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Advanced Glycation End Product 3 (AGE3) Increases Apoptosis and the Expression of Sclerostin by Stimulating TGF-β Expression and Secretion in Osteocyte-Like MLO-Y4-A2 Cells

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Abstract

Advanced glycation end products (AGEs) cause bone fragility due to deterioration in bone quality. We previously reported that AGE3 induced apoptosis and inhibited differentiation via increased transforming growth factor (TGF)-β signaling in osteoblastic cells. Additionally, we demonstrated that AGE3 increased apoptosis and sclerostin expression and decreased receptor activator of nuclear factor-κB ligand (RANKL) expression in osteocyte-like cells. However, it remains unclear whether TGF-β signaling is involved in the effects of AGEs on apoptosis and the expression of sclerostin and RANKL in osteocytes. Effects of AGE3 on apoptosis of mouse osteocyte-like MLO-Y4-A2 cells were examined by DNA fragmentation ELISA. Expression of TGF-β, sclerostin, and RANKL was evaluated using real-time PCR, Western blotting, and ELISA kits. To block TGF-β signaling, we used SD208, a TGF-β type I receptor kinase inhibitor. AGE3 (200 µg/mL) significantly increased apoptosis and mRNA expression of Sost, the gene encoding sclerostin, and decreased Rankl mRNA expression in MLO-Y4-A2 cells. AGE3 significantly increased the expression of TGF-β. Co-incubation of SD208 with AGE3 significantly rescued AGE3-induced apoptosis in a dose-dependent manner. Moreover, SD208 restored AGE3-increased mRNA and protein expression of sclerostin. In contrast, SD208 did not affect AGE3-decreased mRNA and protein expression of RANKL. These findings suggest that AGE3 increases apoptosis and sclerostin expression through increasing TGF-β expression in osteocytes, and that AGE3 decreases RANKL expression independent of TGF-β signaling.

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Abbreviations

T2DM:

Type 2 Diabetes mellitus

BMD:

Bone mineral density

AGEs:

Advanced glycation end products

TGF-β:

Transforming growth factor-β

RANKL:

Receptor activator of Nuclear factor-κB ligand

BSA:

Bovine serum albumin

α-MEM:

α-minimum essential medium

OPG:

Osteoprotegerin

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Acknowledgements

MN, IK, and TY were responsible for designing and conducting the study. MN performed the experiments and analyzed the data. AT, MY, KT, and TS contributed equipment/materials. MN and IK wrote the paper. All authors approved the final version. IK takes responsibility for the integrity of the data analysis. The authors thank Keiko Nagira for technical assistance.

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Authors and Affiliations

  1. Internal Medicine 1, Shimane University Faculty of Medicine, 89-1, Enya-cho, Izumo, Shimane, 693-8501, Japan

    Masakazu Notsu, Ippei Kanazawa, Ayumu Takeno, Maki Yokomoto-Umakoshi, Ken-ichiro Tanaka, Toru Yamaguchi & Toshitsugu Sugimoto

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  1. Masakazu Notsu
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  2. Ippei Kanazawa
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  3. Ayumu Takeno
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Correspondence to Ippei Kanazawa.

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Masakazu Notsu, Ippei Kanazawa, Ayumu Takeno, Maki Yokomoto-Umakoshi, Ken-ichiro Tanaka, Toru Yamaguchi, and Toshitsugu Sugimoto have no potential conflict of interest to disclose.

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Notsu, M., Kanazawa, I., Takeno, A. et al. Advanced Glycation End Product 3 (AGE3) Increases Apoptosis and the Expression of Sclerostin by Stimulating TGF-β Expression and Secretion in Osteocyte-Like MLO-Y4-A2 Cells. Calcif Tissue Int 100, 402–411 (2017). https://doi.org/10.1007/s00223-017-0243-x

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