Advanced Glycation End Product Precursors Impair Epidermal Growth Factor Receptor Signaling

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.

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.

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.

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.

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.