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Am J Physiol Endocrinol Metab 294: E589-E599, 2008. First published January 2, 2008; doi:10.1152/ajpendo.00705.2007
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RNA suppression of ERK2 leads to collapse of mitochondrial membrane potential with acute oxidative stress in human lens epithelial cells

James M. Flynn,1 Deborah A. Lannigan,2 David E. Clark,2 Margaret H. Garner,1 and Patrick R. Cammarata1

1Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas; and 2Center for Cell Signaling and the Department of Microbiology, University of Virginia, School of Medicine, Charlottesville, Virginia

Submitted 1 November 2007 ; accepted in final form 28 December 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
17β-Estradiol (E2) reduces oxidative stress-induced depolarization of mitochondrial membrane potential (MMP) in cultured human lens epithelial cells (HLE-B3). The mechanism by which the nongenomic effects of E2 contributed to the protection against mitochondrial membrane depolarization was investigated. Mitochondrial membrane integrity is regulated by phosphorylation of BAD, and it is known that phosphorylation of Ser112 inactivates BAD and prevents its participation in the mitochondrial death pathway. We found that E2 rapidly increased both the phosphorylation of ERK2 and Ser112 in BAD. Ser112 is phosphorylated by p90 ribosomal S6 kinase (RSK), a Ser/Thr kinase, which is a downstream effector of ERK1/2. Inhibition of RSK by the RSK-specific inhibitor SL0101 did not reduce the level of E2-induced phosphorylation of Ser112. Silencing BAD using small interfering RNA did not alter mitochondrial membrane depolarization elicited by peroxide insult. However, under the same conditions, silencing ERK2 dramatically increased membrane depolarization compared with the control small interfering RNA. Therefore, ERK2, functioning through a BAD-independent mechanism regulates MMP in humans lens epithelial cells. We propose that estrogen-induced activation of ERK2 acts to protect cells from acute oxidative stress. Moreover, despite the fact that ERK2 plays a regulatory role in mitochondrial membrane potential, estrogen was found to block mitochondrial membrane depolarization via an ERK-independent mechanism.

17β-estradiol; mitochondrial membrane potential; mitochondrial permeability transition

OXIDATIVE STRESS CAUSES PROFOUND INJURY to a diverse number of intracellular macromolecules in eukaryotes, including lipid peroxidation, protein alteration, breakage of covalent bonds of carbohydrates, and cleavage of DNA strands. Mitochondria are particularly susceptible to oxidative damage with consequent depolarization of mitochondrial membrane potential (8, 10, 24). Hyperoxic oxygen uncouples mitochondrial electron transport in human lens epithelial cells (HLE-B3) cells. This uncoupling of electron transport increases the levels of reactive oxygen species (ROS; Ref. 22) and decreases cellular ATP production. Similarly, hydrogen peroxide (H2O2) directly collapses mitochondrial membrane potential ({Delta}{Psi}m) in a variety of cell types, causing decreases and increases in ATP and ROS production, respectively (12).

Pretreatment of cells with estrogen prevents most of the mitochondrial changes elicited by oxidative stress. Estradiol blocks membrane oxidation at physiological concentrations (17). Estrogen treatment reduces lipid peroxidation induced by glutamate and attenuates the increase in intracellular peroxide induced by bolus H2O2 addition (16) or by mitochondrial toxins (41). Mitochondria play a central role in the generation of biological forms of energy and also in the production of ROS. The damage observed in mitochondria from disease and/or experimental insults such as H2O2 lead to deficiency in ATP production, as well as a concomitant increase in production of ROS, overwhelming cellular antioxidant defense systems. Under conditions of oxidative stress, mitochondria undergo a catastrophic loss of the impermeability of the inner mitochondrial membrane that causes a complete collapse of mitochondrial membrane potential ({Delta}{Psi}m), a process termed permeability transition (31). 17β-Estradiol (E2) and 17{alpha}-estradiol increase the amount of Ca2+ or H2O2 needed to collapse mitochondrial membrane potential in human lens epithelial cells, effectively stabilizing mitochondrial integrity and preserving function under pathogenic conditions (42). This effect does not require prolonged exposure to the estrogens, which suggests a nongenomic action by the estrogens. More of the mitochondrial population retains {Delta}{Psi}m and continues to function at a given Ca2+ load. Such response readily explains the preservation of ATP levels by estrogens during H2O2 exposure, as well as the repression of cell death via either necrosis and/or apoptosis.

