PTEN在COX-2影响BMP9诱导间充质干细胞成骨分化中的作用研究

Our previous data has demonstrated that bone morphogenic protein 9 (BMP9) is one of the most potent member of BMPs to commit mesenchymal stem cells (MSCs) into osteo-progenitor lineage, although the detail mechanism remain unclear. BMP9 can induce the expression of cycloxygenase-2 (COX-2) in MSCs, and COX-2 can affects the osteogenesis induced by BMP9 greatly in MSCs. It has been reported that COX-2 is important for osteogenesis through the production of PGE2, which can activate PKA to produce more cAMP to promote the osteogenesis or bone fracture healing process by binding with EP2 and EP4 receptors. But, the mechanism of COX-2 affects the osteogenesis induced by BMP9 is unknown. Because PI3K/Akt is important for BMPs mediated osteogenesis and expression of COX-2, which can be negative regulated by PTEN, so we speculate that PTEN maybe involved in BMP9 induced osteogenesis in MSCs. With further investigation, we found the BMP9 can induce the expression of PTEN in MSCs, but PTEN specific inhibitor can enhance the ALP activity in MSCs induced by BMP9 with a concentration dependent fashion. Knockdown of COX-2 can reduce the ALP activity induced by BMP9 in MSCs, which can be fully reversed by PTEN inhibitor VO-OHpic, and can only be partly reversed by PGE2. This suggest that the effect of COX-2 on BMP9 induced osteogenesis is partly due to the production of PGE2, COX-2 may fulfill this function through other ways. With Western blot analysis, we found that over-expression of COX-2 combine with BMP9 can decrease the total protein level of PTEN, but increase the phosphorylation of PTEN, which can reduce the function of PTEN; knockdown of COX-2 combine with BMP9 can increase the total protein level of PTEN and decrease the phosphorylation of PTEN. These results indicate that PTEN is important for BMP9 to fulfill its function, and the effect of COX-2 on BMP9 induced osteogenesis in MSCs maybe result from the regulation of PTEN by COX-2. This proposal will follow a serial of well-established experiments to validate the importance of PTEN in the process of BMP9 induced osteogenesiss in MSCs, and analysis the possible mechanism of how COX-2 regulates the functional balance of PTEN in BMP9 induced osteogenesis in MSCs. With the practice of this proposal, we can expansion the knowledge of COX-2 in osteogenesis, and clarify the role of PTEN in BMP9 induced osteogenesis. It will accelerate the development of bone tissue engineering project, which will benefit the treatment or recover of bone fracture, bone defect and bone un-union diseases.

BMP9是目前诱导间充质干细胞（MSCs）成骨分化能力最强的BMPs成员之一。课题组研究表明：BMP9能诱导COX-2表达，且COX-2对BMP9诱导MSCs成骨分化有重要影响，但影响机制不清楚；BMP9能诱导PTEN表达，但PTEN抑制剂却增强BMP9诱导MSCs碱性磷酸酶（ALP）活性增加；COX-2沉默表达抑制BMP9诱导MSCs ALP活性增加，PTEN抑制剂可逆转此作用，但PGE2仅能部分逆转；BMP9合并COX-2过表达促进PTEN磷酸化，但BMP9合并COX-2沉默表达却明显抑制PTEN磷酸化。课题组推测：PTEN在BMP9诱导MSCs成骨分化中有重要作用，COX-2通过调节PTEN的功能平衡实现对BMP9作用的影响。本研究利用多种实验技术和方法，阐明PTEN在BMP9诱导MSC成骨分化过程中的作用；分析在BMP9诱导MSCs成骨分化中，COX-2调节PTEN水平的可能机制。

*中文摘要 (对项目的背景、主要研究内容、重要结果、关键数据及其科学意义等做简单概述，800字以内)：.BMP9是诱导干细胞骨分化能力最强的因子之一，具有很好的临床应用前景。但是，到目前为止BMP9诱导干细胞骨分化的机制尚不清楚。课题组前期研究发现，BMP9在干细胞中能明显诱导COX-2表达，抑制或沉默COX-2则减弱BMP9的成骨诱导能力。提示，COX-2在BMP9诱导骨分化过程中具有重要作用。同时，COX-2抑制剂因胃肠道不良反应较少，在临床广泛使用。但是，长期使用COX-2抑制剂可能对骨损伤修复产生抑制作用，目前尚无相关预防措施。本研究发现，COX-2对BMP9诱导干细胞骨分化的调节作用与PTEN有关：BMP9能同时抑制PTEN表达而促进COX-2表达，抑制COX-2增加PTEN表达，抑制PTEN则使COX-2表达增加；同时，抑制PTEN能促进BMP9诱导干细胞骨分化，但这种作用可被COX-2沉默所取消。提示，BMP9能通过抑制PTEN功能而促进干细胞骨分化，但这种作用是通过PTEN影响COX-2表达而实现的。另外， BMP9能激活Wnt/β-catenin信号，COX-2本身是Wnt/β-catenin信号的下游靶基因。但是，BMP9如何激活Wnt/β-catenin信号，以及与COX-2是否有关目前仍不清楚。课题组发现，抑制PTEN能增强BMP9对Wnt/β-catenin信号的活化作用，但抑制COX-2则减弱BMP9的这种作用。提示，COX-2与Wnt/β-catenin信号之间存在正反馈调节，而且COX-2可能通过PGE2和非PGE2途径实现对该信号的调节。该研究结果对于深入诠释BMP9诱导干细胞骨分化的机制，以及全面了解COX-2的生理和病理功能具有重要意义。同时，本研究也发现ATRA能阻止RSG诱导干细胞向脂肪细胞分化，而定向骨分化；抑制COX-2能减弱BMP9诱导干细胞骨分化的功能，但ATRA可逆转或取消COX-2抑制剂骨分化的抑制作用。这一发现提示，ATRA可能在临床有预防NSAIDs骨毒性的作用，可提高相关骨疾病的治疗效果。