Table_1_Triiodothyronine Potentiates BMP9-Induced Osteogenesis in Mesenchymal Stem Cells Through the Activation of AMPK/p38 Signaling.DOCX

Thyroid hormone (TH), triiodothyronine (T3), and thyroxine (T4), which are released from the thyroid, control many cellular processes in various cell types. It is worth noting that TH plays a complex role in skeletal metabolic balance, and few studies have investigated whether TH exerts any effects on osteogenesis in bone mesenchymal stem cells (MSCs). We explored the effects of T3 on bone morphogenetic protein 9 (BMP9)-induced osteogenesis, which process is considered the most important in the osteogenic differentiation of C3H10T1/2 cells. In vitro osteogenesis was analyzed by alkaline phosphatase (ALP) activity and staining, bone mineralisation, and osteocalcin and osteopontin expression. Fetal limb explant cultures and ectopic MSC implantation further confirmed the role of T3. Finally, we examined the effect of AMPK/p38 signaling on the osteoblastic differentiation. T3 synergizes with BMP9 to enhance osteogenic marker expression induced by BMP9. Furthermore, T3 promotes BMP9-induced bone formation by fetal limb explant cultures and ectopic MSC implantation. Co-treatment with BMP9 and T3 can promote AMPK and p38 phosphorylation, and pretreatment with the AMPK inhibitor compound C and siRNA can abolish phosphorylation of p38 and BMP9+T3-induced ALP activity. Our results suggest that BMP9 and T3 promote osteogenic differentiation at least partially via the activation of the AMPK/p38 signaling pathway.

甲状腺（thyroid）分泌的甲状腺激素（TH）、三碘甲状腺原氨酸（T3）与甲状腺素（T4），可调控多种细胞类型的诸多细胞生命活动。值得注意的是，甲状腺激素在骨骼代谢平衡中发挥着复杂作用，目前鲜有研究探讨甲状腺激素对骨髓间充质干细胞（MSCs）成骨作用的影响。本研究聚焦于T3对骨形态发生蛋白9（BMP9）诱导的成骨作用的调控效应，而该过程被认为是C3H10T1/2细胞成骨分化过程中最为核心的环节。研究通过碱性磷酸酶（ALP）活性检测与染色、骨矿化分析以及骨钙蛋白、骨桥蛋白表达检测，对体外成骨过程进行了系统评估。此外，借助胎肢外植体培养与异位骨髓间充质干细胞移植实验，进一步验证了T3的生物学功能。最后，本研究考察了AMPK/p38信号通路在成骨细胞分化中的调控作用。实验结果表明，T3可与BMP9协同增强BMP9诱导的成骨标志物表达。进一步实验证实，T3可通过胎肢外植体培养与异位骨髓间充质干细胞移植模型，促进BMP9诱导的骨形成。联合施加BMP9与T3能够促进AMPK与p38的磷酸化；而预先采用AMPK抑制剂Compound C与小干扰RNA（siRNA）进行预处理，则可阻断p38的磷酸化以及BMP9与T3联合诱导的碱性磷酸酶活性升高。本研究结果提示，BMP9与T3至少可通过激活AMPK/p38信号通路，协同促进成骨细胞分化。