What is the likely mechanism by which estrogen (at concentrations higher than 1 nM) exerts its protection on unhealthy or aged mitochondria against oxidative insult? Warner and Gustafsson (44) recently stated, "The nature and location of the (estrogen) receptor might have a profound effect on its affinity for E2 and this might explain why many rapid effects of E2 are observed at concentrations higher than 1 nM, which is the concentration of E2 at which maximal activity of the nuclear receptor is achieved." In addition to maintaining mitochondrial integrity, estrogen may oppose or protect against the toxic action of H2O2 (oxidative stress) by interacting at the level of signal transduction. H2O2 activates signaling pathways such as the stress-activated protein kinase/JNK pathway in human lens epithelial cells (25) and the p38 pathway in human leukemia cells (49). While activation of these "stress pathways" causes apoptosis (27), suppression of these stress pathways either via direct inhibition or by stimulation of "survival" pathways like the phosphatidylinositol 3-kinase (PI3K)-Akt pathway regulates apoptotic progression (45).

Estrogen activation of "survival" pathways represents a potential mechanism for protection against H2O2-induced apoptosis. Ovarian hormones elicit Akt and ERK phosphorylation in explants of the cerebral cortex (36). E2 activates (i.e., phosphorylates) ERK and stabilizes mitochondrial membrane potential when human and GSH-depleted bovine lens cells are exposed to H2O2 stress (28). The work of Moor et al. (28) established a correlative association between estradiol-stimulated activation of the MAPK signaling pathway and protection of mitochondrial membrane potential in an ocular cell culture model.

Mitochondria have an important function in some apoptotic cascades (1, 11, 15, 20). The regulation of mitochondrial membrane integrity and the release of cytochrome c from mitochondria during apoptosis are processes that are controlled by the Bcl-2 family, of which BAD is one of several proapoptotic proteins (18, 40). BAD resides in the cytoplasm of healthy cells (18) and in response to an apoptotic stimulus translocates to the mitochondria and promotes cytochrome c release. The mechanism of the translocation of BAD to the mitochondrial outer membrane is known, as the cellular localization of BAD is under regulation by phosphorylation and dephosphorylation. Survival factors (protein kinases such as MAPK, Akt, PKA, and others) keep BAD in a phosphorylated state and situated in the cytoplasm (11). The Akt and ERK survival pathways play important roles by keeping BAD in the cytoplasm where it cannot trigger Bax translocation. Bax translocation subsequently causes a pore to form via oligomerization or opens a channel called a voltage-dependent anion channel (VDAC) by direct interaction (33, 35). Tsuruta et al. (39) explained how the Akt pathway can suppress Bax translocation to mitochondria.

The stimulation of the mitogenic MAPK and PI3K pathways inhibits the apoptotic activity of the BAD protein by promoting phosphorylation at serine sites 112 and 136, respectively. Phosphorylation at these sites results in the binding of BAD to 14-3-3 proteins and the inhibition of BAD binding to Bcl-2 and Bcl-xL (46). The prevention of BAD binding to Bcl-2 proteins in the mitochondria and their subsequent inability to interact with the permeability transition pore suggest a plausible mechanism by which estradiol leads to a stabilization of {Delta}{Psi}m.

In this study, we demonstrate the pathway responsible for BAD phosphorylation in virally transformed human lens epithelial cells after stimulation by estradiol. Furthermore, through the use of RNA interference of ERK and BAD, we distinguish the function of MAPK from that of p90 ribosomal S6 kinase (RSK). We accomplish this by determining whether estrogen-mediated protection of mitochondrial membrane potential involves primarily ERK activation, BAD inactivation, or a combination of both ERK and BAD. Understanding how estrogen modulates signal transduction pathways will further establish the mechanism by which estrogen protects mitochondria from damage by oxidative stress.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 GRANTS
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Materials. E2 [1,3,5(10)-estratrien-3,17β-diolE2] was purchased from Steraloids (Newport, RI). Estrogen was dissolved in 100% ethanol to a stock concentration of 10 mM. Estrogen was added to cell cultures to a final concentration of 1 µM. Control cells received an equivalent volume of ethanol. H2O2 was purchased from Fisher Scientific (Fair Lawn, NJ) and added to cell cultures to a final concentration of 100 µM.

