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Am J Physiol Endocrinol Metab 295: E637-E647, 2008. First published June 24, 2008; doi:10.1152/ajpendo.90407.2008
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Role of wild-type estrogen receptor-β in mitochondrial cytoprotection of cultured normal male and female human lens epithelial cells

J. M. Flynn,1 S. D. Dimitrijevich,2 M. Younes,3 G. Skliris,4 L. C. Murphy,4 and P. R. Cammarata1

1Departments of Cell Biology and Genetics and 2Integrative Physiology, University of North Texas Health Science Center, Fort Worth; 3Department of Pathology, Baylor College of Medicine, Houston, Texas; and 4Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 30 April 2008 ; accepted in final form 19 June 2008


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISSCUSION
 REFERENCES
 
The influence of sexual category as a modifier of cellular function is underinvestigated. Whether sex differences affect estrogen-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from postmortem male and female donors. Experimental indicators assessed included differences in estrogen receptor-β (ERβ) isoform expression, receptor localization in mitochondria, and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound JC-1 after nHLE were exposed to peroxide. The impact of wild-type ERβ (wtERβ1) was also assessed using wtERβ1 siRNA to suppress expression. A triple-primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtERβ1, -β2, and -β5) from the total ERβ message pool in male and female cell cultures. Irrespective of sex, nHLE express wtERβ1 and the ERβ2 and ERβ5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria as well as nuclear staining in both cell populations. The ERβ2 and ERβ5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with E2 (1 µM) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtERβ1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of E2. Thus, whereas the estrogen-mediated prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtERβ1 dependent.

THE ACTION OF ESTROGEN AND ESTROGEN RECEPTORS (ERs) is generally regarded to function via the activation of genes through specific estrogen promoters. In recent years, a number of nongenomic actions of estrogen that have been shown to affect numerous tissues in the body have been defined (25, 39, 40, 45). Particularly relevant to the studies described herein, estrogen has been shown to protect cells against oxidative stress through the rapid and nongenomic activation of prosurvival signaling pathways without the necessity of initiation of genes through nuclear translocation of the receptor (21, 43). In 1996, the sequence of a new ER was reported, and since then, ERβ has provided new insights into the actions of estrogen (34).

We recently reported on the expression and subcellular distribution of both ER{alpha} and ERβ in SV-40-transformed cultured human lens epithelial cells (8). In a related study (9), we used immunofluorescence and affinity-purified antisera to demonstrate that the full-length (i.e., wild-type) ERβ isoform is localized in both the mitochondria and nucleus of lens cells, whereas other isoforms, lacking the ligand-binding domain but retaining the DNA binding domain, fail to shuttle and localize to the mitochondria. The wild-type (wt)ERβ1 has been reported to have cardioprotective capabilities (35).

Loss of mitochondrial membrane potential results in the release of apoptotic factors as well as prevention of mitochondrial energy production. The process of mitochondrial permeability transition is mediated by the opening of the permeability transition pore (1, 20, 42). The mitochondrial permeability transition pore is formed from the coupling of adenosine nucleotide translocase and voltage-dependent anion channel on the inner and outer mitochondrial membranes, respectively. Numerous other laboratories aside from our own (8, 9) have shown that ERs are localized to the mitochondria (11, 22, 25, 41, 45).

Estrogen itself may also have antioxidant function because of its chemical structure (33). A number of plant-derived compounds of similar structure to estrogen, the phytoestrogens, have been shown to act as antioxidants (7). 17β-Estradiol (E2) modulates the degree of oxidative stress-induced depolarization of mitochondrial membrane potential in human lens epithelial (HLE)-B3 cells, following H2O2 insult, by activation of mitogen-activated protein kinase (17). We propose that estrogens, which behave as potent biologically active and selective mitochondrial protective compounds, may influence mitochondrial function through interaction with wtERβ.

The study described herein examined the role of sex differences in the mechanism of estrogen-mediated cytoprotection against oxidative stress using normal secondary cultures of male and female human lens epithelial cells. Furthermore, the question of whether mitochondrial-associated wtERβ1 plays a role in the cytoprotection mechanism was resolved by RNA suppression using specific small interfering (si)RNA to the wild-type receptor. We also demonstrated the differential subcellular localization of the wtERβ1 to mitochondria, compared with the isoform variants ERβ2 and ERβ5 to the nucleus and cytosol, respectively, and evaluated the relative expression of the wtERβ1 and the two ERβ isoform variants.

Numerous clinical studies have concluded that women who maintain adequate levels of estrogen show reduced risk for cataractogenesis. Both the Framingham and Blue Mountain Eye Studies indicated that premenopausal women exhibited a reduced risk for cataract compared with men of the same age group (44, 46).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISSCUSION
 REFERENCES
 
Materials. 1,3,5(10)-Estratrien-3,17β-estradiol was purchased from Steraloids (Newport, RI). The hormone was prepared in 100% ethanol and dissolved to a final stock concentration of 1 mM and used at a dilution of 1:1,000. Thirty percent hydrogen peroxide solution was prepared into a 25-mM stock with water. JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazololcarbocyanine iodide) was obtained from Sigma-Aldrich Chemical (St. Louis, MO). Antibody against ERβ used in the initial immunocytochemistry experiments was obtained from Affinity Bioreagents (Golden, CO). The antibodies used in the Western blot for ERβ (H-150) as well as Actin (H-300), which served as a lane-loading control, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Affinity-purified ERβ isoform specific antibodies and secondary antibodies are described below. All other reagents were obtained from commercially available sources.

