Abstract
Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.
Highlights
MT3 Is Significantly Upregulated in bone morphogenetic protein 4 (BMP4) Treated C2C12 Cells
The MT protein family consists of four main isoforms; among them, MT4 is considered to be expressed in the epithelial tissue [29]
The results showed that BMP4 treatment highly induced reactive oxygen species (ROS) production, while MT3 overexpression inhibited the generation of ROS, and MT3 knockdown accelerated ROS production (Figure 5A–D), which indicated that MT3 played a positive role in reducing oxidative stress during BMP4-induced osteoblast differentiation
Summary
Osteoblast differentiation is a process involved in bone tissue self-renewal throughout the period of a whole lifetime, and its dysfunction leads to skeletal disorders such as osteoporosis [1], a disease caused by systemic bone mass reduction [2]. Mesenchymal stem cells and skeletal muscle cells play important roles in bone homeostasis maintenance, as they can differentiate into osteoblastic-like cells and affect bone remodeling [3,4]. During this process, several signaling pathways such as bone morphogenetic protein (BMP), Wnt, Notch, and Hedgehog all contribute greatly to the differentiation of osteoblasts [5]. Effective therapeutic strategies targeting osteoblast differentiation with fewer side effects will contribute greatly to the control of osteogenesisrelated disorders
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