The MEK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (UO126) was purchased from Cell Signaling Technology (Beverly, MA). H-89 was obtained from Calbiochem/EMD chemicals (Gibbstown, NJ). PMA and KT5720 were purchased from Sigma-Aldrich (St. Louis, MO). SL0101 was obtained from Toronto Research Chemicals (North York, ON, Canada). All other chemicals and reagents were of analytical grade and were obtained from commercially available sources.

Cell culture. HLE-B3 cells, a human lens epithelial cell line immortalized by the SV-40 virus (3), were obtained from U. Andley (Washington University School of Medicine, Department of Ophthalmology, St. Louis, MO). Cells were maintained in MEM containing 5.5 mM glucose supplemented with 20% FBS (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, nonessential amino acids, and 0.02 g/l gentamycin solution (Sigma Chemical) and maintained at 37°C and 5% CO2-95% O2. All experiments were performed with monolayers of HLE-B3 cells that did not exceed passage 22 (5). To deplete the cell cultures of estrogens, cells were maintained in 20% charcoal dextran-stripped FBS (CSFBS; Gemini Bio-Products, Woodland, CA) MEM for 24–48 h then switched to 2% CSFBS MEM for 18 h with a final medium change to serum-free MEM on the day of the experiments as described previously (28). In select experiments, cells were pretreated with estrogen overnight with 2% CSFBS in MEM followed by a medium change to 0.5% (CSFBS) MEM for 12–18 h with the addition of fresh estrogen the next morning before experimentation.

Western blot analysis and antibodies. Total cell lysates were collected from HLE-B3 cultures after treatments by rinsing adherent cells with ice-cold 1x PBS, pH 7.4, and then adding lysis buffer [25 mM HEPES, pH 7.4, 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)] directly to cell monolayers for 30 min at 4°C. Lysates were collected and sonicated for 5 s, and a portion of the sample was removed for determination of protein concentration. Protein concentration was determined using the EZQ protein quantification kit from Invitrogen (Carlsbad, CA); 3x SDS (Laemmli) buffer was added to the lysates, which were subsequently boiled for 3 min; and the proteins were resolved by electrophoresis on 10% SDS-polyacrylamide gels (20 µg protein/lane). Proteins were then transferred to nitrocellulose (Scheicher and Schuell, Keene, NH).

For the experiments examining the activity of p90 RSK substrates, an alternative hot protein extraction method was used as described by Henrich et. al. (21). Cells were rinsed in PBS, and the culture dishes with attached cell monolayers were placed on a hot plate set to 100°C and lysed with hot lysis buffer (~100°C). The lysis buffer consists of 0.12 M Tris·HCl (pH 6.8), 4% SDS, and 20% glycerol. The cell lysates were immediately scraped into a 1.6-ml tube, sonicated, and snap frozen in liquid nitrogen.

For Western blot analysis, nitrocellulose membranes were blocked with 0.1% BSA and 0.02% Tween-20 in Tris-buffered saline (TTBS) for 60 min. These membranes were probed overnight at 4°C with primary antibodies. The blots were then rinsed in TTBS (4x with 5-min washes) and incubated in either goat anti-rabbit horseradish peroxidase conjugate or goat anti-mouse horseradish peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Required concentrations of antibodies were determined according to the manufacturer's suggested protocols. Blots were again rinsed in TTBS (4 x 5 min washes), and proteins were detected using a SuperSignal west pico chemiluminescent kit from Pierce (Rockford, IL). Probed membranes were exposed to Kodak BioMax Light Film (Kodak Scientific Imaging, Rochester, NY).

Primary antibodies from Cell Signaling Technology used in the study were rabbit anti-p44/42 MAPK, mouse anti-phospho-p44/42 MAPK (Thr202/Tyr204), anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), anti-Akt, rabbit anti-BAD, rabbit anti-p42 MAPK, anti-phospho-p90 RSK (Thr359/Ser363), and rabbit anti-phospho-BAD (Ser112). Rabbit anti-phospho-BAD (Ser155) and rabbit anti-phospho BAD (Ser136) were procured from Upstate Cell Signaling (Lake Placid, NY), and rabbit anti-VDAC was from Affinity Bioreagents (Golden, CO).

