S100 Calcium-Binding Protein A16 Suppresses the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells via Inhibiting SMAD Family Member 4 Signaling. S100 Calcium-Binding Protein A16 Suppresses the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells via Inhibiting SMAD Family Member 4 Signaling

Osteogenesis is a complex process of bone formation regulated by various factors, yet its underlying molecular mechanisms remain incompletely understood. This study aimed to investigate the role of S100A16, a novel member of the S100 protein family, in the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and uncover a novel Smad4-mitogen-activated protein kinase (MAPK)/Jun N-terminal kinase (JNK) signaling axis. We herein evaluated the expression level of S100A16 in bone tissues and BMSCs from ovariectomized rats and then examined the impact of S100A16 silencing on osteogenic differentiation. Increased S100A16 expression was observed in bone tissues and BMSCs from ovariectomized rats, and S100A16 silencing promoted osteogenic differentiation. Further transcriptomic sequencing revealed that the Smad4 pathway was involved in S100A16 silencing-induced osteogenesis. The results of our Western blot analysis showed that S100A16 overexpression not only downregulated Smad4 but also activated MAPK/JNK signaling, which was validated by treatment with MAPK and JNK inhibitors U0126 and SP600125. Overall, this study elucidated the novel regulatory factors influencing osteogenic differentiation and provided mechanistic insights that could aid in the development of targeted therapeutic strategies for osteoporosis patients. Overall design: To investigate the function S100A16 in the regulation of osteogenesis, we isolated primary bone marrow stem cells from rats. After transfection of S100A16-specific siRNA during osteogenic differentiation, we performed gene expression profiling analysis using data obtained from RNA-seq of S100A16 downregulated cells or control cells.

骨生成（Osteogenesis）是受多种因素调控的复杂骨形成过程，但其潜在分子机制尚未完全阐明。本研究旨在探讨S100蛋白家族新型成员S100A16在大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）成骨分化中的作用，并揭示一条全新的Smad4-丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）/Jun N末端激酶（Jun N-terminal kinase，JNK）信号轴。本研究首先检测了去卵巢大鼠骨组织及BMSCs中S100A16的表达水平，随后探讨了S100A16沉默对成骨分化的影响。研究发现，去卵巢大鼠骨组织及BMSCs中S100A16的表达水平显著升高，且S100A16沉默可促进成骨分化。进一步的转录组测序分析显示，Smad4通路参与了S100A16沉默介导的成骨过程。蛋白质免疫印迹（Western blot）实验结果表明，S100A16过表达不仅可下调Smad4的表达水平，还能激活MAPK/JNK信号通路；该结论通过MAPK抑制剂U0126与JNK抑制剂SP600125的干预实验得到了验证。综上，本研究阐明了影响成骨分化的新型调控因子，并为骨质疏松症患者的靶向治疗策略开发提供了关键的机制学依据。实验设计：为探究S100A16对成骨过程的调控功能，本研究从大鼠体内分离得到原代骨髓间充质干细胞。在成骨分化阶段转染S100A16特异性小干扰RNA（small interfering RNA，siRNA）后，通过对S100A16下调组与对照组细胞进行RNA测序（RNA-seq），获取转录组数据并开展基因表达谱分析。