J Korean Med Sci.  2021 Sep;36(37):e239. 10.3346/jkms.2021.36.e239.

Effects of Advanced Glycation End Products on Differentiation and Function of Osteoblasts and Osteoclasts

• Park SY
• Choi KH
• Jun JE
• Chung HY

• KMID: 2520365
• DOI: http://doi.org/10.3346/jkms.2021.36.e239

Abstract

Background
Risk of fragility fractures increases in patients with diabetes mellitus, independent of bone mineral density. In the present study, the effects of advanced glycation end products (AGEs) on differentiation and function of osteoblasts and osteoclasts were investigated.
Methods
AGEs and 25 mM glucose were administered to marrow-derived macrophages and MCT3T3-E1 cells. The effects of AGEs on osteoclast differentiation was investigated using tartrate-resistant acid phosphatase (TRAP) assay. The effects of AGEs on osteoblast differentiation was investigated using alkaline phosphatase (ALP) activity and bone nodule formation assays. Expression of osteoclast-specific and osteoblast-specific genes and effects on cell signaling pathways associated with cell differentiation were analyzed using reverse transcription polymerase chain reaction and western blotting.
Results
AGEs significantly decreased TRAP-positive multinucleated cell formation in receptor activator of nuclear factor-κB ligand-induced marrow-derived macrophages in a dose-dependent manner. AGEs suppressed the expression of osteoclast-specific genes, JNK, p38, AKT, intercellular adhesion molecule 1, and lymphocyte function-associated antigen 1 in marrow-derived macrophages. AGEs decreased ALP activity and showed a tendency to decrease bone nodule formation in MC3T3-E1 cells. AGEs suppressed the expression of osteoblast-specific genes, lysyl hydroxylase and lysyl oxidase in MC3T3-E1 cells.
Conclusion
AGEs suppressed differentiation and function of osteoclasts and osteoblasts, and collagen cross-linking activity. It suggests that AGE may induce bone fragility through low bone turnover and deterioration of bone quality.

Keyword

Advanced Glycation End Products; Osteoclasts; Osteoblasts; Diabetes mellitus; Osteoporosis

Figure

• 

  Fig. 1 RAGE expression in MC3T3-E1 cells, BMM cells, and marrow-derived MNCs using reverse transcription polymerase chain reaction. RAGE was expressed on all cells. The grouping of gels/blots cropped from different parts of the same gel.RAGE = receptor for advanced glycated end products, BMM = bone marrow mononuclear, MNC = multinucleated cell.

• 

  Fig. 2 Effects of AGEs on osteoclast and osteoblast differentiation. (A) TRAP-positive MNCs in RANKL-induced marrow macrophage cell cultures. Bone marrow mononuclear cells were incubated for three days with 30 ng/mL M-CSF, 70 ng/mL RANKL, 25 mM glucose, 25 mM mannitol, and 1, 10, or 100 ug/mL of AGEs (*P < 0.05). (B) ALP activities in MC3T3-E1 cell cultures. MC3TC-E1 cells were incubated for eight days with 50 ug/mL ascorbic acid, 10 mM β-GP, 25 mM glucose, 25 mM mannitol, and 1, 10, or 100 ug/mL of AGEs (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Bone nodule formations in MC3T3-E1 cell cultures. MC3TC-E1 cells were incubated in 24-well plates (5,000 cells/well) for 14 days with 50 ug/mL ascorbic acid, 10 mM β-GP, 25 mM glucose, 25 mM mannitol, and 1, 10, or 100 ug/mL of AGEs (*P < 0.05, **P < 0.01).TRAP = tartrate-resistant acid phosphatase, MNC = multinucleated cell, M-CSF = macrophage colony-stimulating factor, RANKL = receptor activator of nuclear factor-κB ligand, AGE = advanced glycation end product, ALP = alkaline phosphatase.

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  Fig. 3 Effects of AGEs on osteoclast-specific genes (A) and activation of the mitogen activated protein kinase signaling pathway (B) in RANKL-induced marrow macrophages. The osteoclast-specific genes such as RANK, TRAP, MMP-9, cathepsin K, and NFATc1 were measured by reverse transcription polymerase chain reaction. JNK, p38, ERK, AKT and β-actin were measured by western blots. The grouping of gels/blots cropped from different parts of the same gel.NC = negative control, PC = positive control, G = 25 mM glucose, M = 25 mM mannitol, AGE = advanced glycated end product, RANKL = receptor activator of nuclear factor-κB ligand, TRAP = tartrate-resistant acid phosphatase, MMP-9 = matrix metalloproteinases-9, NFATc1 = nuclear factor of activated T-cells cytoplasmic 1.

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  Fig. 4 Effects of AGEs on ICAM-1 and LFA-1 expression in marrow macrophages. Expressions of ICAM-1, LFA-1α, and LFA-1β genes were measured by reverse transcription polymerase chain reaction. The grouping of gels/blots cropped from different parts of the same gel.NC=negative control, PC=positive control, G=25mM glucose, M=25mM mannitol, AGE = advanced glycated end product, ICAM-1 = intercellular adhesion molecule 1, LFA-1 = lymphocyte function-associated antigen 1.

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  Fig. 5 Effects of AGEs on osteoblast-specific genes (A) and expression of LH and LOX genes (B) in MC3T3-E1 cells. The grouping of gels/blots cropped from different parts of the same gel. The osteoblast-specific genes, LH and LOX genes were measured by reverse transcription polymerase chain reaction.NC = negative control, PC = positive control, G = 25 mM glucose, M = 25 mM mannitol, AGE = advanced glycated end product, LH = lysyl hydroxylase, LOX = lysyl oxidase.

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