低氧诱导因子1α（HIF-1α）的SUMO化修饰调控对髓核细胞氧张力耐受的影响及机制研究

Hypoxia inducible factor-1α (HIF-1α) is constitutively expressed in nucleus pulposus (NP) cells and shows a characteristic of normoxic stabilization, which indicates a metabolic adaptation to the intervertebral disc (IVD) microenvironment. Post-translational modification by members of small ubiquition-related modifier (SUMO) family regulates the stability and transactivation of HIF-1α， but the regulatory mechanism of the SUMO pathway has not been fully studied in NP cells. In this study rat NP cells will be cultured under hypoxic (2%O2) or normoxic (21%O2) conditions, and expressions of SUMO E1 activating enzymes, E2 conjugating enzymes, as well as sentrin-specific proteases (SENPs) will be evaluated. We assume that NP cells probably shift the balance between sumoylation and de-sumoylation in response to varied oxygen tensions. For modulating the SUMO pathway in NP cells, SENP1 specific siRNA and pCDNA/SENP1 plasmid are created. Gain- and loss-of-function experiments will be performed to investigate the relationship between HIF-1α sumoylation, HIF-1α protein stability, and the transcriptional activity of HIF-1α target genes e.g. VEGF, Glut-1. It is probable that NP cells stabilize the expression of HIF-1α by modulating the SUMO pathways, maintain a reasonable transcriptional activity of its downstream targets, thus gaining the tolerance to the local oxemic state. Our study will for the first time support the viewpoint that SUMO signaling is critical for the post-translational modification of HIF-1α in NP cells, suggesting any impairment of the sumoylation or de-sumoylation mechanism is potentially involved in the pathogenesis of IVD degeneration. By identifying the key element in the SUMO pathways, our study will also support the feasibility of modulating the SUMO signaling for IVD regeneration.

髓核细胞具有组成性表达低氧诱导因子-1α（HIF-1α）并耐受氧张力波动的特性。蛋白水平的类泛素（SUMO）化修饰机制与HIF-1α的转录后稳定及生物学效应密切相关。本研究观察正常氧与低氧状态下髓核细胞内SUMO化E1激活酶、E2连接酶和SUMO特异性蛋白酶的表达差异，探讨氧张力波动环境中髓核细胞SUMO化修饰活性的变化规律。继而强化或抑制髓核细胞SUMO化信号，观察HIF-1α的SUMO化水平与HIF-1α蛋白稳定性之间的量效关系，并结合HIF-1α下游信号Glut-1、VEGF的激活水平，综合评价SUMO化修饰调控对髓核细胞应答氧张力波动能力的影响。从"细胞调控SUMO化活性→维持HIF-1α蛋白稳定→适应氧张力波动"角度阐明髓核细胞对局部氧浓度变化的应答与耐受机制，为理解椎间盘退行性变的细胞学病因，以及通过干预蛋白SUMO化修饰信号来防治椎间盘退变打开崭新思路。

椎间盘退变的生物学修复依赖深入认识椎间盘内的低氧微环境。纤维环细胞诱导性表达HIF-1α,髓核细胞稳定表达并激活HIF-1α,两者如何及为何差异性的调控HIF-1α仍无定论。类泛素化修饰(sumoylation)信号的发现为深入认识蛋白翻译后的精细调控开辟崭新道路。SUMO化修饰后的HIF-1α稳定性下降。本课题假说SUMO修饰信号可能参与椎间盘细胞内HIF-1α的精细调控。我们发现. （1）椎间盘从胚胎发育到衰老退变的整个自然史中髓核与纤维环细胞都与循环系统隔离，提示髓核与纤维环细胞对HIF-1α的差异性调控可能不归因于对低氧环境的后天性适应。（2）在大鼠髓核与纤维环细胞内SUMO-1和SUMO-2/3，SUMO化修饰E1激活酶SAE1和SAE2，E2连接酶UBC9，去SUMO化关键酶SENP1主要在细胞核内表达。低氧时髓核细胞一过性上调SUMO-1、SUMO-2/3、SAE2、UBC9,纤维环细胞上调SUMO-1，下调SUMO-2/3、SAE1、SAE2、UBC9，而SENP1在髓核与纤维环细胞内都上调,提示低氧环境下髓核细胞倾向于维持SUMO化修饰的整体平衡，而纤维环细胞倾向于强化去SUMO化修饰。（3）髓核细胞内沉默SENP1后HIF-1α蛋白表达下降，HIF-1α的mRNA表达下降，HIF-1α下游靶基因VEGF、GLUT-1的转录激活下降，维持细胞低代谢的PDK1表达下降，提示SENP1在转录及蛋白水平都可能参与调控髓核细胞内HIF-1α。但SUMO-1与HIF-1α的无明显结合，而髓核与纤维环细胞内SUMO-1都与RanGAP1大量结合，提示椎间盘细胞内SUMO分子直接作用于HIF-1α蛋白的效应微弱，可能主要通过SUMO化修饰其他信号（如PHD、NFkB等）间接调控HIF-1α转录。（4）髓核与纤维环细胞在低氧环境下细胞增殖、周期分布、凋亡、老化等活性指标无明显丢失，在SENP1和HIF-1α下调的髓核细胞内低氧并未诱导显著的凋亡，提示髓核与纤维环细胞差异性调控HIF-1α的目的可能不是适应性的在低氧环境下维持细胞活力。. SUMO化修饰信号参与调控椎间盘细胞内HIF-1α的表达与转录激活,且低氧环境下髓核与纤维环细胞具有各自独特的SUMO化信号调控及HIF-1α效应模式，因而为从翻译后修饰角度理解椎间盘退变的分子信号基础打开崭新思路。