Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway

Abstract

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells.

Keywords: advanced glycation end products (AGEs); autophagy; cell proliferation; extracellular-signal-regulated kinase (ERK); osteoblast; receptor for advanced glycation end products (RAGE).

FIGURE 1.

Effect of AGE-BSA on cell viability and proliferation in hFOB 1.19 cells, and autophagy is involved in the effect of AGE-BSA. A, cells were treated with AGE-BSA at various concentrations for 0, 24, 48, or 72 h, and cell viability was estimated using MTT assay. Values represent mean ± S.E. of at least three independent experiments. *, p < 0.05 versus control; **, p < 0.01 versus control. B, cells were treated with BSA at various concentrations for 0, 24, 48, or 72 h, and cell viability was estimated using MTT assay. Values represent mean ± S.E. of at least three independent experiments. C and D, cells were treated with or without 3-MA (5 mm) incubated with AGE-BSA (150 μg/ml) for 0, 24, 48, or 72 h. C, cell viability was estimated using MTT assay; D, cell proliferation was estimated using a BrdU kit. Values represent mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; #, p < 0.05 versus AGE-BSA treatment; ##, p < 0.01 versus AGE-BSA treatment. E, cells were treated with or without 3-MA (5 mm) incubated with AGE-BSA (150 μg/ml) for 24, 48, or 72 h; cell death was estimated using a cell death detection ELISA^Plus. Values represent mean ± S.E. of at least three independent experiments.

FIGURE 2.

Autophagy as a protective response against high concentrations of AGE-BSA-induced apoptosis in hFOB 1.19 cells. A–C, cells preincubated with or without Z-DEVD-fmk (25 μm) for 1 h were treated with or without 3-MA (5 mm) incubated with AGE-BSA (200 μg/ml) for 24 h. A, cell viability was estimated using MTT assay. B, cell death was estimated using a cell death detection ELISA^Plus. Values represent mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment. C, cell apoptosis detection using annexin V-FITC/PI kit. Viable cells (annexin V⁻/PI⁻), early apoptotic cells (annexin V⁺/PI⁻), late apoptotic cells (annexin V⁻/PI⁺), and necrotic cells (annexin V⁺/PI⁺) are located in the bottom left, bottom right, top right, and top left quadrants, respectively. The numbers in each quadrant represent the percentage of cells.

FIGURE 3.

Effect of AGE-BSA on the expression of autophagy related protein beclin-1, LC3, and p62 in hFOB 1.19 cells. A, Western blotting showing beclin-1, LC3, and p62 protein levels in hFOB 1.19 cells treated with AGE-BSA (150 μg/ml) for 0, 0.5, 1, 3, 6, 12, 24, or 48 h. B, Western blotting showing beclin-1, LC3, and p62 protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. C, Western blotting showing beclin-1, LC3, and p62 protein levels in hFOB 1.19 cells treated with or without 3-MA (5 mm) incubated with AGE-BSA (150 μg/ml) or BSA (150 μg/ml) for 24 h. D–F, quantitative analysis of LC3, beclin-1, and p62 protein expression. Each bar represents mean ± S.E. of at least thee independent experiments. *, p < 0.05 versus control; **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment.

FIGURE 4.

AGE-BSA (150 μg/ml)-induced autophagy in hFOB 1.19 cells, and the autophagy was blocked by 3-MA. A, transmission electron microscopy showing autophagosomes (bold arrows) in hFOB 1.19 cells treated with or without 3-MA (5 mm) incubated with AGE-BSA (150 μg/ml) for 24 h. C, quantitation of autophagosomes. Each bar represents the mean ± S.E. of three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment. B, fluorescence microscopy showing endogenous LC3 (green fluorescence), exogenous EGFP-LC3 (green fluorescence), and nucleus (blue fluorescence) in hFOB 1.19 cells treated with or without 3-MA (5 mm) incubated with AGE-BSA (150 μg/ml) for 24 h. D, quantitation of LC3 puncta. Each bar represents the mean ± S.E. of three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment. E, quantitation of EGFP-LC3 puncta. Each bar represents the mean ± S.E. of three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment.

FIGURE 5.

RAGE/Raf/MEK/ERK pathway is involved in AGE-BSA induced autophagy. A, Western blotting showing RAGE protein levels in hFOB 1.19 cells treated with AGE-BSA at various concentrations for 24 h. B, quantitative analysis of RAGE protein expression. Each bar represents mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control. C, Western blotting showing RAGE protein levels in hFOB 1.19 cells infected lentiviral with control shRNA or RAGE shRNA for 24 h. D, quantitative analysis of RAGE protein expression. Each bar represents mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus control shRNA treatment. E, Western blotting showing Raf/MEK/ERK pathway relative protein (c-Raf, MEK1/2, or ERK1/2) levels and autophagy relative protein (Beclin-1, LC3, or p62) levels in hFOB 1.19 cells treated with or without PD98059 (50 μm), control shRNA, or RAGE shRNA incubated with or without AGE-BSA (150 μg/ml) for 24 h. F and G, quantitative analysis of c-Raf, MEK1/2, ERK1/2, LC3, beclin-1, or p62 protein expression. Each bar represents mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment.

FIGURE 6.

RAGE shRNA and PD98059 attenuated the effect of AGE-BSA on cell proliferation in hFOB 1.19 cells. A and B, cells were treated with or without PD98059 (50 μm) or RAGE shRNA incubated with AGE-BSA (150 μg/ml) for 24 h. A, cell viability was estimated using MTT assay; B, cell proliferation was estimated using a BrdU kit. Values represent mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment.

FIGURE 7.

3-MA, RAGE shRNA and PD98059 attenuated the effect of AGE-BSA on the expression of a complex set of bone formation parameters (ALP and OCN) and bone resorption parameters (OPG and RANKL) in hFOB 1.19 cells. A–D, real time RT-PCR showing RANKL (A), OPG (B), OCN (C), or ALP (D) mRNA levels in hFOB 1.19 cells treated with or without 3-MA(5 mm), PD98059 (50 μm), or RAGE shRNA incubated with AGE-BSA (150 μg/ml) for 24, 48, 72 h. Values represent mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment. E, Western blotting showing RANKL, OPG, or OCN protein levels in hFOB 1.19 cells treated with or without 3-MA (5 mm), PD98059 (50 μm), or RAGE shRNA incubated with AGE-BSA (150 μg/ml) for 24, 48, and 72 h. F–H, quantitative analysis of RANKL, OPG, or OCN protein expression. Each bar represents mean ± S.E. of at least three independent experiments. **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment. I, ALP activity kit showing ALP activity levels in hFOB 1.19 cells treated with or without 3-MA (5 mm), PD98059 (50 μm), or RAGE shRNA incubated with AGE-BSA (150 μg/ml) for 24, 48, and 72 h. Values represent mean ± S.E. of at least three independent experiments. *, p < 0.05 versus control; **, p < 0.01 versus control; ##, p < 0.01 versus AGE-BSA treatment.