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Complications

Advanced Glycation End Product Precursors Impair Epidermal Growth Factor Receptor Signaling

  1. Manuel Portero-Otín1,
  2. Reinald Pamplona1,
  3. Maria Josep Bellmunt1,
  4. Maria Cristina Ruiz1,
  5. Joan Prat1,
  6. Robert Salvayre2 and
  7. Anne Nègre-Salvayre2
  1. 1Metabolic Pathophysiology Research Group, Department of Basic Medical Sciences, University of Lleida, Lleida, Spain
  2. 2Institut National de Santé et de Recherche Médicale (INSERM) U466 and Biochemistry Department, Centre Hospitalier Universitaire Rangueil, University Paul Sabatier, Toulouse, France
    Diabetes 2002 May; 51(5): 1535-1542. https://doi.org/10.2337/diabetes.51.5.1535
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    • FIG. 1.
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      FIG. 1.

      EGFR tyrosine phosphorylation is inhibited by GO and MGO. Cells were starved for 16 h in RPMI-1640 containing 0.5% FCS and then incubated with GO or MGO at the indicated times and concentrations. Cell lysates were analyzed by Western blotting (SDS-PAGE on 7.5% polyacrylamide gels) and probed with anti-phosphotyrosine (anti-PY) or anti-EGFR. Data shown are representative of four experiments. A and B: Time course of inhibition of EGF-induced EGFR autophosphorylation on ECV304 cells, either control or incubated with 5 mmol/l GO (A) or MGO (B) for the indicated time and stimulated by EGF (10 nmol/l for 15 min). C and D: Dose response of EGFR tyrosine phosphorylation in ECV304 cells (C) and B82K+ fibroblasts (D) incubated (or not) for 6 h with GO at the indicated concentrations and stimulated by EGF (10 nmol/l for 15 min). The arrow indicates the presence of anti-EGFR reactive high-molecular-weight aggregates.

    • FIG. 2.
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      FIG. 2.

      GO and MGO alter EGFR downstream signaling. A: Inhibition of EGF-induced recruitment of PLCγ1 on EGFR. ECV304 cells were incubated with GO (5 mmol/l for 6 h) and then stimulated by EGF (10 nmol/l for 15 min). The cells were lysed in solubilization buffer (which allows coimmunoprecipitation of SH2-proteins bound to EGFR) and (1.5 mg cell protein) immunoprecipitated with anti-EGFR antibody overnight at 4°C. Anti-EGFR immunoprecipitates were recovered on protein G-Sepharose (1 h at 4°C), washed, eluted, and analyzed by SDS-PAGE. The spots were revealed by immunoblotting with anti-phosphotyrosine, anti-EGFR, and anti-PLCγ1. Data shown is representative of three experiments. B and C: Time course of inhibition of ERK activation subsequent to EGF stimulation. ECV304 cells, preincubated at variable periods of time with 5 mmol/l GO (B) or MGO (C), were stimulated with EGF (10 nmol/l for 15 min), and ERK activation was investigated by Western blot using antibodies against activated ERK (anti-phosphorylated ERK1/ERK2 [anti-pERK]) and total ERK (anti-ERK1/ERK2 [anti-ERK]). These data are representative of three separate experiments.

    • FIG. 3.
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      FIG. 3.

      Altered PTPase activity and EGFR dephosphorylation in cells incubated with GO and MGO. A: PTPase activity was evaluated in ECV304 cells preincubated (or not) with GO (0.5 and 5 mmol/l) for 5 h. PTPase activities were measured as described in research design and methods by dephosphorylation of [33P]poly-Glu-Tyr. Data are expressed as percent of PTPase activation versus the basal control. Results are the means ± SD from four separate experiments. **P < 0.001 vs. unstimulated cells (control) (ANOVA). B: Inhibition of EGFR dephosphorylation by MGO. EGF (10 nmol/l) was added to ECV304 cells on ice. After binding on ice for 15 min, the cells were washed, chased at 37°C in EGF-free medium for the time periods indicated, and lysed in solubilizing buffer. After equilibration of protein concentration, cell lysates were analyzed by Western blot with an anti-phosphotyrosine antibody and developed by ECL as described in the text. These data are representative of three separate experiments.

