Subcellular distribution of native estrogen receptor alpha and beta subtypes in cultured human lens epithelial cells

Abstract

The expression and intracellular localization of the estrogen receptor (ER) subtypes, ERalpha and ERbeta were examined in cultured human lens epithelial cells (HLE-B3). ERalpha and ERbeta mRNA expression was evaluated by coupled reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. Subcellular localization of ERalpha and ERbeta was determined on formaldehyde-fixed, Saponin-permeabilized cells using conventional immunofluorescence techniques, as well as immunodetection of differential cellular components after subjecting the cultured cells to fractionation by sucrose gradient centrifugation. Using RT-PCR, ER species specific 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 286-bp (ERalpha) and 167-bp (ERbeta) PCR products were verified by sequence analysis. Southern blot analysis using internal oligonucleotides directed to specific primer pairs for ERalpha and ERbeta, respectively, further confirmed the authenticity of the PCR products. HLE-B3 cells expressed ERalpha and ERbeta in association with the nucleus and ERbeta in the mitochondria. That the mitochondrial-enriched subfraction correlated with the presence of the ERbeta subtype was confirmed by Western blot analysis. The differential subcellular partitioning of ERalpha and ERbeta subtypes lends a new aspect to the estrogen signalling system wherein mitochondrial stabilization, possibly through ER binding, may play a causal role in the maintenance of cellular integrity. The occurrence of ER subtypes in human lens epithelial cells suggests that estrogen plays a role in the physiology of the lens. The comparative nuclear and mitochondrial distribution of ERalpha and ERbeta raises the interesting probability that the two receptors, dependent upon subcellular localization, may have differential cytoprotective potential, and by inference, disparity in their mechanisms of action.