College of Pharmacy and Research Institute of Pharmaceutical Sciences, Chonnam National University, Gwangju 61186, Korea.
School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325000, China.
Int J Mol Sci. 2021 Apr 21;22(9):4312. doi: 10.3390/ijms22094312.
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.
金属硫蛋白（MTs）是富含半胱氨酸的细胞内蛋白质，其表达在应激条件下增强。MTs 被认为具有调节生物体内氧化还原平衡的能力；然而，其在调节成骨细胞分化中的作用尚不清楚。在这项研究中，我们发现金属硫蛋白家族成员 MT3 的表达在 BMP4 处理的 C2C12 成肌细胞分化过程中特异性地上调。在 C2C12 细胞中转染 MT3 过表达质粒增强了其向成骨细胞的分化，并上调了骨特异性转录因子 runt 相关基因 2（Runx2）、成骨特异性转录因子（Osterix）和远隔同源盒 5（Dlx5）的蛋白表达。此外，MT3 敲低则产生相反的效果。进一步的研究表明，过表达 MT3 减少了 BMP4 处理的 C2C12 细胞中活性氧（ROS）的产生，而 MT3 沉默则增强了 ROS 的产生。用抗氧化剂 N-乙酰半胱氨酸处理 C2C12 细胞也促进了成骨细胞分化，并上调了 Runx2/Osterix/Dlx5，而 ROS 生成剂安密妥钠处理则产生相反的效果。最后，安密妥钠处理抑制了 MT3 过表达的 C2C12 细胞中的成骨细胞分化和 Runx2/Osterix/Dlx5 表达。这些发现确定了 MT3 在成骨细胞分化中的作用，并表明 MT3 在成骨研究领域可能具有有趣的潜力。
