Synergistic stimulating effect of 2-hydroxymelatonin and BMP-4 on osteogenic differentiation in vitro
Introduction
Osteoporosis is a skeletal disease characterized by low bone mass and microstructural degradation of bone tissues that enhances bone fragility and increase fracture risk [1]. Globally, it has been estimated that more than 200 million people suffer from osteoporosis [2]. Melatonin is widely studied in order to help prevent this disease. Indeed, melatonin has a significant role in enhancing the growth of bone. A number of studies have demonstrated the effects of melatonin on bone remodeling, osteoporosis and dentine formation [[3], [4], [5]].
The current body of knowledge includes some studies on the mechanism of melatonin-mediated osteogenic differentiation. For example, melatonin regulates Osterix protein expression in bone morphogenetic protein (BMP)-4-induced C2C12 cells [6]. In addition, melatonin activates the AMP-activated protein kinase (AMPK)/β-catenin signaling pathway in BMP-9-induced mesenchymal stem cells [7]. Although some literature has proven that melatonin can promote osteogenic differentiation, there have been no reports on melatonin metabolites that can improve bone metabolic activity in osteoporosis.
The profiles of melatonin metabolites have been mainly studied in plants [8]. Among these, 2-hydroxymelatonin is a metabolite that is produced when melatonin 2-hydroxylase catalyzes melatonin [9]. In plants, 2-hydroxymelatonin modulates non-enzymatic antioxidant and nutritional conditions and enhances the resistance to diverse external abiotic stresses under biological toxicity [10,11]. Interestingly, 2-hydroxymelatonin also occurs in animals, however biological roles of 2-hydroxymelatonin have not been elucidated yet. The present study, therefore, examined 2-hydroxymelatonin to evaluate its potential use in bone regeneration with respect to osteoblast differentiation.
Here, we report that 2-hydroxymelatonin, in combination with BMP-4, enhanced alkaline phosphatase (ALP) activity. This study demonstrated that 2-hydroxymelatonin induces osteoblast differentiation-related genes such as ALP, Runx2, and Osterix in the presence of BMP-4. Enhancement of osteoblast differentiation via the synergistic effect of 2-hydroxymelatonin and BMP-4 may contribute to the prevention of osteoporosis.
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Cell culture and induction of osteoblast differentiation
C2C12, a preliminary source cell derived from mice, was cultivated using Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA, USA) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics (Thermo Fisher Scientific, Waltham, MA, USA) in a 37 °C, 5% CO2 incubator. In order to induce the differentiation of the osteoblasts, the same growth medium was used until the cells filled the plate, and BMP-4 (500 ng/mL) was also added.
Reagents
2-Hydroxymelatonin was purchased from Santa
2-Hydroxymelatonin has no effects on cell proliferation and viability
Prior to the analysis of the osteogenic effects of 2-hydroxymelatonin, viability and cytotoxicity on C2C12 cells were examined using the MTT assay. After treatment with 2-hydroxymelatonin at various concentrations for 24 or 48 h, MTT assay was performed. In the result, 2-hydroxymelatonin has no significant toxic effects even at high concentrations (>100 μM) compared to the control group (Fig. 1A).
Synergy effect of 2-hydroxymelatonin and BMP-4 on osteoblast differentiation of C2C12 cells.
To
Discussion
In human, melatonin is metabolized in the liver by cytochrome P450 enzyme, CYP1A2 to various metabolites including 6-hydroxymelatonin, 2-hydroxymelatonin, 4-hydroxymelatonin, and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) [15]. Although 6-hydroxymelatonin is a chief metabolite in human unlike 2-hydroymelatonin is predominant in plants, most of 6-hydroxymelatonin is subsequently conjugated with sulfuric acid or glucuronic acid excreted in the urine as an inactive form [16]. On the other
Acknowledgments
This paper was supported by Wonkwang University in 2020.
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These authors contributed equally to this work.