Cell culture. Whole globes, donor tissue from eye banks, were incubated in serum-free Dulbecco's minimal essential medium (DMEM) with 20% antibiotics (solution containing 10,000 units of penicillin and 10 mg streptomycin/ml; Sigma) at 4°C for 30 min followed by additional 30-min incubation in a similar solution containing 10% antibiotics. The globes were rinsed in sterile PBS, pH 7.4. The lenses were carefully excised from the ciliary processes, and a slit was cut across the lens capsule at the equator and the capsule peeled off, discarding the cortex/nucleus. Remaining interior lens fragments were removed from the capsule by careful irrigation with serum-free DMEM. Thereafter, the cells were cultured in tissue culture flasks and subsequently plated onto collagen-coated coverslips or tissue culture plastic in 10% FBS MEM for experimentation.

HLE-B3 cells were obtained from Dr. Usha Andley (Department of Ophthalmology, Washington University School of Medicine, St. Louis, MO). HLE-B3 is an SV-40-transformed cell line-derived human lens epithelium (2). HLE-B3 cells and MCF7 (clone 89) breast carcinoma cells were grown on tissue culture plastic under 37°C incubation with 5% CO2. All experiments using HLE-B3 cells did not exceed passage 21. Both of these cell lines were maintained in 20% FBS MEM. At least 48 h prior to any experimentation, the cell lines were switched to charcoal-stripped FBS MEM to remove hormones present in the serum, as described by Moor et al. (30).

Triple-primer PCR. Triple-primer PCR was carried out as described previously (28), except that signals of the resulting PCR products were quantified by densitometry following autoradiography, using the Quantity One software (version 4.2; Bio-Rad, Hercules, CA). The triple-primer PCR technique has been validated extensively (9, 23, 27) and is designed to measure quantitatively the relative expression of truncated transcripts, such that the ratio of the triple-primer PCR products is related directly to the initial ratio of the input cDNAs. The upper primer ERβ1U (5'-CGATGCTTTGGTTTGGGTGAT-3'; sense, located in exon 7, positions 1,400–1,420, Genbank accession no. AB006590) anneals to ERβ1 and several isoform variants of ERβ that are alternatively spliced after exon 7. Two lower primers are used to distinguish between the wtERβ1 and the alternatively spliced transcripts: 1) ERβ1L (5'-GCCCTCTTTGCTTTTACTGTC-3'; antisense, located in exon 8, position 1,667–1,648, Genbank accession no. AB006590) will detect ERβ1 only and generates a 268-bp PCR fragment; 2) ERβ2L (5'-CTTTAGGCCACCGAGTTGATT-3'; antisense, located in ERβ2 extra sequences, positions 1,933–1,913, Genbank accession no. AB051428) will detect ERβ2, ERβ4, and ERβ5 transcripts, which are distinguished from each other by the size of PCR product, that are 214, 529, and 295 bp, respectively. For each sample, ERβ1, ERβ2, and ERβ5 signals are expressed as a percentage of the sum of all signals measured (ERβ1 + ERβ2 + ERβ5 signals). Three independent triple-primer PCRs were carried out on each cDNA sample, and the mean of the relative signals was calculated as means ± SD.

JC-1 mitochondrial membrane potential staining. Cells were initially seeded onto 35-mm dishes and allowed to grow to semiconfluence. The cells were then pretreated with the addition of estrogen (1 µM) or an ethanol vehicle of equal volume as described previously (30, 37) to determine the state of mitochondrial membrane potential. JC-1 is a potentiometric dye that exhibits a membrane potential-dependent loss as J-aggregates (polarized mitochondria) transition to JC-1 monomers (depolarized mitochondria), as indicated by a fluorescence emission shift from red to green. Therefore, mitochondrial depolarization can be indicated by an increase in the green/red fluorescence intensity ratio.

To stain the cells, monolayers were rinsed with DMEM without phenol red (Sigma-Aldrich). Cell monolayers were incubated with DMEM containing 10% serum and 5 µg/ml JC-1 at 37°C for 30 min. Cells were then rinsed two times with DMEM, and images were obtained using a x10 objective on a Zeiss LSM410 confocal microscope set to excitation at 488 nm and detection at 510- to 525- (green) and 590-nm (red) channels using a dual band-pass filter. Images were then statistically analyzed for both the red and green fluorescent intensity using metamorph software (see below).

Immunocytochemistry. Cells were seeded onto coverslips and maintained in MEM with 20% fetal bovine calf serum for 24 h at 37°C, 5% CO2. Cells were labeled with 200 nM Mitotrack-633 (Molecular Probes, Eugene, OR) for 45 min according to the manufacturer's protocol. For immunofluorescent labeling of estrogen receptors, cells were treated according to previously published methods (14). The cells were fixed in 1% paraformaldehyde in 0.05 M PBS, pH 7.0, for 30 min at 4°C. After the cells were fixed, they were then rinsed in 0.05 M PBS and 0.05 m PBS, pH 7.0, containing 50 mM NH4Cl (washing buffer) two times at 10 min/rinse.

Cell membranes were permeabilized by incubation in 0.05% saponin in 0.05 M PBS for 20 min followed by 2% BSA PBS solution for blocking. Coverslips were subsequently exposed to rabbit polyclonal antibodies generated against synthetic peptides corresponding to the first 150 residues of the amino terminus of ERβ (PA1-311; Affinity Bioreagents, Golden, CO) or to the carboxy terminus of ERβ isoforms 1 (18 amino acids), 2 (18 amino acids), and 5 (10 amino acids), as published previously (9). These antibodies were previously used to demonstrate expression of ERβ isoforms in human breast cancer tissue (12). The antibodies were used at a dilution of 1:50 in blocking buffer overnight at 4°C. After rinsing, coverslips were incubated with Alexa 488-labeled secondary antibodies (4 µg/ml, goat anti-rabbit IgG; Molecular Probes) in blocking buffer for 60 min at room temperature and subsequently rinsed again. Cells were mounted with ProLong Antifade kit (Molecular Probes) on glass slides. Multiple donors were examined for each receptor isoform and for whether isoform distribution changed with male or female lens epithelial cells. Controls consisted of incubation with rabbit IgG at 4 µg/ml under the same experimental conditions. Slides were imaged with a Zeiss LSM410 confocal microscope and set to excitation/emission of 488/565 (for Alexa 448-labeled antibodies) and 633/665–700 nm (for Mitotrack 633-labeled mitochondria). All measure bars indicate 30 µM.

