Subcellular Distribution of Native Estrogen Receptor beta Isoform Variants in Virally–transformed and Normal Human Lens Epithelial Cells in Culture.

Abstract

Abstract: : Purpose: A number of variants of the wild type (WT) estrogen receptor beta (ERß1) coexist in a wide range of tissues. In the human these include, together with others, the expression of several isoforms (ERß2–ERß5) 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 WT and variant isoforms of ERß and, furthermore, identify the subcellular localization of the WT ERß1 and ERß isoform variants in HLE–B3 and normal human lens epithelial cells (nHLE). Methods:ERß isoform mRNA expression was evaluated by coupled RT–PCR. Subcellular localization of ERß isoforms was determined on formaldehyde–fixed, Saponin–permeabilized cells using conventional immunofluorescence techniques and affinity purified polyclonal antibodies specific for ERß1 as well as to two of the carboxy terminus isoforms (ß2 and ß5). Results:Using RT–PCR, specific estrogen receptor primers distinguished mRNA from total RNA extracted from HLE–B3 cells, as well as from human breast adenocarcinoma cells (MCF–7), which provided a positive control. The PCR products corresponded to wild type ERß1 as well as to the isoform variants ß2 and ß5. Confocal microscopy and immunofluorescence revealed ERß2 was associated exclusively with the nucleus in both HLE–B3 and nHLE cell cultures. Prominent immunostaining of ERß1 appeared in the mitochondria (along with weaker staining in the nucleus) of both HLE–B3 and nHLE cells as authenticated by colocalization with Mitotrack–633. ERß5 immunostaining was equivalent to background controls. Conclusions:The differential subcellular partitioning of ERß1 and ERß2 isoforms lends a new aspect to the estrogen signaling system. We speculate that one action of estrogen might be to shuttle WT ERß1 (which retains an intact ligand binding domain or LBD) to the mitochondria or bend the receptor into a conformation allowing it to bind to the mitochondrial membrane. The ERß2 variant, which has no functional LBD, but does retain a normal DNA binding domain, associates with the nucleus and does so without the need of estrogen binding. The comparative mitochondrial and nuclear distribution of ERß1 and ERß2 raises the probability that the WT receptor and variant isoform may have differential cytoprotective potential, and by inference, disparity in their mode of interaction, the former being primarily non–genomic and the latter being genomic.

Keywords: receptors: pharmacology/physiology • mitochondria • antioxidants