Abstract
Purpose::
Wild type estrogen receptor beta (wtER-ß1) and its splice variants (ER-ß2-5) coexist in human lens, as well as in cultured SV-40 transformed human lens epithelial cells (HLE-B3) (Exp Eye Res. 81:165-175; 2005). 17-beta estradiol (E2) modulates the degree of oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in HLE-B3 cells, following H2O2 insult, by activation of mitogen-activated protein kinase (MAPK) (Mitochondrion 5:235-247; 2005). This study resolved whether gender played a role in the protection mechanism based upon differences in ER-ß isoform expression, receptor localization in mitochondria and response to estrogen-mediated mitochondrial protection against oxidative stress employing cultured populations of normal male and female human lens epithelial (nHLE) cells.
Methods::
: nHLE cell cultures were prepared from explants of post-mortum male and female donors across a wide age distribution. A triple primer PCR assay (Exp Eye Res. 81:165-175; 2005) was used to determine the proportional distribution of the receptor isoforms (wtER-ß1, ß2 and ß5) from the total ER-ß message pool in male and female cell cultures. Subcellular localization of ERß isoforms was determined using conventional immunofluorescence techniques and affinity purified polyclonal antibodies specific for wtER-ß1, ß2 and ß5. To examine changes in MMP, the potentiometric fluorescent compound, JC-1, was used after cell cultures were exposed to peroxide ± pretreatment with E2.
Results::
Male and female nHLE cells express wtER-ß1, ER-ß2 and ER-ß5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in both the peripheral mitochondrial arrays and perinuclear mitochondria (along with weaker internal nuclear staining) of both male and female nHLE cells. The ER-ß2 and ER-ß5 isoforms were distributed in the cytosol; no association with the mitochondria was detected. Both male and female nHLE cells treated with E2 (1µM) showed similar levels of protection against oxidative stress.
Conclusions::
While we have yet to establish whether wtER-ß1 (in mitochondria) plays a definitive role in the E2-mediated mitochondrial protection mechanism, these observations establish that prevention of depolarization of the MMP must be regarded as gender-independent.
Keywords: receptors • mitochondria • antioxidants