To examine the level of receptor colocalization with the mitochondria, the confocal images were separated into individual red and green channels using metamorph image software (version 6.1). These images were then analyzed using the colocalization function of ImageJ software (version 1.36b). The threshold for significant colocalization was set at 50, and the resulting images generated show only pixels that meet threshold.

siRNA knockdown of ERβ. To silence the expression of ERβ, a series of four On-Target siRNA duplexes were obtained from Dharmacon (Lafayette, CO), designed against ERβ mRNA. The sequences and designations of these duplex are as follows: ERβ siRNA no. 1: sense 3'-GGA AAU GCG UAG AAG GAA UUU-5', antisense 5'-AUU CCU UCU ACG CAU UUC CUU-3'; ERβ siRNA no. 2: sense 3'-UUC AAG GUU UCG AGA GUU AUU-5', antisense 5'-UAA CUC UCG AAA CCU UGA AUU-3'; ERβ siRNA no. 3: sense 3'-GCA CGG CUC CAU AUA CAU AUU-5', antisense 5'-UAU GUA UAU GGA GCC GUG CUU-3'; and ERβ siRNA no. 4: sense 3'-GAA CCC ACA GUC UCA GUG AUU-5', antisense 5'-UCA CUG AGA CUG UGG GUU CUU-3'. In addition to this set of duplexes, a nonspecific scrambled duplex was purchased from Dharmacon to use as a control against erroneous knockdown of our protein of interest.

The procedure for the preparation of cell transfections was adapted from methodology described by Arnold et al. (3). Cells were plated into 35-mm tissue culture dishes at 50% confluence in 20% charcoal-stripped FBS MEM. The cells were then allowed to attach to the plate overnight. The cells were switched into 10% charcoal-stripped FBS MEM on the morning of the transfection. In a 1.6-ml eppendorph tube, 300 µl of serum-free MEM was combined with 6 µl of TransIT-TKO transfection reagent (Mirus Bio, Madison, WI). This solution was allowed to incubate at room temperature for 5 min. siRNA was added to the tube to achieve a final concentration of 30 nM in the culture dish and allowed to incubate for 15 min. The solution was then immediately added onto the cells and placed in an incubator for 24 h. After 24 h, the medium was changed and cells were subsequently allowed to grow out for another 48 h.

At 72 h posttransfection, the cells were again transfected using the same procedure as described above to achieve maximal knockdown of ERβ. Due to what was found to be slow turnover of the ER protein within mitochondria of the lens cells, this was the most effective method to limit ERβ expression (i.e., localization) within mitochondria. After the second 72-h period, the cells were collected for assay.

Traditional PCR. After siRNA treatments the cells were scrapped from 60-mm dishes, and the RNA was extracted using GE Healthcare's Illustra-RNAspin Mini Isolation kit (Buckinghamshire, UK). The RNA content of the samples was determined using 260/280 nm on an Ultrospec 2100 Pro Spectrophotometer (Amersham Bioscience, Cambridge, UK). Ten micrograms of RNA was taken and used in a reverse transcriptase reaction with reagents from Applied Biosystems's High-Capacity Reverse Transcription kit (Foster City, CA). The resulting cDNA produced was used for a PCR reaction using Stratagene's 2x PCR master mix (Cedar Creek, TX), with primers against ERβ and actin. The sequence to the ERβ primers was designed to include the full transcript of the wtERβ1 mRNA transcript, as described previously (9). The sequence for the ER primers is as follows: the upper primer is 5'-CGATGCTTTGGTTTGGGTGAT-3' and the lower primer is 5'-GCCCTCTTTGCTTTTACTGTC-3', which yields a single band of 268 bp. Actin was used as a housekeeper gene with primers as follows: upper primer 5'-GTACAGGGATAGCACAGCCT-3' and lower primer 5'-CATCCTCACCCTGAAGTACC-3'. PCR products were run on a 2% agarose gel. Images of the gels were digitally recorded with a Flurochem Digital Imaging System (Alpha Innotech, San Leondro, CA) under transmitted UV illumination.

In a separate set of experiments, RT-PCR analysis was performed to evaluate whether silencing the expression of wtERβ1 (see below) influenced the relative expression of its splice variants ERβ2 and ERβ5. PCR primers were prepared to the human wtERβ1, ERβ2, and ERβ5, as reported previously (9).

Western blotting. Cell lysates from Western blot analysis were collected by initially rinsing the cultures with ice-cold PBS (pH 7.4). The cells were lysed using buffer consisting of 25 mM HEPES, 0.25 NaCl, 0.5% Igepal (NP-40), 0.2% Triton X-100, 1 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 10 mM NaF, 0.1 mM Na3VO4, and a cocktail of protease inhibitors (Sigma-Aldrich). The buffer was allowed to incubate for 15 min on ice. The lysate was collected and sonicated for 10 s. Protein content of the samples was determined using the Bio-Rad protein assay kit. The lysates were combined with 3x SDS laemmli buffer and boiled for 3 min. The proteins were run on 10% SDS polyacrylamide gels loaded with 20 µg of protein per lane and transferred to PVDF membranes. The membranes were blocked in 1% BSA Tween-Tris-buffered saline for 1 h. The blots were incubated overnight in blocking buffer with rabbit polyclonal antibodies against either actin (H-300, 1:1,500 dilution; Santa Cruz Biotechnology) or ERβ (H-150, 1:750 dilution; Santa Cruz Biotechnology). The blots were subsequently washed four times in Tween-Tris-buffered saline for 5 min. After being washed, the blots were incubated in goat anti-rabbit HRP-linked secondary antibody (1:20,000 dilution; Santa Cruz Biotechnology). The blots were washed again four times for 5 min/wash and developed with Pico West super signal chemilumiesence kit (Pierce, Rockford, IL). The blots were imaged and digitally recorded on a Flurochem Digital Imaging System (Alpha Innotech, San Leondro, CA).

