Department of Orthopedics, Wuxi First People's Hospital Affiliated to Nanjing Medical University, Wuxi City, Jiangsu 214023, China.
Department of Orthopedics, Wuxi First People's Hospital Affiliated to Nanjing Medical University, Wuxi City, Jiangsu 214023, China.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):646-51. doi: 10.1016/j.bbrc.2014.06.030. Epub 2014 Jun 16.
Precartilaginous stem cells (PSCs) are adult stem cells which could initiate chondrocytes and bone growth. In the current study, we purified PSCs from the neonate mice' perichondrial mesenchyme through immunomagnetic beads with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Mouse PSCs were seeded and cultured, and their phenotype was confirmed by FGFR-3 over-expression. Transforming growth factor-β (TGF-β) was added to induce PSCs differentiation. TGF-β increased mRNA expression of chondrogenesis-related genes (collagen type II, Sox 9, and aggrecan) in the cultured PSCs, which was abolished by TGF-β receptor II (TGFRII) lentiviral shRNA depletion. TGF-β induced AKT activation in mouse PSCs, while the PI3K/AKT inhibitor (LY294002) and the AKT specific inhibitors (perifosine and MK-2206) largely suppressed TGF-β-induced collagen II, Sox 9, and aggrecan mRNA expression. Meanwhile, the mTOR complex 1 (mTORC1) blocker RAD001 or the mTORC1/2 dual inhibitor AZD-2014 also alleviated TGF-β-induced chondrogenesis-associated genes expression. Further, lentiviral shRNA depletion of SIN1 (a mTORC2 component) or mTOR inhibited TGF-β's effect in the mouse PSCs. In conclusion, our evidence suggests that TGF-β induces the expression of chondrogenesis-related genes through TGFRII-AKT-mTOR signaling in cultured mouse PSCs.
软骨前体细胞（PSCs）是一种成体干细胞，能够启动软骨细胞和骨生长。在本研究中，我们通过纤维母细胞生长因子受体 3（FGFR-3）抗体的免疫磁珠从新生鼠的软骨膜间质中纯化 PSCs。将小鼠 PSCs 接种和培养，并通过 FGFR-3 过表达来确认其表型。添加转化生长因子-β（TGF-β）以诱导 PSCs 分化。TGF-β增加了培养的 PSCs 中软骨形成相关基因（II 型胶原、Sox9 和聚集蛋白聚糖）的 mRNA 表达，而这种表达被 TGF-β 受体 II（TGFRII）慢病毒 shRNA 耗竭所消除。TGF-β激活了小鼠 PSCs 中的 AKT，而 PI3K/AKT 抑制剂（LY294002）和 AKT 特异性抑制剂（perifosine 和 MK-2206）则大大抑制了 TGF-β诱导的 II 型胶原、Sox9 和聚集蛋白聚糖 mRNA 的表达。同时，mTOR 复合物 1（mTORC1）抑制剂 RAD001 或 mTORC1/2 双重抑制剂 AZD-2014 也减轻了 TGF-β诱导的软骨形成相关基因的表达。此外，通过慢病毒 shRNA 耗竭 SIN1（mTORC2 的一个组成部分）或 mTOR 抑制了 TGF-β在小鼠 PSCs 中的作用。总之，我们的证据表明，TGF-β 通过 TGFRII-AKT-mTOR 信号通路诱导培养的小鼠 PSCs 中软骨形成相关基因的表达。
