Andley U P, Song Z, Wawrousek E F, Bassnett S
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1998 Nov 20;273(47):31252-61. doi: 10.1074/jbc.273.47.31252.
alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction.
αA-晶体蛋白（αA）是小热休克蛋白（sHSP）家族的成员，具有在体外防止变性蛋白质聚集的能力。晶状体上皮细胞表达相对较低水平的αA，但在分化的纤维细胞中，αA是最丰富的可溶性蛋白质。αA基因敲除小鼠的晶状体在幼年时就会出现混浊，这意味着αA在维持纤维细胞透明度方面起着关键作用。然而，α-晶体蛋白在晶状体上皮中的功能尚不清楚。为了研究αA在晶状体上皮细胞中的生理功能，我们使用了以下两种系统：αA基因敲除（αA(-/-)）小鼠晶状体上皮细胞和过表达αA的人晶状体上皮细胞。与野生型细胞相比，αA(-/-)小鼠晶状体上皮细胞的生长速率降低了50%。通过对碘化丙啶染色细胞进行荧光激活细胞分选分析来测量细胞周期动力学，结果表明αA(-/-)细胞在G2/M期相对缺乏。将小鼠晶状体上皮细胞暴露于生理水平的紫外线A（UVA）会导致培养物中凋亡细胞数量增加。照射4小时后，αA(-/-)培养物中的凋亡细胞比例比野生型增加了40倍。在缺乏αA的细胞中，UVA暴露会改变F-肌动蛋白，但在表达αA的细胞中肌动蛋白受到保护。通过用哺乳动物表达载体构建体pCI-neoαA转染延长寿命的人晶状体上皮细胞，产生了稳定转染的过表达人αA的细胞系。通过克隆存活测定，过表达αA的细胞对UVA应激具有抗性。暴露于UVA或热应激后，αA仍保留在细胞质中，这表明与其他sHSP不同，αA的保护作用与其重新定位到细胞核无关。这些结果表明，αA在晶状体中除了其在折射方面已得到充分表征的作用外，还具有重要的细胞功能。