Determination of estrogen-induced pSer112-BAD by fast activated cell-based ELISA. An ELISA-based assay was used to confirm the phosphorylation of BAD at the serine 112 phosphorylation site. The Fast Activated Cell-Based ELISA (FACE) kit was obtained from Active Motif (Carlsbad, CA). The FACE method can specifically monitor both the serine 112 phosphorylated form of BAD and compare it with total BAD content. HLE-B3 cells were cultured in 96-well plates in 20% CSFBS MEM. The cells were then placed in 2% CSFBS overnight. Before the bolus addition of 1 µM E2, the media were changed to serum-free FBS MEM. Wells were set up in triplicate for 0, 5, 15, 30, 60, and 90 m for both phosphorylated BAD and total BAD. After estrogen treatment, the cells were immediately fixed to the 96-well plate. The cells were then treated according to the manufacturer's directions for the ELISA reaction. After plate development, optical density at 450 nm was determined using a Molecular Devices Spectramax 190 (Sunnyvale, CA).

PKA activity assay. PKA activity was determined using an ELISA-based PKA activity assay kit from Assay Designs (Ann Arbor, MI). The assay is based on the phosphorylation of a synthetic peptide by PKA in the cell lysates, and the synthetic PKA substrate is detected by a phospho-specific antibody. Briefly, HLE-B3 cells were grown on 100-mm dishes in 20% CSFBS MEM. The media were changed to 2% CSFBS MEM overnight and then into serum-free MEM the day of the experiment. E2 was added to the cultures at a final concentration of 1 µM for a period of 0, 5, 15, 30, or 90 min. Cells were then rinsed one time with ice-cold PBS and lysed immediately. The protein concentration in the cell lysates was determined and normalized by appropriate volume adjustment. The ELISA reactions were performed in triplicate according to the manufacturer's directions using 1.75 µg of crude protein sample per reaction. Once the reaction was completed, the microplate was read for optical density at 450 nm on a Molecular Devices Spectramax 190.

JC-1 stain mitochondrial membrane analysis and image analysis. After experimental treatments, cells were stained with the cationic dye 5,5',6,6'-tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR) as described previously (14, 28, 29) to visualize the state of mitochondrial membrane potential. JC-1 is a potentiometric dye that exhibits a membrane potential dependent loss as J-aggregates (polarized mitochondria) to accumulation of JC-1 monomers (depolarized mitochondria) as indicated by fluorescence emission shift from red to green (34). That is, mitochondrial depolarization is indicated by an increase in the green-to-red fluorescence intensity ratio.

The cells were stained using the following procedure. Monolayers were rinsed one time with serum-free 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. After this incubation, cells were again rinsed two times with the serum-free DMEM and multiple images were obtained using a x10 objective on a confocal microscope (Zeiss LSM410) excited at 488 nm set to simultaneously detect green emissions (510–525 nm) and red emissions (590 nm) channels using a dual band-pass filter.

For the experiments involving small interfering RNA (siRNA), cells were preloaded with the JC-1 dye before bolus addition of peroxide and immediately imaged on the confocal microscope. Sequential images from a randomly chosen field of cells were taken every 150 s for a 50-min time sequence. Data were collected from three individual plates of cells, each having been treated with either double-stranded, nontargeted RNA, siRNA ERK2, or siRNA BAD. These images were then compiled into a time-lapsed stack of 20 frames each. The fluorescent channels of the image stacks were individually analyzed with image software as described in Statistical analysis. The resulting graphic depiction for each experimental treatment represents 20 time points of mitochondrial membrane potential. Each time point is based on the mean of the green-to-red fluorescence ratio from three random fields of cells gathered from three individual cell populations.

For siRNA experiments that examined the influence of estrogen on mitochondrial protection after siRNA ERK2 treatment, cells were treated for RNA suppression; however, 18 h before the image analysis the cells were treated with 1 µM E2 or ethanol vehicle. The cells were then stained and imaged.

Silencing RNA of signaling proteins. RNA suppression was used to disrupt the mRNA production of the signaling proteins, ERK2 and BAD siRNA. Cell Signaling Technology's SignalSilence kits specific for human BAD and p42 MAPK were used in these experiments (23). The cells were initially planted into 35-mm dishes at 50% confluence in 20% FBS MEM. The media were replaced 24 h later with 1.3 ml 5% FBS MEM. The transfection solution was prepared according to manufacturer's instructions. Briefly, 6 µl of TransIT-TKO transfection reagent from Mirus Bio (Madison, WI) were added to 300 µl of serum-free MEM and left to stand for 5 min at room temperature. The desired molarity of duplex siRNA was added to the transfection solution, and it was again left to to stand for 5 min at room temperature. The appropriate siRNA containing solution was subsequently added to each of three plates of cells and maintained at 37°C and 5% CO2-95% O2 for 24 h. The media were subsequently replaced with fresh 20% FBS MEM. The transfection efficiency was determined by examining fluorescein-conjugated control siRNA-treated cells under a confocal microscope set to observe both fluorescent and transmitted channels. The cells were then counted using ImageJ software (described in Statistical analysis).

