Tamm E R, Russell P, Johnson D H, Piatigorsky J
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Invest Ophthalmol Vis Sci. 1996 Nov;37(12):2402-13.
Oxidative stress and other forms of injury to trabecular meshwork (TM) cells may contribute to changes seen with age and primary open-angle glaucoma. This study was designed to investigate if TM expresses alpha B-crystallin, a small heat-shock protein with chaperone activity, and whether it might be overexpressed under stress conditions.
The TM from human and monkey eyes, as well as organ and primary cell cultures derived from these eyes, were investigated for alpha B-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultures were stressed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mumol for 1 hour). Semiquantitation of alpha B-crystallin messenger RNA (mRNA) or protein was obtained by densitometry.
In both species, alpha B-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochemistry of fresh samples showed that alpha B-crystallin was expressed predominantly in the cribriform area. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures from human TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of alpha B-crystallin mRNA in Northern blots. In monkey TM cultures, a 0.8-kb band was observed, which comigrated with lens alpha B-crystallin. In both species, heat shock caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in alpha B-crystallin mRNA also was observed after oxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observed. Western dot blot analysis showed a 3.4-fold increase in protein 24 hours after heat shock and a 20-fold increase after 48 hours. No constitutive mRNA expression and only a minimal increase 4 hours after heat shock could be observed in simian virus 40 transformed cell lines from human TM.
Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated with various stress conditions.
小梁网（TM）细胞的氧化应激及其他形式的损伤可能与年龄相关变化及原发性开角型青光眼有关。本研究旨在调查TM是否表达αB-晶状体蛋白（一种具有伴侣活性的小分子热休克蛋白），以及在应激条件下其表达是否会上调。
通过免疫组织化学、二维凝胶电泳、Northern印迹和Western印迹分析，研究人眼和猴眼的TM以及源自这些眼睛的器官和原代细胞培养物中的αB-晶状体蛋白。TM细胞培养物通过热休克（44℃，15分钟）或过氧化氢（200μmol，1小时）进行应激处理。通过光密度测定法对αB-晶状体蛋白信使核糖核酸（mRNA）或蛋白质进行半定量分析。
在两个物种中，通过二维凝胶电泳结合Western印迹分析，均可在新鲜和培养的TM中检测到αB-晶状体蛋白。新鲜样本的免疫组织化学显示，αB-晶状体蛋白主要在筛板区域表达。在4至7天的器官培养物中，蛋白质表达增强。人TM细胞的原代培养物在Northern印迹中表达两种大小（约0.8和1.1kb）的αB-晶状体蛋白mRNA。在猴TM培养物中，观察到一条0.8kb的条带，其与晶状体αB-晶状体蛋白迁移位置相同。在两个物种中，热休克均导致αB-晶状体蛋白mRNA显著增加，4小时后达到峰值。氧化应激后也观察到αB-晶状体蛋白mRNA增加；然而，mRNA诱导的起始较慢。热休克后，但氧化应激后未观察到，mRNA迁移率出现短暂变化。Western斑点印迹分析显示，热休克后24小时蛋白质增加3.4倍，48小时后增加20倍。在人TM的猿猴病毒40转化细胞系中，未观察到组成型mRNA表达，热休克4小时后仅观察到极小的增加。
αB-晶状体蛋白的过表达可能是TM预防与各种应激条件相关的细胞损伤的重要机制。
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