Expression and regulation of α-, β-, and γ-crystallins in mammalian lens epithelial cells
Expression and regulation of α-, β-, and γ-crystallins in mammalian lens epithelial cells
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DOI：
10.1167/iovs.04-0423
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发表时间：
2004-10-01
影响因子：
4.4
通讯作者：
Beebe, DC
中科院分区：
文献类型：
--
作者：
Wang, XH;Garcia, CM;Beebe, DC
目的。在哺乳动物晶状体中，β -和γ -晶体蛋白家族的表达被认为仅限于纤维细胞。然而，一些研究在人晶状体上皮细胞中检测到这些蛋白或其mrna。为了解决这种明显的差异，我们检测了14种晶体蛋白mrna，并测定了晶体蛋白在晶状体上皮细胞中的表达和亚细胞分布。利用P3大鼠晶状体上皮在基础培养基或添加FGF2中培养0或20小时或4或7天的mRNA，采用实时荧光定量PCR分析转录产物水平。使用β - ab1 -、γ -、α -和α -晶体蛋白抗体对成年小鼠、人、牛、兔和大鼠晶状体上皮细胞和纤维细胞中提取的蛋白进行Western blot分析。将大鼠晶状体或晶状体上皮快速固定在原位、死后30分钟或从晶状体上剥离后，用免疫染色和共聚焦显微镜观察晶体蛋白在细胞内的分布。在培养的晶状体上皮中检测到晶状体蛋白基因的四种表达模式。编码大多数β -和-结晶蛋白的转录本是可检测的，在某些情况下，在外植体时大量存在。在基础或fgf补充培养基中培养的P3上皮中，晶体蛋白水平的变化通常反映了其mrna的变化。成人、小鼠、大鼠、兔和牛晶状体上皮细胞中含有丰富的β - ab1 -和γ -晶体蛋白。在成人晶状体上皮细胞的不同亚细胞位置发现α -、β -和γ -晶体蛋白。这些蛋白在纤维细胞分化过程中以及晶状体上皮死亡和/或剥离后显著地重新定位。β ab1 -和γ -晶体蛋白在成年哺乳动物晶状体上皮细胞中通常含量丰富。复杂的转录和降解程序调节晶状体上皮细胞在应激后、不同年龄和细胞分化过程中晶状体上皮细胞中晶体蛋白mrna的积累。由于晶体蛋白在分化或应激过程中选择性地定位于不同的亚细胞区室，它们可能起保护晶状体细胞免受损伤的作用。应激后，大多数α -和α -结晶蛋白亚基不在同一个大分子复合物中。
PURPOSE. In the mammalian lens, the expression of the beta- and gamma-crystallin families is thought to be limited to fiber cells. However, several studies detected these proteins or their mRNAs in human lens epithelial cells. To resolve this apparent discrepancy, 14 crystallin mRNAs were examined and the expression and subcellular distribution of selected crystallin proteins in lens epithelial cells determined.METHODS. Transcript levels were analyzed by quantitative realtime PCR using mRNA from P3 rat lens epithelia cultured for 0 or 20 hours or 4 or 7 days in basal medium or with added FGF2. Antibodies to betaB1-, gammaS-, alphaA-, and alphaB-crystallins were used for Western blot analysis of proteins extracted from adult mouse, human, bovine, rabbit, and rat lens epithelial and fiber cells. Rat lenses or lens epithelia were rapidly fixed in situ, 30 minutes after death, or after dissection from the lens, and the intracellular distributions of crystallins were examined by immunostaining and confocal microscopy.RESULTS. Four patterns of crystallin gene expression were detected in cultured lens epithelia. Transcripts encoding most beta- and gamma-crystallins were detectable and, in some cases, abundant at the time of explantation. Changes in crystallin protein levels in P3 epithelia cultured in basal or FGF-supplemented medium generally reflected the changes in their mRNAs. betaB1- and gammaS-crystallins were abundant in adult human, mouse, rat, rabbit, and bovine lens epithelial cells. The alpha-, beta- and gamma-crystallins were found in distinct subcellular locations in adult lens epithelial cells. These proteins dramatically relocalized during fiber cell differentiation and after death and/or dissection of the lens epithelium.CONCLUSIONS. betaB1- and gammaS-crystallins are normally abundant in adult mammalian lens epithelial cells. Complex programs of transcription and degradation regulate the accumulation of crystallin mRNAs in lens epithelial cells after stress, at different ages, and during cell differentiation. Because crystallins selectively localize in distinct subcellular compartments during differentiation or stress, they may function to protect lens cells from injury. After stress, most alphaA- and alphaB-crystallin subunits are not in the same macromolecular complexes.