Image analysis and statistical analysis. The images from JC-1 analysis were taken and separated into individual red and green channels using MetaMorph image software. The background fluorescence was removed from each image. The fluorescence intensity signal from each image was quantified for the entire image (16). The ratio of these values was used in statistical analysis. All statistical differences between treatments were calculated with a two-way ANOVA test using Graphpad Prism (version 5.00).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISSCUSION
 REFERENCES
 
Expression of ERβ isoforms from male and female normal lens epithelial cells cultures. To determine the relative expression of the different isoforms of ERβ, the triple-primer PCR method was employed. Briefly, this method is a coupled PCR reaction that can simultaneously monitor the relative expression of the wild-type ERβ1, ERβ2, and ERβ5 isoforms expressed in lens epithelial cells. The result from these reactions is summarized in Table 1 and illustrates the average percentage ± SD of the total ERβ message pool. The triple-primer PCR data represent three independent PCR reactions from individual lenses, and samples were generated from multiple male and female donors (refer to Table 1 for specific details).


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  		Table 1. Distribution of ERβ isoforms

 
The normal male and female HLE (nHLE) secondary cell cultures reveal a remarkably similar distribution of the wild-type ERβ1, ERβ2, and ERβ5 isoforms. The wild-type receptor and ERβ2 are equally expressed in the normal cells irrespective of sex. Moreover, the relative expression pattern of the ERβ isoform cDNAs in nHLE, reported in this study, is similar to that of human lens tissue (9), supporting the notion that the cell culture model represents a valid physiological surrogate. The proportional distribution of wtERβ1, -β2, and -β5 PCR products differed between the normal lens epithelial cells and the SV-40-transformed lens epithelial cell line. HLE-B3 showed an expression pattern of the receptor isoforms that paralleled that of the neoplastic breast carcinoma cell line in that HLE-B3 cells exhibit reduced wild-type receptor and an elevated expression of ERβ5.

17β-Estradiol protects against mitochondrial membrane depolarization after exposure to acute oxidative stress and is sex independent. Previous studies from this laboratory have established an estrogen-mediated mechanism of mitochondrial cytoprotection using HLE-B3 cells, a virally transformed cell line (30, 31, 43). This is the first report of estrogen-mediated cytoprotection in normal lens epithelial cells.

Secondary cultures of normal lens epithelial cells and HLE-B3 cells were plated onto 35-mm tissue culture dishes. The cells were pretreated with either an ethanol vehicle control or 1 µM 17β-estradiol for 24 h prior to peroxide exposure and subsequently exposed to 12.5, 25, 50, or 100 µM peroxide for 2 h. The cells were then stained with JC-1 and imaged using a confocal microscope. The normal lens epithelial cells showed a significant increase in depolarization across the peroxide dose range (Fig. 1A). This increase in the green to red ratio was significantly reduced (i.e., prevented depolarization) in doses >25 µM H2O2 with the addition of estrogen prior to oxidative stress. (n = 6, P < 0.01).


Figure 1
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  		Fig. 1. Mitochondrial protection after estrogen treatment in normal and transformed lens epithelial cells. Both normal lens epithelial (nHLE) cells (A) and the virally transformed human lens epithelial cells (HLE-B3; B) were maintained in serum-free medium with and without pretreatment with 1 µM of 17β-estradiol followed by bolus addition of peroxide from 12.5 to 100 µM for 2 h and subsequently stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazololcarbocyanine iodide (JC-1). Confocal images of each treatment were taken immediately after JC-1 staining (n = 6; means ± SD). Red and green fluorescent channels were quantified and the ratio statistically analyzed as described in EXPERIMENTAL PROCEDURES.

 
The HLE-B3-transformed cell line was far less responsive to oxidative stress. The cell line only showed a significant increase in depolarization at 100 µM H2O2 for 2 h (Fig. 1B). HLE-B3 cells also showed significant protection against mitochondrial membrane potential with prior estrogen treatment (n = 6, P < 0.001). These data were taken as the standard with further experiments. That is, 100 µM was used as the peroxide dose to promote mitochondrial depolarization, because at this dose, irrespective of whether the virally transformed cell line or normal cells were being examined, a significant increase in depolarization was secured and protection with estrogen was certain.

ERβ localization to the mitochondria of male and female normal lens epithelial cells. Figure 2A illustrates a common feature of normal lens epithelial cells stained for total ERβ (green fluorescence) and counterstained for mitochondria (red fluorescence). The cells were typified by tracks of mitochondrial arrays extending outward from the perinuclear region to the cell periphery. The ER colocalized with the mitochondrial arrays indicated by the arrows, which indicate the merge of the green and red fluorescent stains (bar = 30 µM).


Figure 2
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  		Fig. 2. Control images for estrogen receptor (ER) localization. A: confocal image of nHLE cells stained for both mitochondria (red channel; stained with Mitotracker 633) and ERβ (green channel; ERβ primary antibody and Alexa 488 secondary antibody). The arrows indicate points where tracks of mitochondria that colocalize with the wild-type (wt)ERβ form arrays that extend outward from the perinuclear region. Bar = 30 µm. B, top: typical images of IgG serum controls replacing the primary antibodies and demonstrating that with normal rabbit serum only background fluorescence was detected. Cells were counterstained with Mitotracker 633 for mitochondria. B, bottom: typical images of cells incubated with affinity-purified specific antibodies to wtERβ1, ERβ2, and ERβ5. Cells were counterstained with Mitotracker 633. Bar = 30 µm.