Statistical analysis. Confocal JC-1 images were analyzed with MetaMorph image analysis software (version 6.1; Molecular Devices, Downingtown, PA) as previously described by Flynn and Cammarata (14). The entire field of a randomly captured image was utilized for quantification of average fluorescence after the individual fluorescence channels for red (590 nm) and green (510–525 nm) were adjusted for background fluorescence, and then the total signal was evaluated as green-to-red ratio. These ratio values were then used to run statistical tests.

The images from the siRNA experiments were analyzed with ImageJ (v1.36b; National Institutes of Health, Bethesda, MD). The percentage of transfection was calculated using the cell counter plug-in to count the number cells exhibiting a positive fluorescein signal from the total cell population captured in the image.

For the green-to-red ratio calculation, each field of cells (3 fields/siRNA) had a unique starting ratio. To correct for this, the lowest value for each field was subtracted from every value obtained for that field and the SD of the mean of three fields was calculated. Data were graphed and analyzed using PRISM 4 software (GraphPad, San Diego, CA). To test for significance between treatments over the time course, a two-way ANOVA was performed with each time point. As the green-to-red ratio in each treatment began to deviate, the point where they become significantly different was determined using Bonferroni posttests. In all cases, once a treatment significantly deviated from other treatments subsequent time points likewise remained significantly different from other treatments.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Estrogen protects the mitochondria from peroxide-induced depolarization. {Delta}{Psi}m was determined in HLE-B3 cells using the fluorescent cationic indicator JC-1. JC-1 exhibits potential-dependent accumulation of monomers (emission maximum = ~525 nm, green) instead of aggregates (emission maximum = ~590 nm, red) as the mitochondrial permeability transition pore opens (i.e., depolarizes). Polarized and depolarized mitochondria display red and green fluorescence, respectively. Mitochondrial depolarization is indicated by an increase in the green/red fluorescence intensity ratio.

Based on the green-to-red ratio of JC-1, pretreatment of HLE-B3 cells for 3 or 24 h with E2 provides similar protection against the mitochondrial depolarization induced by H2O2 (Fig. 1). These data support our hypothesis that a rapid, nongenomic mechanism of defense takes place after a relatively brief estrogen preincubation. However, it is very possible that genomic mechanisms also play a role in contributing to the overall protective effect.


Figure 1
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  		Fig. 1. 5,5',6,6'-Tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) images comparing HLE-B3 cells preincubated with estradiol for 3 vs. 24 h before bolus peroxide addition indicate that the protective mechanism is, in part, nongenomic. Note that an equal level of protection against depolarization is afforded irrespective of the duration of estrogen preincubation compared with the significant mitochondrial depolarization that occurs with bolus addition of peroxide in the absence of estrogen preincubation. Bar graphs are based on images taken from 8 randomly chosen fields.

 
ERK2 but not BAD is responsible for mitochondrial protection. Our data below show that estrogen increases the phosphorylation of MAPK and Ser112-BAD. We tested whether pMAPK or pSer112-BAD was important to the mechanism by which estrogen prevents the collapse of mitochondrial membrane potential during oxidative stress. ERK2 and BAD were silenced using specific siRNA. An optimal transfection period of 24 h was established for knockdown of ERK2 or BAD (Fig. 2A). Silencing BAD expression did not alter the levels of ERK2. Moreover, transfection of HLE-B3 cells with ERK2-specific siRNA reduced ERK2 protein expression without affecting BAD expression levels. After 24 h of RNA suppression, HLE-B3 cells were stained with 5 µg/ml JC-1 for 30 min, administered a bolus 100 µM H2O2, and photographed 50 min later. These images show an increase in green fluorescence for the ERK siRNA-treated cells relative to the BAD siRNA-treated cells and mock-transfected cells (Fig. 2B). The greater relative extent of green fluorescence shows that silencing ERK2 enhances depolarization compared with knockdown of BAD. The green-to-red ratio was calculated from serial stacks of confocal images, taken over 50 min after the introduction of peroxide to the transfected cells. The normalized results for three fields of cells for each siRNA (Fig. 2C) demonstrate the significant increase in mitochondrial membrane depolarization that occurs when ERK expression is silenced. Silencing BAD had little effect on peroxide-induced mitochondrial depolarization. We conclude that ERK2 regulates mitochondrial membrane potential.


