Crif1调控Nrf2-ARE信号通路促进BMSCs抗辐射损伤机制研究

Low radiation sensitivity is one of the important characteristics of bone marrow-derived mesenchymal stem cells (BMSCs) in potential clinical applications, but the mechanism is not clear. Radiation cell will produce mass of reactive oxygen species (ROS), which make kelch 2 like ECH2 associated protein1(Keap1) isomerizes and start Keap1- nuclear factor erythroid-2-related factor 2(Nrf2)/ Antioxidant Response Element (ARE) pathway and induced oxidative stress and autophagy for damage repair. Recently research showed that CR6-interacting factor 1 (Crif1) can reduce the sensitivity of cells to ROS thorough regulating Nrf2 stability. In the cytosol, Crif1 physically interacts with both N- and C-terminal regions of NRF2 and promotes NRF2 ubiquitination and subsequent proteasome-mediated NRF2 protein degradation, however, the regulation of Nrf2 in nucleus still needs to be studied. Since our preliminary studies had shown that Crif1 of BMSCs migrates from cytosol into the nucleus after radiation, and its expression negatively related with Nrf2 and the autophagy level. We suggest that, at resting stage, Crif1 ubiquitinates cytosolic NRF2 collaborating with Keap1.After radiation damage, Crif1 modulates oxidative stress and autophagy by regulating the Nrf2-ARE signaling pathway. Therefore we sought to assess synergistic effects of Crif1 and Keap1, functions and subcellular localization of Crif1 and Crif1 modulate nucleus levels of Nrf2 in irradiated and non-irradiated BMSCs to explore the mechanism of Crif1 in resistance to radiation damage of BMSCs.

辐射敏感性低是BMSCs临床应用潜力的重要特性之一，但其机制未明。辐射后细胞产生大量的ROS使Keap1异构启动Nrf2-ARE信号通路诱导氧化应激与自噬促进损伤修复。最新发现，Crif1可调控Nrf2稳定性降低细胞对ROS的敏感性，其N'、C'端均能与Nrf2结合，胞浆内介导后者泛素化（类似Keap1）,但其对Nrf2核内的调控有待研究。本室预实验结果显示，BMSCs辐射后表达Crif1先下降后升高，与Nrf2水平、自噬均成负相关，且表达升高后出现核内聚集。推测：静息态时，BMSCs胞浆内Crif1协同Keap1泛素化Nrf2，辐射损伤后，Crif1通过调节Nrf2-ARE信号通路，从而调控辐射损伤后氧化应激、自噬。为此，本项目拟从Crif1与Keap1协同作用、辐射前后Cirf1亚细胞定位及功能、Crif1核内调节Nrf2作用三方面研究Crif1对BMSCs抗辐射损伤作用机制。

辐射敏感性低是BMSCs临床应用潜力的重要特性之一，但其机制未明。主要研究内容：辐射后Crif1表达水平升高在BMSCs分化失衡中的作用及其机制研究；Crif1在辐射诱导BMSCs衰老中的作用机制研究。我们阐明了辐射致BMSCs成脂分化增强的机制主要通过上调Crif1的表达水平进而与PKA-α相互作用促进CREB的磷酸化，从而启动成脂分化关键基因的转录表达，是对BMSCs细胞辐射应激反应机制研究及辐射耐受机制研究的重要补充。辐射可导致红骨髓比例下降和黄骨髓比例上升，造血能力减弱，我们的发现从造血微环境重要组成细胞—BMSCs成脂分化增加出发解释了辐射致骨髓脂肪化和造血能力下降的分子机制。同时，该研究有助于从PKA-α及Crif1的抑制剂筛选出发，寻找促造血恢复的抗辐射药物。阐明了辐射后BMSCs抵抗衰老的部分机制：通过上调Crif1的表达并与PKC-δ相互作用介导Nrf2 S40磷酸化和减少Nrf2的泛素化降解，从而启动II相解毒酶的转录表达，最终清除细胞内ROS。该发现有助于进一步解释BMSCs辐射相对耐受的分子机制。造血功能能否恢复是急性放射病患者救治的关键，我们在该项目资助下证实：Crif1的表达水平上升有利于BMSCs抵抗辐射致细胞衰老，增强BMSCs的造血支持功能；同时我们还发现Crif1的持续高表达不利于成骨分化和造血微环境的重建。因此，我们的研究提示：在最佳时间窗调控Crif1的表达水平，既防止BMSCs的衰老又逆转BMSCs的定向分化失衡，将最大可能的促进辐射后造血微环境的恢复和重建造血系统。