Cammarata Patrick R, Flynn James, Gottipati Srinivas, Chu Shaoyou, Dimitrijevich Slobadan, Younes Mamoun, Skliris George, Murphy Leigh C
Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Exp Eye Res. 2005 Aug;81(2):165-75. doi: 10.1016/j.exer.2005.01.019.
A number of variants of the wild-type (wt) estrogen receptor beta (ERbeta-1) coexist in a wide range of tissues. In the human these include, together with others, the expression of several isoforms (ERbeta-2-ERbeta-5) due to alternative splicing of exons encoding the carboxy terminus. In this study, we determined whether virally transformed cell cultures of human lens epithelial cells (HLE-B3) express both full length (or wt) and variant isoforms of ERbeta in comparison to normal secondary cultures of human lens epithelial cells (nHLE) and furthermore, identify the subcellular localization of the wtERbeta-1 and ERbeta isoform variants in HLE-B3 and nHLE cells, as well as from human breast adenocarcinoma cells (MCF-7) which provided a positive control. ERbeta isoform mRNA expression was evaluated by coupled RT-PCR. Subcellular localization of ERbeta isoforms was determined on formaldehyde-fixed, Saponin-permeabilized cells using conventional immunofluorescence techniques and affinity purified polyclonal antibodies specific for ERbeta-1 as well as to two of the truncated carboxy terminus isoforms (beta-2 and beta-5). Total RNA was extracted from HLE-B3 and nHLE cells and lens tissue, as well as from human breast adenocarcinoma cells (MCF-7) and subjected to RT-PCR using specific estrogen receptor primers intended to distinguish ERbeta-1-ERbeta-5 mRNA. The PCR products corresponded to wtERbeta-1 as well as to the isoform variants beta-2 and beta-5. The proportional distribution of wtERbeta-1, beta-2 and beta-5 PCR products differed between the normal lens epithelial cells and the SV-40 transformed lens epithelial cell line; the nHLE being similar to lens tissue with respect to relative expression of ERbeta isoform cDNAs. Confocal microscopy and immunofluorescence revealed ERbeta-2 was distributed throughout the cytosol and was associated with the nucleus of all cells examined, although sporadic immunostaining was observed with the nuclei of MCF-7. Prominent immunostaining of ERbeta-1 appeared in the mitochondria (along with weaker staining in the nucleus) of all cell types as authenticated by co-localization with Mitotrack-633. ERbeta-5 immunostaining was diffuse in the cytosol and also associated with the nuclei of all cell types. The differential subcellular partitioning of ERbeta-1 to the mitochondria and ERbeta-2 to the nucleus suggests a new aspect of regulation and function of the estrogen signalling system.
野生型（wt）雌激素受体β（ERβ-1）的多种变体共存于多种组织中。在人类中，除其他变体外，由于编码羧基末端的外显子发生选择性剪接，还存在几种异构体（ERβ-2至ERβ-5）的表达。在本研究中，我们比较了人晶状体上皮细胞（HLE-B3）的病毒转化细胞培养物与正常人晶状体上皮细胞（nHLE）的二次培养物中是否表达全长（或wt）和ERβ异构体变体，此外，还确定了wtERβ-1和ERβ异构体变体在HLE-B3和nHLE细胞中的亚细胞定位，以及来自人乳腺腺癌细胞（MCF-7）的亚细胞定位，后者作为阳性对照。通过偶联RT-PCR评估ERβ异构体mRNA的表达。使用常规免疫荧光技术和针对ERβ-1以及两种截短的羧基末端异构体（β-2和β-5）的亲和纯化多克隆抗体，在甲醛固定、皂素通透的细胞上确定ERβ异构体的亚细胞定位。从HLE-B3和nHLE细胞、晶状体组织以及人乳腺腺癌细胞（MCF-7）中提取总RNA，并使用旨在区分ERβ-1至ERβ-5 mRNA的特异性雌激素受体引物进行RT-PCR。PCR产物对应于wtERβ-1以及异构体变体β-2和β-5。wtERβ-1、β-2和β-5 PCR产物的比例分布在正常晶状体上皮细胞和SV-40转化的晶状体上皮细胞系之间有所不同；nHLE在ERβ异构体cDNA的相对表达方面与晶状体组织相似。共聚焦显微镜和免疫荧光显示，ERβ-2分布于所有检测细胞的整个细胞质中，并与细胞核相关，尽管在MCF-7细胞核中观察到散在的免疫染色。通过与Mitotrack-633共定位证实，ERβ-1在所有细胞类型的线粒体中均有明显免疫染色（细胞核中染色较弱）。ERβ-5免疫染色在细胞质中呈弥漫性，也与所有细胞类型的细胞核相关。ERβ-1在线粒体中的差异亚细胞分布以及ERβ-2在细胞核中的差异亚细胞分布提示了雌激素信号系统调控和功能的一个新方面。