 
Affinity-purified antibodies specific for wtERβ1, ERβ2, and ERβ5 were employed to examine the cellular distribution of the ERβ isoforms. Figure 2B, top, represents control images of the immunocytochemistry reactions with both the red and green channels merged. These are typical control images stemming from cell incubations with normal rabbit serum and demonstrate minimal nonspecific binding of fluorescently labeled secondary antibody. Figure 2B, bottom, represents typical merged images of cells stained with ERβ isoform-specific primary antibodies and Alexa 488 fluorescent secondary antibody. Note that each primary antibody shows a different and distinct staining pattern in normal human lens epithelial cells.

The sex specificity for the distribution and differential localization for each ERβ isoform was examined next. Figure 3 illustrates the typical staining pattern for normal female (top) and male (bottom) lens epithelial cells with the wtERβ1 isoform. Both males and females show a similar distribution of mitochondrial arrays within the cytoplasm (red; Fig. 3, left). ERβ1 showed a similar staining pattern, irrespective of sex, with some nuclear localization and a pattern of receptor staining extending outward from the nucleus. The merge of the red and green fluorescence channels indicates colocalization with mitochondria. The images were examined with the use of ImageJ software, which quantifies and compares each image pixel to verify mitochondrial colocalization. The software was set with threshold parameters that accepted pixels showing only a high level of colocalization. The resulting peak colocalization image (Fig. 3, right) is a binary image that shows all colocalized pixels as white and pixels that do not meet the threshold standards as black. Both male and female normal lens cell cultures show considerable mitochondrial colocalization.


Figure 3
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  		Fig. 3. Localization of wtERβ1 in mitochondria of male and female nHLE cells. nHLE cells from both male and female donors were labeled with affinity-purified antibody against ERβ1 (green) and counterstained with Mitotracker 633 (red). Mitochondria distributed throughout the cytoplasm but were relatively condensed in the perinuclear region. wtERβ1 was localized in mitochondria, as well as nucleus, of male and female nHLE cells. Software analysis of the merged image revealed pixels that met threshold conditions for high levels of colocalization (far right). Bar = 30 µm.

 
The distribution of the ERβ2 isoform shows primarily nuclear staining with faint cytoplasmic staining (Fig. 4). No merge of the mitochondrial and the ERβ2 fluorescence stains was apparent. This observation was confirmed with the ImageJ software analysis, which revealed little to no peak colocalization of ERβ2 and mitochondria in either male or female lens epithelial cells.


Figure 4
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  		Fig. 4. Localization of ERβ2 in nucleus of male and female nHLE cells. nHLE cells from both male and female donors were labeled with affinity-purified antibody against ERβ2 (green) and counterstained with Mitotracker 633 (red). ERβ2 was localized in the nucleus of male and female nHLE cells. Software analysis of the merged image revealed no mitochondrial colocalization. Bar = 30 µm.

 
Last, the ERβ5 isoform was examined for subcellular localization (Fig. 5). Male or female lens epithelial cells indicated faint diffuse cytosolic staining throughout the cell with some nuclear staining. The merged images show no evidence of colocalization of the mitochondria with the ERβ5 isoform variant. Analysis with the ImageJ software confirmed the lack of mitochondrial colocalization.


Figure 5
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  		Fig. 5. Localization of ERβ5 in cytoplasm of male and female nHLE cells. nHLE cells from both male and female donors were labeled with affinity-purified antibody against ERβ5 (green) and counterstained with Mitotracker 633 (red). The ERβ5 isoform shows diffuse staining throughout the cell's cytoplasm and some nuclear staining of male and female nHLE cells. Software analysis shows no colocalization with the mitochondria. Bar = 30 µm.

 
Estrogen-mediated cytoprotection in male and female normal lens epithelial cells. We next examined whether there was a sex specificity to the estrogen-mediated cytoprotection mechanism by comparing normal male and female lens epithelium, employing the potentiometric dye JC-1. Male and female lens epithelial cells were cultured from lens explants, as described in EXPERIMENTAL PROCEDURES. The cells were preincubated with either an ethanol vehicle or 1 µM 17β-estradiol for 24 h and thereafter exposed to 100 µM H2O2 for 2 h prior to JC-1 staining. Both male and female cells displayed significant loss of mitochondrial membrane potential between control and peroxide-treated cells (Fig. 6), which was significantly prevented, in either sex, with the administration of estrogen (n = 6, means ± SD, P < 0.001). These data indicate that the estrogen-mediated mechanism for protection of mitochondria against acute oxidative stress is sex independent.


Figure 6
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  		Fig. 6. Mitochondrial protection after estrogen treatment with male and female lens epithelial cells. To examine whether estradiol's protection of the mitochondria is sex related, secondary cultures of normal male and female lens epithelial cells were exposed to acute oxidative stress. Cells were maintained in serum-free medium with and without pretreatment with 1 µM of 17β-estradiol followed by bolus addition of 100 µM peroxide for 2 h and subsequently stained with JC-1. The prior addition of 17β-estradiol equally prevents the collapse of membrane potential against peroxide exposure in cell populations of both male and female donors. +Significant prevention of depolarization of peroxide-treated cells with estrogen treatment. *Significant increase in the level of mitochondrial membrane depolarization relative to control cells (n = 6, means ± SD, P < 0.001).

