软骨酸性蛋白CRTAC1介导的钙离子调控在年龄相关性白内障发病机制中的作用

Age-related cataract (ARC) is the major cause of blindness. Oxidative stress, a hallmark for aging in various cells, plays a key role in the progression of ARC, and may be used as a therapeutic target for ARC. The abnormal accumulation of calcium, which is capable of inducing mitochondrial production of ROS, in lens epithelial cells (LECs) is closely related to the development of ARC. Our previous data showed that the expression of Cartilage Acidic Protein 1 (CRTAC1), a secreted protein predicted to bind to calcium based on its domains, is upregulated in ARC. Knockdown of CRTAC1 results in decreased intracellular calcium and ROS levels, suggesting an important regulatory role of CRTAC1 on calcium induced ROS production. However, the molecular function of CRTAC1 remains unknown. We propose that CRTAC1 is capable of directly or indirectly regulating the calcium flux in LECs to alter mitochondria metabolism, and consequently induce ROS generation, leading to the aggravated development of ARC. This study is to reveal the role of CRTAC1 in ARC development, including how CRTAC1 transcription is induced during ARC, the mechanism of calcium flux regulation by CRTAC1 (such as whether it can transport calcium by direct binding to calcium, or form calcium permeable pore like amyloid beta, or alter the function of calcium channels or transporters), and the downstream signaling pathways, mitochondrial metabolism and ROS production. Furthermore, we will explore the potential therapeutic value of CRTAC1 interference in ARC animal models. These studies will provide a new insight into ARC pathogenesis, and may suggest a new strategy for ARC treatment.

年龄相关性白内障（ARC）是最常见的致盲性眼病。氧化应激在ARC中起着关键作用。钙离子是氧化应激反应的一个重要调控因素。我们前期研究证明软骨酸性蛋白（CRTAC1）是ARC晶状体中表达增加最显著基因之一，其能造成人晶状体上皮细胞（HLECs）内钙离子与活性氧（ROS）水平上升，但其功能至今仍不明确。我们推测CRTAC1可能通过直接或间接对钙离子水平的调节，参与了晶状体微环境的氧化应激反应调控，从而促进ARC发生。本项目旨在研究CRTAC1对ARC的发病机制：检测ARC中调控CRTAC1上游的转录因子改变；研究CRTAC1下游对HLECs内钙离子浓度的3种可能的调节机制（直接结合并转运钙离子、形成细胞膜上钙离子透过孔、或与钙离子通道或转运蛋白相互作用），继而在线粒体代谢、信号转导通路及ROS生成中的调控作用；利用动物模型探索CRTAC1的干预作用。期望为ARC的预防及早期干预提供新的视角。

年龄相关性白内障（ARC）是最常见的致盲性眼病。氧化应激在ARC的发展机制中起着关键作用。钙离子是氧化应激反应的一个重要调控因素。前期研究证明软骨酸性蛋白（CRTAC1）是ARC晶状体中表达增加最显著基因之一，其能造成人晶状体上皮细胞（HLECs）内钙离子与活性氧（ROS）水平上升，但其功能至今仍不明确。本研究初步阐明了CRTAC1对ARC的发病机制。（1）通过生物信息学分析及荧光素酶报告基因检测结果表明NF-κB可能为CRTAC1的转录因子，通过NF-κB抑制剂PDTC，NF-κB激活剂TNF-α，ROS抑制剂NAC，p38激动剂anisomycin和p38抑制剂SB203580探究NF-kB/CRTAC1/p38信号轴，并揭示了ROS、NF-κB p65和CRTAC1之间的关系。（2）临床组织检测和体外实验结果提示CRTAC1可能与白内障的细胞焦亡有关，CRTAC1通过ROS信号途径介导UVB引起的细胞焦亡，UVB照射后升高的炎性因子IL-1β和IL-18的水平以及细胞焦亡相关蛋白的表达受到CRTAC1下调的抑制。过表达CRTAC1促进上述分子表达，并且通过ROS的抑制剂能够阻止CRTAC1的过表达作用。（3）阐明ASC-exo与CRTAC1的相互影响，ASC-exo通过上调miR-10a-5p的表达水平，下调CRTAC1的表达水平，从而减弱了UVB诱导的HLECs凋亡、ROS生成和Ca2+水平，提示ASC-exo可能成为治疗白内障的新的治疗手段。（4）阐明了TRIM69通过负向调节P53抑制白内障发生的作用机制。在白内障囊膜样本中TRIM69的表达下降，而过表达TRIM69可以通过诱导p53蛋白泛素化修饰缓解UVB损伤造成的HLECs细胞凋亡和ROS的释放。另外我们发现FoxO3a是TRIM69的潜在转录因子，并被UVB损伤激活，为后续研究白内障发机制提供了重要基础。