Department of Pharmacology, College of Pharmacy, Chongqing Medical University, No. 1 Yixueyuan Road, Yuzhong District, Chongqing, 400016, People's Republic of China.
Chongqing Key Laboratory for Biochemistry and Molecular Pharmacology, Chongqing, 400016, People's Republic of China.
Tissue Eng Regen Med. 2023 Aug;20(5):705-723. doi: 10.1007/s13770-023-00526-z. Epub 2023 Apr 3.
All-trans retinoic acid (ATRA) promotes the osteogenic differentiation induced by bone morphogenetic protein 9 (BMP9), but the intrinsic relationship between BMP9 and ATRA keeps unknown. Herein, we investigated the effect of Cyp26b1, a critical enzyme of ATRA degradation, on the BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs), and unveiled possible mechanism through which BMP9 regulates the expression of Cyp26b1.
ATRA content was detected with ELISA and HPLC-MS/MS. PCR, Western blot, and histochemical staining were used to assay the osteogenic markers. Fetal limbs culture, cranial defect repair model, and micro-computed tomographic were used to evaluate the quality of bone formation. IP and ChIP assay were used to explore possible mechanism.
We found that the protein level of Cyp26b1 was increased with age, whereas the ATRA content decreased. The osteogenic markers induced by BMP9 were increased by inhibiting or silencing Cyp26b1 but reduced by exogenous Cyp26b1. The BMP9-induced bone formation was enhanced by inhibiting Cyp26b1. The cranial defect repair was promoted by BMP9, which was strengthened by silencing Cyp26b1 and reduced by exogenous Cyp26b1. Mechanically, Cyp26b1 was reduced by BMP9, which was enhanced by activating Wnt/β-catenin, and reduced by inhibiting this pathway. β-catenin interacts with Smad1/5/9, and both were recruited at the promoter of Cyp26b1.
Our findings suggested the BMP9-induced osteoblastic differentiation was mediated by activating retinoic acid signalling, viadown-regulating Cyp26b1. Meanwhile, Cyp26b1 may be a novel potential therapeutic target for the treatment of bone-related diseases or accelerating bone-tissue engineering.
全反式维甲酸（ATRA）促进骨形态发生蛋白 9（BMP9）诱导的成骨分化，但 BMP9 与 ATRA 之间的内在关系尚不清楚。在此，我们研究了关键 ATRA 降解酶 Cyp26b1 对间充质干细胞（MSCs）中 BMP9 诱导的成骨分化的影响，并揭示了 BMP9 调节 Cyp26b1 表达的可能机制。
采用 ELISA 和 HPLC-MS/MS 检测 ATRA 含量。采用 PCR、Western blot 和组织化学染色检测成骨标志物。胎肢培养、颅缺损修复模型和微计算机断层扫描用于评估骨形成质量。采用 IP 和 ChIP assay 探索可能的机制。
我们发现 Cyp26b1 蛋白水平随年龄增加而升高，而 ATRA 含量降低。BMP9 抑制或沉默 Cyp26b1 可增加成骨标志物的诱导，而外源性 Cyp26b1 则减少。抑制 Cyp26b1 可增强 BMP9 诱导的骨形成。BMP9 促进颅缺损修复，抑制 Cyp26b1 可增强该作用，外源性 Cyp26b1 则减弱该作用。机制上，BMP9 降低 Cyp26b1，激活 Wnt/β-catenin 可增强该作用，抑制该通路则降低 Cyp26b1。β-catenin 与 Smad1/5/9 相互作用，两者均募集于 Cyp26b1 启动子。
我们的研究结果表明，BMP9 诱导的成骨分化是通过激活维甲酸信号通路，下调 Cyp26b1 来介导的。同时，Cyp26b1 可能是治疗骨相关疾病或加速骨组织工程的一种新的潜在治疗靶点。