 
siRNA knockdown of ERβ prevents 17β-estradiol from protecting against peroxide-induced loss of mitochondrial membrane potential. The role of wtERβ1 in the estrogen-mediated cytoprotection pathway has yet to be fully elucidated. To determine whether the mitochondrial localization of wtERβ1 positions the receptor such that it plays some role in the cytoprotection pathway, we tested whether suppressing wtERβ1 expression limited estrogen's otherwise inherent capacity to prevent mitochondrial permeability transition with peroxide stress. A series of four commercially available siRNA duplexes designed against ERβ mRNA was used in these experiments. Using transfection methodology adapted from Arnold et al. (3), HLE-B3 cells were transfected with either siRNA against ERβ mRNA or nonspecific siRNA, which served as a negative control (refer to EXPERIMENTAL PROCEDURES for transfection details). At termination of the second round of transfection, total RNA was extracted from cells transfected with nonspecific siRNA or with siRNA-ERβ. RT-PCR was first performed to determine whether ERβ mRNA was suppressed by one or more of the siRNA-ERβ duplexes. Figure 7A shows the resulting PCR reactions of ERβ expression with the four duplexes directed against ERβ mRNA and the control, nonspecific siRNA duplex. Of the four available duplexes, only siRNA duplex no. 4 markedly lowered ERβ mRNA compared with cells transfected with nonspecific siRNA. Actin was used as a housekeeping gene to account for lane loading.


Figure 7
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  		Fig. 7. RNA suppression of wtERβ. A: RT-PCR of HLE-B3 cells treated with ERβ small interfering RNA (siRNA). Cells were transfected as described in EXPERIMENTAL PROCEDURES with a nonspecific, scrambled siRNA duplex or 1 of 4 siRNA duplexes designed against ERβ (nos. 1–4). Of the 4 available duplexes, only siRNA duplex no. 4 markedly lowered ERβ mRNA compared with cells transfected with nonspecific siRNA. Actin was used as a housekeeper gene for equal lane loading. B: Western blot of HLE-B3 cells treated with ERβ siRNA. Cells were transfected in the same manner as in Fig. 7A. The cells were treated with either a mock transfection, nonspecific (ns) siRNA, siRNA duplex no. 1, or siRNA duplex no. 4. Only siRNA-ERβ duplex no. 4 (lane 4) dramatically reduced ERβ protein levels. Actin was used to monitor lane loading for the SDS-PAGE gels. C: mitochondrial protection assay after siRNA treatment against ERβ. Once the optimal conditions were established for suppression of ERβ, the HLE-B3 cells were tested for their response to acute oxidative stress with or without the prior addition of estrogen. The transfected cells were pretreated with 1 µM 17β-estradiol for 24 h followed by the bolus addition of 100 µM peroxide for 1 h. Cells were then immediately stained with JC-1 and imaged. *Statistical analysis of these images showed that there was a significant increase in mitochondrial depolarization after peroxide exposure compared with control cells in all siRNA treatments. +Cells treated with the nonspecific siRNA control duplex showed significant protection after preincubation with estrogen compared with the cells treated with peroxide alone, as did the cells treated with siRNA duplex no. 1. Cells treated with the siRNA duplex no. 4, which significantly suppressed the ERβ on the level of mRNA (A) and protein (B), failed to protect against mitochondrial depolarization as initiated by the peroxide insult subsequent to 17β-estradiol preincubation (n = 6, means ± SD, P < 0.001). D: expression of mRNA's encoding ERβ variants using reverse transcription-PCR after RNA suppression of wtERβ. Total cDNAs were prepared from RNA from SV-40-transformed HLE cells. Top (from left to right): control nonspecific siRNA duplex: lane 1, wtERβ1 (Murphy primers); lane 2, ERβ2 (Moore primers); lane 3, ERβ5 (Moore primers); lane 4, actin. Bottom (from left to right): siRNA duplex no. 4: lane 1, wtERβ1 (Murphy primers); lane 2, ERβ2 (Moore primers); lane 3, ERβ5 (Moore primers); lane 4, actin. PCR primer sequences were designed as reported in Ref. 9.

 
We reconfirmed the suppression of ERβ by siRNA-ERβ duplex no. 4 by repeating the transfection procedure and extracting equal amounts of protein (20 µg/lane) for Western blot analysis and probing with ERβ antibody (see Fig. 7B). The first lane (loading from left to right) was a mock transfection that represented no siRNA into the transfection solution and showed the highest level of ERβ with ERβ antibody. The second and third lanes illustrate the nonspecific control siRNA and siRNA duplex no. 1 directed against ERβ, respectively. Comparison of these lanes demonstrates that duplex no. 1 was ineffective at suppressing ERβ protein within the HLE-B3 cells. siRNA-ERβ duplex no. 4 (lane 4) dramatically reduced ERβ protein levels. Actin was again used to monitor lane loading for the SDS-PAGE gels.

Having established the conditions for efficient suppression of the receptor, the transfection was again repeated and the response of HLE-B3 cells to oxidative stress assessed with or without the presence of 17β-estradiol, using JC-1 analysis. HLE-B3 cells were transfected with either nonspecific siRNA, duplex no. 1 siRNA, or duplex no. 4 siRNA. Control cells were treated with neither peroxide nor estradiol addition. The green-to-red ratios were equivalent for control cells, irrespective of whether nonspecific siRNA, siRNA-ERβ duplex no. 1, or siRNA-ERβ duplex no. 4 was used in the transfection treatment, indicating that the transfection treatment was not the cause of increased mitochondrial depolarization. The addition of bolus peroxide caused significant depolarization in the HLE-B3 cells irrespective of transfection treatment. Those cells transfected with nonspecific siRNA and the siRNA-ERβ duplex no. 1 displayed significant protection against mitochondrial permeability transition if treated with estrogen prior to peroxide insult. The cells transfected with siRNA-ERβ duplex no. 4 lost the capability to prevent mitochondrial depolarization by estrogen intervention (Fig. 7C).

