James Eric R, Robertson Lorie, Ehlert Erich, Fitzgerald Patrick, Droin Nathalie, Green Douglas R
Department of Ophthalmology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.
Invest Ophthalmol Vis Sci. 2003 Dec;44(12):5269-76. doi: 10.1167/iovs.03-0401.
The purpose of this study was to determine whether lens epithelial cells (LECs) contain a glucocorticoid receptor (GR) that is transcriptionally active and that is able to induce production of known glucocorticoid-inducible proteins.
Protein and mRNA were obtained from human, rabbit, and bovine lens epithelia and from cultured human lens epithelial cells (B3, hLECs) and rabbit lens epithelial cells (N/N1003A, rLECs). Paraffin-embedded sections were prepared from human lenses for immunohistochemical localization of GR. RT-PCR was performed to amplify portions of GR, and the products were sequenced. Protein samples were analyzed by Western blot. hLECs and rLECs were transfected with pTAT3-luc and assayed for luciferase activity after treatment with dexamethasone (Dex) and/or RU486. Dex-treated LECs were also analyzed by quantitative real-time PCR and by Western blot for expression of specific mRNA and proteins.
By PCR and sequencing, products consistent with GR sequences were obtained from human, rabbit, and bovine lenses and from hLECs and rLECs. The complete GRalpha sequence was obtained from rLECs and was found to be 89% identical with human GR. A 1757-bp 3' fragment of bovine GRalpha cDNA was also amplified from bovine lens. By Western blot, bands of approximately 94 kDa, the expected size of GR, were identified from human, rabbit, and bovine lens samples and from hLECs and rLECs, using anti-GR antibodies. Anti-GR antisera localized GR to both the cytosol of anterior and bow region LECs and to the nuclei of epithelial and early-differentiating lens fiber cells. Luciferase expression was induced in pTAT3-luc-transfected rLECs and hLECs by Dex treatment and this expression was partially (rLECs) or completely (hLECs) blocked by pretreatment with RU486. mRNA levels for type-1 glucocorticoid-induced target genes and also mRNA and protein levels for type-2 genes were upregulated after Dex exposure.
The data confirm the existence of GR in hLECs, indicate that GR is present in rLECs, and resolve the controversy over the presence of GR in bovine lens. The GRalpha in hLECs and rLECs was shown to be transcriptionally active and the expression levels in hLECs of mRNAs and proteins known to be regulated by glucocorticoids were modified in these cells by glucocorticoid treatment.
本研究旨在确定晶状体上皮细胞（LEC）是否含有具有转录活性且能够诱导已知糖皮质激素诱导蛋白产生的糖皮质激素受体（GR）。
从人、兔和牛的晶状体上皮以及培养的人晶状体上皮细胞（B3、hLEC）和兔晶状体上皮细胞（N/N1003A、rLEC）中获取蛋白质和mRNA。从人晶状体制备石蜡包埋切片用于GR的免疫组织化学定位。进行RT-PCR扩增GR的部分片段，并对产物进行测序。蛋白质样品通过蛋白质印迹法分析。用pTAT3-luc转染hLEC和rLEC，用地塞米松（Dex）和/或RU486处理后检测荧光素酶活性。对Dex处理的LEC也通过定量实时PCR和蛋白质印迹法分析特定mRNA和蛋白质的表达。
通过PCR和测序，从人、兔和牛的晶状体以及hLEC和rLEC中获得了与GR序列一致的产物。从rLEC中获得了完整的GRα序列，发现其与人GR的同源性为89%。还从牛晶状体中扩增出了牛GRα cDNA的1757 bp 3'片段。通过蛋白质印迹法，使用抗GR抗体从人、兔和牛的晶状体样品以及hLEC和rLEC中鉴定出了预期大小约为94 kDa的GR条带。抗GR抗血清将GR定位到前房和弓形区域LEC的细胞质以及上皮和早期分化的晶状体纤维细胞的细胞核中。Dex处理可诱导pTAT3-luc转染的rLEC和hLEC中的荧光素酶表达，且这种表达被RU486预处理部分（rLEC）或完全（hLEC）阻断。Dex暴露后，1型糖皮质激素诱导的靶基因的mRNA水平以及2型基因的mRNA和蛋白质水平均上调。
数据证实hLEC中存在GR，表明rLEC中存在GR，并解决了关于牛晶状体中GR存在与否的争议。已证明hLEC和rLEC中的GRα具有转录活性，糖皮质激素处理可改变这些细胞中已知受糖皮质激素调节的mRNA和蛋白质在hLEC中的表达水平。
