Andley U P, Song Z, Wawrousek E F, Fleming T P, Bassnett S
Departments of Ophthalmology and Visual Sciences, Biochemistry & Molecular Biophysics, Genetics, and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2000 Nov 24;275(47):36823-31. doi: 10.1074/jbc.M004233200.
alphaA- and alphaB-crystallins are molecular chaperones expressed at low levels in lens epithelial cells, and their expression increases dramatically during differentiation to lens fibers. However, the functions of alphaA- and alphaB-crystallins in lens epithelial cells have not been studied in detail. In this study, the relative ability of alphaA- and alphaB-crystallin, in protecting lens epithelial cells from apoptotic cell death was determined. The introduction of alphaA-crystallin in the transformed human lens epithelial (HLE) B-3 lens epithelial cell line (which expresses low endogenous levels of alphaB-crystallin) led to a nearly complete protection of cell death induced by staurosporine, Fas monoclonal antibody, or the cytokine tumor necrosis factor alpha. To further study the relative protective activities of alphaA- and alphaB-crystallins, we created a cell line derived from alphaA-/-alphaB-/- double knockout mouse lens epithelia by infecting primary cells with Ad12-SV40 hybrid virus. The transformed cell line alphaAalphaBKO1 derived from alphaA/alphaB double knockout cells was transfected with alphaA- or alphaB-crystallin cDNA contained in pCIneo mammalian expression vector. Cells expressing different amounts of either alphaA-crystallin or alphaB-crystallin were isolated. The ability of alphaA- or alphaB-crystallin to confer protection from apoptotic cell death was determined by annexin labeling and flow cytometry of staurosporine- or UVA- treated cells. The results indicate that the anti-apoptotic activity of alphaA-crystallin was two to three-fold higher than that of alphaB-crystallin. Our work suggests that comparing the in vitro annexin labeling of lens epithelial cells is an effective way to measure the protective activity of alphaA- and alphaB-crystallin. Since the expression of alphaA-crystallin is largely restricted to the lens, its greater protective effect against apoptosis suggests that it may play a significant role in protecting lens epithelial cells from stress.
αA-晶状体蛋白和αB-晶状体蛋白是在晶状体上皮细胞中低水平表达的分子伴侣，在向晶状体纤维细胞分化过程中其表达显著增加。然而，αA-晶状体蛋白和αB-晶状体蛋白在晶状体上皮细胞中的功能尚未得到详细研究。在本研究中，测定了αA-晶状体蛋白和αB-晶状体蛋白保护晶状体上皮细胞免于凋亡性细胞死亡的相对能力。在转化的人晶状体上皮（HLE）B-3细胞系（其αB-晶状体蛋白内源性表达水平低）中引入αA-晶状体蛋白，导致对由星形孢菌素、Fas单克隆抗体或细胞因子肿瘤坏死因子α诱导的细胞死亡几乎完全保护。为了进一步研究αA-晶状体蛋白和αB-晶状体蛋白的相对保护活性，我们通过用Ad12-SV40杂交病毒感染原代细胞，创建了一种源自αA-/-αB-/-双敲除小鼠晶状体上皮的细胞系。用包含在pCIneo哺乳动物表达载体中的αA-或αB-晶状体蛋白cDNA转染源自αA/αB双敲除细胞的转化细胞系αAαBKOI。分离出表达不同量αA-晶状体蛋白或αB-晶状体蛋白的细胞。通过膜联蛋白标记和对经星形孢菌素或紫外线A处理的细胞进行流式细胞术，测定αA-或αB-晶状体蛋白赋予免于凋亡性细胞死亡的保护能力。结果表明，αA-晶状体蛋白的抗凋亡活性比αB-晶状体蛋白高两到三倍。我们的工作表明，比较晶状体上皮细胞的体外膜联蛋白标记是测量αA-晶状体蛋白和αB-晶状体蛋白保护活性的有效方法。由于αA-晶状体蛋白的表达在很大程度上局限于晶状体，其对凋亡的更大保护作用表明它可能在保护晶状体上皮细胞免受应激方面发挥重要作用。