To test the specificity of siRNA duplex no. 4, coupled RT-PCR was performed on total RNA extracted from HLE-B3 cells, with specific primers designed to identify wild-type ERβ and two of its four alternative splice isoforms, ERβ2 and ERβ5, subsequent to transfection with either siRNA duplex no. 4 or nonspecific siRNA. mRNA was detected for wild-type ERβ1, ERβ2, and ERβ5 (Fig. 7D). ERβ2 and ERβ5, as well as wild-type ERβ1 mRNA, was markedly lowered consequent to transfection with siRNA duplex no. 4 compared with cells transfected with nonspecific siRNA.


   DISSCUSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISSCUSION
REFERENCES
 
There is a distinct need to study issues specific to women, including the scientific and clinical importance of analyzing data separately for males and females. The use of primary cells harvested from normal human lens epithelial cells derived from male and female donor lens offers the unique advantage of providing homogeneous cell populations, thus allowing the acquisition of unequivocal sex-related data. The rationale driving these studies is that estrogen protects mitochondria against damage from oxidative stress. Our conceptual framework dictates a mechanistic model of rapid nongenomic activation of specific phosphoproteins of survival signal transduction pathways, which leads to the stabilization of mitochondria consequential to oxidative insult. The studies reported herein were designed to determine whether sex differences play a crucial role in the estrogen-mediated mitochondrial protective mechanism (17).

Mitochondria are susceptible to damage by oxidative insult. Although the precise mechanism of injury is unknown, one of the known outcomes is loss of mitochondrial membrane potential (43). Two questions are addressed in this report. 1) Is protection against loss of mitochondrial membrane potential in the face of oxidative stress sex dependent or sex independent? 2) Does the mitochondrial-associated wtERβ1 play a role in the estrogen-mediated protection of the lens epithelial cell from oxidative damage? To answer these questions, an analysis of the differential expression and comparative subcellular localization of wtERβ1 and two of its isoform variants, ERβ2 and ERβ5, in cultured normal human male and female lens epithelial cells was undertaken. We evaluated the state of mitochondrial membrane potential, using the potentiometric dye JC-1 to determine whether sex differences play a role in the estrogen-mediated cytoprotection pathway. Moreover, this is the first study of its kind to address whether the wtERβ1 plays a role in the mitochondrial protective mechanism, using specific siRNA suppression of the mitochondrial-associated wild-type receptor.

Our data at the RNA level show that ERβ1, ERβ2, and ERβ5 are coexpressed in normal human male and female cells and in the virally transformed lens cell line. In general, the level of variant isoform expression relative to wtERβ1 expression was similar in normal male and female lens cells in culture. With the lens epithelial cells that had been transformed with SV-40, a significant difference in ERβ isoform expression at the level of RNA was evident. The relative expression of wtERβ1 and ERβ2 mRNA was markedly downregulated compared with the ERβ5 isoform variant. This paralleled the pattern of expression seen in the breast carcinoma cell line used in this study as control and with previous data (9, 27). The reason for this phenomenon and its functional implications as it may pertain to estrogen's capacity to influence mitochondrial protection against depolarization from oxidative stress is unclear to date. Given the data presented in this study, it is conceivable that the altered expression of wtERβ1 and the ERβ isoform variants has the potential to manipulate mitochondrial function.

There are no studies to date of which we are aware that address sex difference-related subcellular distribution of the wtERβ1 and the ERβ isoform variants in normal tissues. In this study, normal male and female lens epithelial cells were stained with affinity-purified isoform-specific antibodies, and distinct staining patterns were evident (Figs. 3–5). The full-length ERβ wild-type isoform was the only receptor isoform to localize to the mitochondria, irrespective of sex. The ERβ2 isoform displayed primarily nuclear staining, in agreement with previous observations that this ERβ isoform is primarily nuclear in rat brain (15) and the virally transformed human lens epithelial cell (9). ERβ2 retains a DNA-binding domain but lacks an intact ligand-binding domain (32). We have previously suggested that ERβ2 may shuttle from the cytosol to the nucleus without the necessity of binding estradiol, whereas wtERβ1, which has both an intact DNA-binding domain and ligand-binding domain, has the capability to associate with 17β-E2 in the cytosol such that formation of the complex, wtERβ1~17β-E2, might be required to shuttle wtERβ1 to nuclear and/or mitochondrial compartments (9). The ERβ5 isoform variant displayed weak staining throughout the cytosol, with some weak nuclear staining in both male and female lens epithelial cells.

We have previously shown that 17β-E2 promotes mitochondrial stabilization in the face of oxidative stress by preventing the loss of impermeability of the inner mitochondrial membrane, as demonstrated by JC-1 analysis and fluorescence microscopy (30), using the virally transformed lens epithelial cell line HLE-B3. Our current data are unique in that we have reconfirmed this observation with normal human male and female lens epithelial cells. There was no apparent sex-related association to estrogen's ability to protect mitochondria against depolarization (Fig. 6). We also can now rule out the possibility that our past result, based upon the use of the virally transformed cell line HLE-B3, may have been dictated by viral transformation.

The biological action of estrogens is mediated by binding to one of two ERs, ER{alpha} and ERβ, both members of the nuclear receptor superfamily, a family of ligand-related transcription factors, reviewed by Matthews and Gustafsson (29). The actions of 17β-E2 occur on binding the ER, and the nuclear pool of these receptors can then transactivate target genes (26). In addition to its role in being a prominent transcription factor, studies of the antioxidant activity of 17β-E2 have demonstrated that estrogens do not necessarily require the classical receptor-dependent mechanism to exert their positive effects (6, 19).