    • FIG. 4.
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      FIG. 4.

      In vitro inhibition of EGFR activation induced by GO and MGO. A: EGFR autophosphorylation was determined on EGFR immunopurified from unstimulated ECV304 (previously incubated in situ without or with 0.5 and 5 mmol/l GO and MGO for 5 h) and incubated in vitro without (control) or with 1 nmol/l EGF for 15 min. The assay was performed in phosphorylation buffer containing 5 μCi/assay of [γ-33P]ATP, as described in research design and methods. After spotting on phosphocellulose membranes, the radioactivity was counted. Data are expressed as percent of the unstimulated control. B: EGFR tyrosine kinase activity was evaluated using the same protocol but in the presence of the poly-Glu-Tyr substrate. Results are the means ± SD from four separate experiments. *P < 0.02, **P < 0.001 vs. unstimulated cells (control) (ANOVA). C: Western blot experiments of EGFR in membranes prepared from A-431 cells and preincubated in phosphorylation buffer with or without 5 mmol/l GO (30 min), then stimulated with 1 nmol/l EGF (15 min), and then blotted with anti-phosphotyrosine and anti-EGFR antibodies. The arrow indicates the presence of anti-EGFR reactive high-molecular-weight aggregates. Data are representative of three separate experiments.

    • FIG. 5.
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      FIG. 5.

      Inhibitory effect of GO and MGO is related to AGE formation in EGFR. A: Derivatization of EGFR free amino groups by GO and MGO. ECV304 cells preincubated with GO or MGO (5 mmol/l for 5 h) were treated without (control) or with EGF (1 nmol/l for 15 min), and EGFR was immunoprecipitated as described above. Free amino groups of EGFR were labeled with [3H]SP, as indicated in research design and methods. EGFR was resolved by SDS-PAGE, and the radioactivity of the 170-kDa bands was counted. Results are the means ± SD from three separate experiments. *P < 0.02 vs. unstimulated cells (control) (ANOVA). B: Detection of CML adducts by anti-CML antibodies in proteins from ECV304 cells preincubated with GO for 5 h at the indicated concentrations. Data are representative of four separate experiments.

    • FIG. 6.
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      FIG. 6.

      Aminoguanidine prevents GO- and MGO-induced inhibition of EGF signaling in ECV304 cells. Cells were preincubated with or without aminoguanidine (5 mmol/l) for 8 h, exposed to GO or MGO (5 mmol/l for 5 h, as indicated), and stimulated with EGF. Analysis of EGFR phosphorylation and ERK phosphorylation was performed as described in research design and methods. Data are representative of four separate experiments.

    • FIG. 7.
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      FIG. 7.

      MGO inhibits PDGFR autophosphorylation. Serum-starved rabbit smooth muscle cells (American Type Culture Collection) were preincubated with 3 mmol/l MGO for 5 h (as indicated) and stimulated with 1 nmol/l PDGF for 20 min. Thereafter, cells were lysed in solubilizing buffer, and equilibrated proteins were separated by SDS-PAGE (7.5%), as described in the legend to Fig. 1. Blots were probed with anti-phosphotyrosine, anti-PDGFR, and anti-phosphorylated ERK (anti-pERK) antibodies and developed by ECL as described. Data are representative of three separate experiments.

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    Advanced Glycation End Product Precursors Impair Epidermal Growth Factor Receptor Signaling
    Manuel Portero-Otín, Reinald Pamplona, Maria Josep Bellmunt, Maria Cristina Ruiz, Joan Prat, Robert Salvayre, Anne Nègre-Salvayre
    Diabetes May 2002, 51 (5) 1535-1542; DOI: 10.2337/diabetes.51.5.1535

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    Advanced Glycation End Product Precursors Impair Epidermal Growth Factor Receptor Signaling
    Manuel Portero-Otín, Reinald Pamplona, Maria Josep Bellmunt, Maria Cristina Ruiz, Joan Prat, Robert Salvayre, Anne Nègre-Salvayre
    Diabetes May 2002, 51 (5) 1535-1542; DOI: 10.2337/diabetes.51.5.1535
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