Qin Qianyi, Yang Haoqing, Guo Runzhi, Zheng Yunfei, Huang Yiping, Jin Luyuan, Fan Zhipeng, Li Weiran
Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing, 100081, China.
Laboratory of Molecular Signalling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing, 100050, China.
Stem Cell Res Ther. 2024 Dec 18;15(1):471. doi: 10.1186/s13287-024-04083-7.
Periodontal ligament stem cell (PDLSC)-based therapy is one of the methods to assist bone regeneration. Understanding the functional regulation of PDLSCs and the mechanisms involved is a crucial issue in bone regeneration. This study aimed to explore the roles of the family with sequence similarity 96 member B (FAM96B) in the functional regulation of PDLSCs.
To assess the osteogenic differentiation of PDLSCs, the alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative calcium analysis, and osteogenic marker detection were conducted. Transplantation PDLSCs under the dorsum of nude mice and into the rat calvarial defects were also performed. Then, FAM96B-overexpressed PDLSCs were used for RNA-sequencing and bioinformatic analysis. To evaluate the ferroptosis of PDLSCs, cytosolic reactive oxygen species (ROS), expression of glutathione peroxidase 4 (GPX4), mitochondrial morphology and functions including the mitochondrial ROS, mitochondria membrane potential, and mitochondrial respiration were detected.
The osteogenic indicators ALP activity, level of mineralization, and osteocalcin expression were decreased in PDLSCs by FAM96B, which demonstrated that FAM96B inhibited the osteogenic differentiation of PDLSCs. FAM96B knockdown promoted the new bone formation of PDLSCs subcutaneously transplanted to the dorsum of nude mice. Then, related biological functions were detected by the RNA-sequencing and the ferroptosis was focused. FAM96B enhanced the cytosolic ROS level and inhibited the expression of GPX4 and mitochondrial functions in PDLSCs. Hence, FAM96B promoted the ferroptosis of PDLSCs. Meanwhile, we found that FAM96B inhibition upregulated the target gene FOS like 1, AP-1 transcription factor subunit (FOSL1) expression and FOSL1 promoted the osteogenic differentiation of PDLSCs in vitro. FOSL1 also promoted the new bone formation of PDLSCs transplanted subcutaneously to the dorsum of nude mice and transplanted into rat calvarial defects. Then, the inhibitory effect of FOSL1 on the ferroptosis was confirmed.
FAM96B depletion promoted the osteogenic differentiation and suppressed the ferroptosis of PDLSCs. FAM96B negatively regulated the downstream gene FOSL1 and FOSL1 promoted the osteogenic differentiation of PDLSCs via the ferroptosis. Hence, our findings provided a foundation for understanding the FAM96B-FOSL1 axis acting as a target for MSC mediated bone regeneration.
基于牙周膜干细胞（PDLSC）的治疗是辅助骨再生的方法之一。了解PDLSCs的功能调节及其相关机制是骨再生中的关键问题。本研究旨在探讨序列相似性96成员B（FAM96B）在PDLSCs功能调节中的作用。
为评估PDLSCs的成骨分化，进行了碱性磷酸酶（ALP）活性测定、茜素红染色、定量钙分析和成骨标志物检测。还将PDLSCs移植到裸鼠背部和大鼠颅骨缺损处。然后，使用过表达FAM96B的PDLSCs进行RNA测序和生物信息学分析。为评估PDLSCs的铁死亡，检测了胞质活性氧（ROS）、谷胱甘肽过氧化物酶4（GPX4）的表达、线粒体形态和功能，包括线粒体ROS、线粒体膜电位和线粒体呼吸。
FAM96B使PDLSCs中的成骨指标ALP活性、矿化水平和骨钙素表达降低，这表明FAM96B抑制了PDLSCs的成骨分化。FAM96B基因敲低促进了皮下移植到裸鼠背部的PDLSCs的新骨形成。然后，通过RNA测序检测相关生物学功能并聚焦于铁死亡。FAM96B增强了PDLSCs中的胞质ROS水平，抑制了GPX4的表达和线粒体功能。因此，FAM96B促进了PDLSCs的铁死亡。同时，我们发现抑制FAM96B上调了靶基因FOS样1、AP-1转录因子亚基（FOSL1）的表达，并且FOSL1在体外促进了PDLSCs的成骨分化。FOSL1还促进了皮下移植到裸鼠背部和移植到大鼠颅骨缺损处的PDLSCs的新骨形成。然后，证实了FOSL1对铁死亡的抑制作用。
FAM96B缺失促进了PDLSCs的成骨分化并抑制了其铁死亡。FAM96B负向调节下游基因FOSL1，并且FOSL1通过铁死亡促进了PDLSCs的成骨分化。因此，我们的研究结果为理解FAM96B-FOSL1轴作为间充质干细胞介导的骨再生靶点提供了基础。
