Zhang Zhuang, Zhou Chaoqing, Yu Lili
Macau University of Science and Technology, Faculty of Chinese Medicine, E205, Avenida Wai Long, Taipa, Macau, 999078, China.
The 2nd People's Hospital of Zhuhai, Zhuhai, China.
BMC Mol Cell Biol. 2024 Dec 18;25(1):26. doi: 10.1186/s12860-024-00523-7.
Cellular senescence is a key driver of decreased bone formation and osteoporosis. Leptin (LEP) has been implicated in cellular senescence and osteogenic differentiation. The aim of this study was to investigate the mechanisms by which LEP mediates cellular senescence and osteogenic differentiation.
C3H10T1/2 cells were treated with etoposide to induce cellular senescence, which was assessed by β-galactosidase staining. Quantitative real-time PCR and western blotting were used to measure the levels of senescence markers p21 and p16, as well as osteogenic differentiation-related genes ALP, COL1A1, and RUNX2. Alkaline phosphatase (ALP) staining and alizarin red S staining were performed to evaluate osteogenic differentiation. The NF-κB pathway and O-GlcNAcylation were assessed by western blotting.
Etoposide treatment increased the number of senescent cells and the levels of p21 and p16, along with elevated LEP expression. These effects were reversed by LEP knockdown. Additionally, LEP knockdown increased ALP staining density and osteoblast mineralization nodules, as well as the mRNA and protein levels of ALP, COL1A1, and RUNX2, indicating that LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells. Mechanistically, LEP knockdown inactivated the NF-κB pathway by inhibiting the nuclear translocation of p65. Furthermore, OGT was found to promote O-GlcNAcylation of LEP at the S50 site.
Our findings demonstrated that O-GlcNAcylation of LEP inactivated the NF-κB pathway by reducing LEP protein levels, thereby inhibiting cellular senescence and promoting osteogenic differentiation in C3H10T1/2 cells. This study may provide a novel therapeutic target for the treatment of osteoporosis.
细胞衰老 是骨形成减少和骨质疏松症的关键驱动因素。瘦素（LEP）与细胞衰老和成骨分化有关。本研究的目的是探讨LEP介导细胞衰老和成骨分化的机制。
用依托泊苷处理C3H10T1/2细胞以诱导细胞衰老，通过β-半乳糖苷酶染色进行评估。采用定量实时PCR和蛋白质免疫印迹法检测衰老标志物p21和p16以及成骨分化相关基因碱性磷酸酶（ALP）、I型胶原蛋白α1链（COL1A1）和 runt相关转录因子2（RUNX2）的水平。进行碱性磷酸酶（ALP）染色和茜素红S染色以评估成骨分化。通过蛋白质免疫印迹法评估核因子κB（NF-κB）信号通路和O-连接的N-乙酰葡糖胺糖基化（O-GlcNAcylation）。
依托泊苷处理增加了衰老细胞的数量以及p21和p16的水平，同时LEP表达升高。LEP基因敲低可逆转这些效应。此外，LEP基因敲低增加了ALP染色密度和成骨细胞矿化结节，以及ALP、COL1A1和RUNX2的mRNA和蛋白质水平，表明LEP基因敲低促进了C3H10T1/2细胞的成骨分化。机制上，LEP基因敲低通过抑制p65的核转位使NF-κB信号通路失活。此外，发现O-连接的N-乙酰葡糖胺转移酶（OGT）促进LEP在第50位丝氨酸（S50）位点的O-GlcNAcylation。
我们的研究结果表明，LEP的O-GlcNAcylation通过降低LEP蛋白水平使NF-κB信号通路失活，从而抑制C3H10T1/2细胞的衰老并促进成骨分化。本研究可能为骨质疏松症的治疗提供一个新的治疗靶点。
