age 35.1 Ϯ 11.7 years; HbA 1c 12.4 Ϯ 2.3%) was collected on the day the diagnosis was made. Four healthy volunteers (three men, one woman; age 31.5 Ϯ 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 Tü bingen, 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 -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