Synergistic stimulating effect of 2-hydroxymelatonin and BMP-4 on osteogenic differentiation in vitro

https://doi.org/10.1016/j.bbrc.2020.04.126Get rights and content

Highlights

  • 2-Hydroxymelatonin enhances osteoblast differentiation of C2C12 cells upon the BMP-4 stimulation.

  • 2-Hydroxymelatonin simulates bone development-related gene expression.

  • 2-Hydroxymelatonin upregulates ALP transcriptional activity via p38/ERK signaling pathway.

Abstract

2-Hydroxymelatonin is a metabolite produced when melatonin 2-hydroxylase catalyzes melatonin. Recent studies have reported the important roles of melatonin in bone metabolism. However, the roles of 2-hydroxymelatonin in bone metabolism remains poorly understood. The purpose of this study is to present evidence of the effect of 2-hydroxymelatonin on osteogenic differentiation in C2C12 cells. In this study, we demonstrated the synergistic stimulating effect of 2-hydroxymelatonin and bone morphogenetic protein (BMP)-4 on osteogenic differentiation in vitro, using alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, qPCR, and luciferase reporter assay. The combination of 2-hydroxymelatonin and BMP-4 revealed a synergistic effect on osteogenic differentiation in vitro. This finding provides evidence that optimal concentrations of both 2-hydroxymelatonin and BMP-4 are beneficial for anabolic effects on bone 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.

Section snippets

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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    1

    These authors contributed equally to this work.

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