Gottipati Srinivas, Cammarata Patrick R
Department of Cell Biology and Genetics, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, USA.
Mol Vis. 2008 May 16;14:898-905.
17 beta-estradiol (17beta-E(2)) protects human lens epithelial cells against oxidative stress by preserving mitochondrial function in part via the non-genomic rapid activation of prosurvival signal transduction pathways. The study described herein examined whether 17beta-E(2) also elicits genomic protection by influencing the expression (and activity) of mitochondrial-associated manganese superoxide dismutase (MnSOD) as a possible parallel mechanism by which 17beta-E(2) protects against oxidative stress.
Virally-transformed human lens epithelial cells (HLE-B3) were pre-incubated with 17beta-E(2), and mRNA or protein lysates were collected over a time course ranging from 90 min to 24 h. Positive expression of lens epithelial cell MnSOD mRNA was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and its levels were monitored by real-time PCR up to 24 h after 17beta-E(2) administration. Western blot analysis was used to examine the pattern of protein expression as influenced by 17beta-E(2) treatment. MnSOD activity as influenced by 17beta-E(2) was determined by measuring enzymatic activity.
A significant rapid increase in the activity of MnSOD was observed with HLE-B3 cells by 90 min post-bolus addition of 17beta-E(2), which returned to control level by 240 min. Neither an increase in MnSOD mRNA nor in protein expression was detected up through 24 h.
These data demonstrate that 17beta-E(2) rapidly and transiently increases the activity of MnSOD but influences neither its mRNA expression nor its protein expression. The results suggest that (estrogen-activated) MnSOD plays an important role against mitochondrial oxidative stress by diminishing reactive oxygen species, thus promoting cell survival.
17β-雌二醇（17β-E₂）通过部分保留线粒体功能，经由促生存信号转导途径的非基因组快速激活，保护人晶状体上皮细胞免受氧化应激。本文所述研究检测了17β-E₂是否也通过影响线粒体相关锰超氧化物歧化酶（MnSOD）的表达（及活性）引发基因组保护，这可能是17β-E₂抵御氧化应激的一种并行机制。
将病毒转化的人晶状体上皮细胞（HLE-B3）与17β-E₂预孵育，在90分钟至24小时的时间范围内收集mRNA或蛋白质裂解物。通过半定量逆转录聚合酶链反应（RT-PCR）测定晶状体上皮细胞MnSOD mRNA的阳性表达，并在给予17β-E₂后24小时内通过实时PCR监测其水平。蛋白质印迹分析用于检测受17β-E₂处理影响的蛋白质表达模式。通过测量酶活性来确定受17β-E₂影响的MnSOD活性。
在向HLE-B3细胞推注添加17β-E₂后90分钟，观察到MnSOD活性显著快速增加，到240分钟时恢复到对照水平。在24小时内均未检测到MnSOD mRNA或蛋白质表达增加。
这些数据表明，17β-E₂迅速且短暂地增加MnSOD的活性，但不影响其mRNA表达或蛋白质表达。结果表明，（雌激素激活的）MnSOD通过减少活性氧对线粒体氧化应激起重要作用，从而促进细胞存活。
