Han Xiao, Wang Xin-Ling, Li Qin, Dong Xiao-Xuan, Zhang Jin-Song, Yan Qi-Chang
Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University; Key Laboratory of Lens Research of Liaoning Province, Eye Hospital of China Medical University, 11 Xinhua Road, Heping District, Shenyang, Liaoning Province, 110005, People's Republic of China.
Graefes Arch Clin Exp Ophthalmol. 2015 Aug;253(8):1279-90. doi: 10.1007/s00417-015-2999-x. Epub 2015 Apr 16.
High blood glucose can induce oxidative damage and result in diabetic cataract. Oxidative stress induces various signal pathways including HIF-1α transcriptional signal to attenuate the damage of lenses. Whether HIF-1α SUMOylation can increase the activation of HIF-1α or if high glucose can affect the SUMOylation of HIF-1α in cultured human lens epithelial cells (HLECs) is still unknown, as well as the function of HIF-1α SUMOylation in oxidative damage induced by high glucose in HLECs. In the present study, we examined SUMO and SUMO E3 (Cbx4 and PIASy) expression induced by high glucose, and investigated SUMO or SUMO E3 overexpression that enhanced HIF-1α SUMOylation in HLECs.
SRA01/04 cells, one kind of human lens epithelial cell line, were addressed in media with 5.5 mmol/l (normal control group), 25 mmol/l (high glucose1 group) and 50 mmol/l (high glucose2 group) final glucose respectively. Expression of SUMO1 ~ 4, Cbx4, PIASy, HIF-1α, GLUT1, and VEGFA were detected in the mRNA and protein levels by RT-PCR and Western blot analysis. Protein expression localization and co-localization were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO overexpression, SUMO E3 overexpression, and Proteasome inhibitor MG132 respectively on the stability and transcriptional activity of HIF-1α were analyzed by immunoblot.
High glucose treatment induced SUMO1-4 expression and enhanced the expression of Cbx4 and PIASy. It also increased the expression of HIF-1α, GLUT1, and VEGFA. The co-localization of HIF-1α and SUMO was mainly in the nucleus induced by high glucose. Further studies showed that SUMO overexpression or SUMO E3 overexpression could enhance HIF-1α stability and transcriptional activity in HLECs. Proteasome inhibitor MG132 protected the stability and transcriptional activity of HIF-1α in the SRA01/04 cells.
HIF-1α SUMOylation affected the stability and transcriptional activity of HIF-1α in cultured human lens epithelial cells; SUMO overexpression or SUMO E3 overexpression enhanced the expression of HIF-1α, which is involved in inhibiting cell apoptosis and protecting lens opacification, and presumably plays a key role in protecting lenses from diabetic cataract.
高血糖可诱导氧化损伤并导致糖尿病性白内障。氧化应激诱导包括HIF-1α转录信号在内的各种信号通路，以减轻晶状体的损伤。HIF-1α的SUMO化是否能增加HIF-1α的激活，或者高糖是否能影响培养的人晶状体上皮细胞（HLECs）中HIF-1α的SUMO化，以及HIF-1α SUMO化在高糖诱导的HLECs氧化损伤中的作用尚不清楚。在本研究中，我们检测了高糖诱导的SUMO和SUMO E3（Cbx4和PIASy）表达，并研究了SUMO或SUMO E3过表达增强HLECs中HIF-1α SUMO化的情况。
将一种人晶状体上皮细胞系SRA01/04细胞分别置于终末葡萄糖浓度为5.5 mmol/l（正常对照组）、25 mmol/l（高糖1组）和50 mmol/l（高糖2组）的培养基中。通过RT-PCR和蛋白质印迹分析检测SUMO1~4、Cbx4、PIASy、HIF-1α、GLUT1和VEGFA在mRNA和蛋白质水平的表达。通过免疫荧光和共免疫荧光检测蛋白质表达定位和共定位。通过免疫印迹分析SUMO过表达、SUMO E3过表达和蛋白酶体抑制剂MG132分别对HIF-1α稳定性和转录活性的影响。
高糖处理诱导SUMO1-4表达，增强Cbx4和PIASy的表达。它还增加了HIF-1α、GLUT1和VEGFA的表达。高糖诱导下，HIF-1α与SUMO的共定位主要在细胞核中。进一步研究表明，SUMO过表达或SUMO E3过表达可增强HLECs中HIF-1α的稳定性和转录活性。蛋白酶体抑制剂MG132保护SRA01/04细胞中HIF-1α的稳定性和转录活性。
HIF-1α SUMO化影响培养的人晶状体上皮细胞中HIF-1α的稳定性和转录活性；SUMO过表达或SUMO E3过表达增强了HIF-1α的表达，这与抑制细胞凋亡和保护晶状体混浊有关，可能在保护晶状体免受糖尿病性白内障方面起关键作用。