In a recent review (40) regarding the role of estrogen, ERs, and mitochondrial protection, the point of view was taken that, "In cell culture systems known to naturally express one of the two known estrogen receptors (ER{alpha} or ERβ), pharmacological strategies that use ER antagonists, such as tamoxifen and ICI 182,780, have supported the requirement of these receptors in mediating the effects of estrogen on cell survival (13, 36). Some studies support the role of ER{alpha} (18), whereas others implicate ERβ in mediating estrogen-induced protection (38)." On the other hand, in a study by Wang et al. (43), the nonfeminizing estrogen 17{alpha}-estradiol (which, reputedly, has a greatly reduced capacity to bind to ER{alpha} or -β) was employed and was reported to be equally as effective as 17β-E2 in the recovery of peroxide-insulted lens cell viability, suggesting that estrogen's protective response may be ER independent. Because of the ongoing controversy as to whether estrogens protect cells against oxidative stress through an ER-dependent or ER-independent mediated mechanism, we decided to reexamine this issue. Other studies (5) have indicated that 17{alpha}-E2 retains ~70% binding capacity to ERβ, as does 17β-E2 at 1 µM, which was the concentration used by Wang et al. (43). Thus, a reevaluation of our past pharmacologically driven study was warranted.

A role for ER{alpha} in estradiol-mediated protection against oxidative stress has been authenticated using ER knockout mice (24). We have concluded that RNA interference and the use of knockout mice represent the best ways to verify the role of ERs in the mitochondrial protection pathway. We recently employed small interfering RNA to conclusively show that ERK2, and more specifically, phosphorylated ERK2, was a key constituent of the estrogen-mediated mitochondrial protection pathway (17). Herein we report that wtERβ1 plays a definitive role in the E2-mediated mitochondrial protection mechanism. These data establish that wtERβ, associated with the mitochondria, is a necessary prerequisite for 17β-E2-mediated cytoprotection such that protection against H2O2 toxicity is likely to be mediated through classic estrogen receptors.

The siRNA duplex used to knockdown ERβ was purchased from a commercially available source with the intent that it be targeted specifically to wtERβ1 because the siRNA duplex no. 4 used to silence the wild-type receptor (see Fig. 7, A and B) is found only in the 3' (COOH-terminal) mRNA part of ERβ1. In principle, there should have been little, if any, influence on the ERβ variant isoforms, since their mRNA does not contain that sequence. Nevertheless, our data indicated that suppression of wtERβ1, in turn, affected the relative levels of ERβ2 and ERβ5 (see Fig. 7D). Such data suggest that wtERβ1 regulates, in an unknown manner, the fate and levels of its splice variants. Unlike wtERβ1, which associates with both the nucleus and mitochondria (Fig. 3), ERβ2 is associated exclusively with the nucleus (Fig. 4), and as such we cannot conclusively rule out the possibility that ERβ2 (or the nuclear form of the ERβ1) provides some genomic contribution to the mitochondrial protection scheme via the upregulation of protective nuclear-encoded mitochondrial proteins. Clearly, more work is needed to fully comprehend the molecular mechanism of estrogen and ERs in mitochondrial protection. However, the specific knockdown of wtERβ1 with subsequent loss of estrogen's ability to prevent mitochondrial depolarization in the face of oxidative stress (refer to Fig. 7C), coupled with the fact that only wtERβ1 localizes to mitochondria (refer to Figs. 3–5), strengthens our contention that the mechanism of estrogen-induced mitochondrial cytoprotection is wtERβ1 dependent.

To summarize, whereas the prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtERβ1 dependent. Baines (4), in an excellent recent minireview, points out that "despite extensive knowledge regarding the triggers and signal transduction networks, the critical targets of the (mitochondrial) protective machinery have remained elusive. Evidence has implicated mitochondria and, in particular, the mitochondrial permeability transition pore as important targets of cardioprotective signaling." Although the precise nature of the mitochondrial-associated wtERβ1 mechanism responsible for protection by estradiols against oxidative stress remains to be determined, we can begin to suggest several possible modes of action. wtERβ1 may provide cytoprotection by directly associating with specific components of the mitochondrial permeability transition pore and by doing so prevent the opening of the mitochondrial membrane pore, thereby preventing mitochondrial permeability transition. In this regard, Chen et al. (10) have demonstrated by electron microscopy that ERβ antibody-linked gold particles localize within the matrix of mitochondria. Alternatively, the wtERβ1 may operate indirectly in the cytoprotection mechanism by "anchoring" sufficient levels of estrogen in the mitochondrial matrix such that estrogen, not necessarily the ER, might directly associate with specific components of the mitochondrial permeability transition pore. We recently reported that, despite the fact that ERK2 plays a regulatory role in maintaining mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism (17). Then again, estrogen's modus operandi might be to activate prosurvival kinases that could, in turn, subsequently phosphorylate (i.e., activate) so-called "end effectors," which subsequently interact with and influence the mitochondrial membrane permeability transition pore.

These data provide mechanistic insight into the actions of estrogen and ERs in mitochondrial protection. The reason premenopausal women exhibit a reduced risk for cataract compared with men of the same age group is not because men exhibit a different isoform distribution of ERs, nor does estrogen offer preferential protection in women over that of men. The prevention of mitochondrial membrane permeability transition is sex independent and clearly involves wtERβ1. Isoform distribution differences or prevention machinery are not an issue for potential therapies designed to exploit mitochondrial protection in both men and women.


   ACKNOWLEDGMENTS
 
This project is taken in part from a dissertation submitted by James M. Flynn to the University of North Texas Health Science Center, Fort Worth, TX, in partial fulfillment of the requirements for the degree Doctor of Philosophy. We also thank Dr. Lawrence X. Oakford for assistance in figure preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. R. Cammarata, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (e-mail: pcammara{at}hsc.unt.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISSCUSION
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