GPx1介导的氧化还原信号调控软骨形成与稳态维持的分子机制

Reactive oxygen species (ROS, 氧自由基)-generated oxidative stress has been considered as a pivotal player in various pathological conditions. However, few antioxidants or redox-based therapeutic strategies have a definitive proof of benefit in clinical trials according to the criteria of evidence-based medicine. Glutathione peroxidases (GPxs, 谷胱甘肽过氧化物还原酶) use glutathione (GSH, 还原性谷胱甘肽) as a source of reducing equivalents to catalyze low molecular ROS, thereby producing oxidized glutathione (GSSG, 氧化型谷胱甘肽). Due to its roles in the reduction of ROS, GPxs were originally known as an antioxidant enzyme. However, there was no significant oxidative stress in four independent GPx1 knockout mouse lines (Merry et al., 2016; Esposito et al., 2000; de Haan et al., 1998; Ho et al., 1997). On the contrary, we demonstrated that suppression of GPx activity or knockdown of GPx1 expression could induce GSH reductive stress, which inhibited chondrogenic differentiation of chondroblast ATDC5 in vitro. These observations indicate GPx1 may transmit ROS signal to GSH/GSSG, rather than an antioxidant enzyme to eliminate ROS. We propose to examine the interaction between ROS, which stimulate chondrocyte differentiation, and thiol peroxidase GPx1, which is induced during chondrogenesis and further explore the molecular mechanisms involved. Firstly, the spatial-temporal dynamics of ROS and GSH/GSSG were detected through in situ, real-time, and quantitative approach by using genetically encoded fluorescent redox sensors at subcellular level. Secondly, the thiol redoxome network was dissected through oxidative isotope-coded affinity tags (oxICAT，巯基氧化修饰同位素标记) quantitative proteomics and bioinformatics approach including functional analysis and molecule networks identification. Finally, the vital nodes were identified through gain and loss of function assay in cartilage formation and degeneration postnatal. In summary, the redox relay properties of GPx1 are verified through carefully examination of the diffusion and reception of H2O2 signaling. Simultaneously, the “GSH thiol peroxidase signal model” is proposed. Then, decipher the redox mechanisms in the decision of chondrocyte fate and the redox signaling which involved in cartilage regeneration. Furthermore, verify the hypothesis that the redox signaling dysfunction could induce both oxidative and reductive stress. Finally, the specific redox biomarker and selective redox modulator in cartilage formation and degeneration are characterized to test the efficiency of this signaling model. As a result, throw light on the prevention and treatment of development deficiency and degenerative disorders or related conditions of cartilage.

氧化应激参与多种病理过程，但基于抗氧化机制的临床实践却缺乏循证医学证据。依据GPx1敲除鼠无氧化应激产生，及申请人发现GPx1抑制导致GSH还原应激引起软骨细胞分化障碍。提示GPx1可能传递ROS信号至GSH电势，而不是仅作为抗氧化酶清除ROS。研究软骨细胞分化依赖表达的GPx1与分化诱导信号H2O2之间的相互作用机制。利用基因编码荧光探针，在亚细胞水平原位、实时、定量检测H2O2与GSH电势的时空动态；通过oxICAT巯基氧化修饰定量蛋白组的系统发现、生物信息数据挖掘、与功能缺失或获得实验相结合，鉴别氧化还原敏感蛋白网络与其中的关键节点。明确GPx1的ROS信号传递作用，提出“GSH巯基过氧化物酶信号模型”，揭示软骨形成与重塑过程中的氧化还原调控机制，验证ROS信号传递障碍是导致氧化、还原应激的关键，发现特异性的氧化还原应激生物标志物或氧化还原调节靶点，为软骨退变相关疾病的诊治提供依据。

GPx1在生长板肥大软骨细胞层中表达，在成软骨细胞诱导分化过程中蛋白表达水平显著增加。PI3k-Akt-mTOR-P70S6、P38与ERK分别在转录后诱导GPx1的表达。GPx1敲低抑制肢芽间充质干细胞成软骨分化。同样GPx活性抑制或GPx1稳定干扰，均能抑制成软骨细胞肥大分化。但是GPx1敲除后却能促进软骨细胞肥大分化。GPx活性抑制或GPx1干扰均能减弱PI3k-Akt-mTOR-P70S6与P38信号，而GPx1敲除却能增强以上信号。基因表达分析显示GPx1敲除后PI3K-Akt、MAPK与Ras通路，以及粘着斑与细胞骨架过程得到富集。提示GPx1对IGF信号通路的增强或减弱，可能是GPx活性抑制或GPx1基因干扰、与GPx1基因敲除促进或抑制截然相反效应的潜在机制。GPx活性抑制、GPx1干扰与GPx1敲除后GPx活性分别降低了40%、60%、90%，相应地GSH/GSSG比例分别升高约10%、30%、40%。说明GPx活性不同程度降低，产生不同程度的GSH还原应激，导致IGF信号活化或抑制。进而发现蛋白磷酸酶PTP1b和PP2R1A，以及oxICAT氧化还原定量蛋白组发现与蛋白翻译核糖体组装、细胞黏附与细胞骨架等相关蛋白存在巯基氧化还原敏感修饰。另外ATAC-seq分析显示PI3K-Akt、MAPK、Ras信号和细胞骨架与黏附得到富集。最后发现随关节软骨生长GPx1从表层逐渐分布于深层，提示GPx1在关节软骨形成与稳态维持中可能发挥重要作用。总之，研究发现IGF信号诱导GPx1在转录后水平表达，同时GPx1也是IGF信号通路的重要调控因子之一。GPx1能提升GSH氧化还原电势、通过巯基氧化还原敏感靶蛋白、调节细胞信号、蛋白翻译、细胞骨架黏附等，发挥传递氧化还原信号作用，调控软骨发育、维持软骨稳态。