Department of Orthopaedics, The First Affiliated Hospital, Dalian Medical University, Xigang, Dalian 116011, China.
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, Lvshunkou, Dalian 116044, China.
Aging (Albany NY). 2023 Oct 19;15(20):11471-11488. doi: 10.18632/aging.205136.
Our study was performed to investigate whether the Wingless and int-1 (Wnt) signaling pathway promotes osteogenic differentiation and inhibits apoptosis in bone marrow mesenchymal stem cells (BMSCs) by regulating telomerase reverse transcriptase (TERT) expression. An in vivo model of osteoporosis (OP) in C57BL/6J mice by bilateral ovariectomy (OVX) and an in vitro model of H2O2-induced BMSCs were established separately. Western blotting was used to detect the expression of the pathway-related proteins TERT, β-catenin, and phosphorylated-glycogen synthase kinase-3beta (p-GSK3β)/GSK3β, the osteogenic-related markers osteopontin (OPN), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (Runx2), and the apoptosis-related indicators B-cell lymphoma-2 (Bcl-2) and BAX. Osteoblastic phenotypes were also evaluated by alkaline phosphatase (ALP) staining and serum ALP activity assays. Osteogenic differentiation phenotypes in mice were verified by H&E staining, micro-CT, and parameter analysis of the femur. Western blotting results showed that the expression of the pathway-related proteins TERT, β-catenin, p-GSK3β/GSK3β was reduced in OVX mice and H2O2-induced BMSCs, accompanied by downregulated protein expression of osteogenic-related markers and antiapoptotic indicators and upregulated protein expression of apoptotic proteins compared to those in the control group. Mechanistic studies showed that the activation of Wnt signaling pathway in BMSCs promoted β-catenin translocation to the nucleus, as verified by immunofluorescence and facilitated colocalization between β-catenin and TERT, as verified by double-labeling immunofluorescence, thereby promoting osteogenic differentiation and reducing apoptosis. In summary, our experiments confirmed that the GSK3β/β-catenin/TERT pathway could regulate the osteogenic differentiation and apoptosis of BMSCs and that TERT might be a promising target for the future treatment of osteoporosis.
我们的研究旨在探讨 Wnt 信号通路是否通过调节端粒酶逆转录酶（TERT）表达来促进骨髓间充质干细胞（BMSCs）的成骨分化并抑制其凋亡。通过双侧卵巢切除术（OVX）建立 C57BL/6J 小鼠骨质疏松（OP）的体内模型，以及 H2O2 诱导的 BMSCs 的体外模型。Western blot 用于检测通路相关蛋白 TERT、β-连环蛋白和磷酸化糖原合酶激酶-3β（p-GSK3β）/GSK3β、成骨相关标志物骨桥蛋白（OPN）、骨形态发生蛋白 2（BMP2）和 runt 相关转录因子 2（Runx2），以及凋亡相关指标 B 细胞淋巴瘤-2（Bcl-2）和 BAX。碱性磷酸酶（ALP）染色和血清 ALP 活性测定也评估成骨表型。通过 H&E 染色、微 CT 和股骨参数分析验证小鼠的成骨分化表型。Western blot 结果表明，OVX 小鼠和 H2O2 诱导的 BMSCs 中通路相关蛋白 TERT、β-连环蛋白、p-GSK3β/GSK3β 的表达减少，与对照组相比，成骨相关标志物和抗凋亡指标的蛋白表达下调，凋亡蛋白的蛋白表达上调。机制研究表明，BMSCs 中 Wnt 信号通路的激活促进了 β-连环蛋白向核内易位，这通过免疫荧光得到验证，并促进了 β-连环蛋白和 TERT 之间的共定位，这通过双标记免疫荧光得到验证，从而促进成骨分化和减少凋亡。综上所述，我们的实验证实，GSK3β/β-连环蛋白/TERT 通路可以调节 BMSCs 的成骨分化和凋亡，TERT 可能是未来治疗骨质疏松症的有前途的靶点。