Figure 2
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  		Fig. 2. A: small interfering RNA (siRNA) for ERK and BAD indicating effective suppression of the ERK and BAD proteins. Western blot analysis of extracts from HLE-B3 cells 24 after mock transfection or transfection with p42 MAPK siRNA and BAD siRNA. Data shown were compiled from parallel plates of cells from those treated with siRNA ERK2 and siRNA BAD but subsequently stained with JC-1 to follow loss of mitochondrial membrane potential (refer to B). Actin serves as control for demonstrating equal lane loading. B: confocal imaging of H2O2-induced mitochondrial membrane depolarization in HLE-B3 cells after ERK and BAD siRNA indicates that cells suppressed for ERK are far more prone to depolarization than are those suppressed for BAD. After 24 h of RNA suppression, cultures were stained with 5 µg/ml JC-1, a collapse of mitochondrial membrane potential ({Delta}{Psi}m)-sensitive dye, for 30 min, administered a bolus of 100 µM H2O2, and photographed 50 min later. Mitochondrial membrane depolarization is indicated by a shift from red to green fluorescence in H2O2-exposed cultures. Note the proportionally increased green fluorescence for the ERK siRNA-treated cells relative to the BAD siRNA-treated cells and mock-transfected cells. These images were taken from a randomly chosen field. Bar = 20 µm. C: serial confocal imaging of H2O2-treated mitochondrial membrane depolarization in HLE-B3 cells after ERK and BAD siRNA demonstrates the significant increase in mitochondrial membrane depolarization when ERK expression is silenced. Sequential images from a randomly chosen field of cells, from 3 individual plates, were taken every 150 s for a 50-min time sequence. Each triplicate set of cell culture plates were treated with either double stranded, nontargeted RNA, siRNA ERK2, or siRNA BAD. These images were then compiled into a time-lapsed stack of 20 frames each. Each point is displayed as the means ± SE. Two-way ANOVA analysis determined that ERK siRNA was significantly different than control after 45 min of peroxide exposure (n = 3; P < 0.05). BAD siRNA was never significantly different from control throughout the time course. D: serial confocal imaging of H2O2-treated mitochondrial membrane depolarization in HLE-B3 cells after ERK siRNA with or without E2 treatment. Sequential images from a random field of cells from 3 individual populations of cells were taken every 150 s throughout a 50-min sequence after the bolus addition of peroxide. HLE-B3 cells were transfected with either nontargeted "control" siRNA or ERK2 siRNA with or without pretreatment with 1 µM E2 for 18 h. Each point is displayed as means ± SE. Two-way ANOVA analysis of the time course revealed that ERK siRNA was significantly different than control after 40 min of peroxide exposure (n = 3; P < 0.05). ERK siRNA and ERK siRNA + estrogen were significantly different by 37.5 min and remained so throughout the duration of peroxide treatment (n = 3; P < 0.05).

 
We tested whether estrogen could still prevent the collapse of mitochondrial membrane potential during oxidative stress despite silencing ERK2 by siRNA (Fig. 2D). The normalized results for three fields of cells demonstrate, as in Fig. 2C, that when ERK expression is silenced, an increase in mitochondrial membrane depolarization is observed. Cells pretreated with estrogen exhibited a propensity to resist membrane depolarization, similar to the serial stacks of confocal images observed with mock-transfected cells in the presence and absence of estrogen. Thus, ERK2 regulates mitochondrial membrane potential, but estrogen blocks mitochondrial membrane depolarization via an ERK-independent mechanism.

Estradiol-induced phosphorylation of ERK, AKT, and BAD. Having demonstrated that BAD is not involved with protection against mitochondrial depolarization or estrogen-mediated protection (Fig. 2, A–D), it was incumbent upon us to justify the rationale of these studies by demonstrating that all the relevant kinases are present and working (or not) in HLE-B3 cells, despite the fact that BAD is not part of the estrogen-depolarization mechanism. Figure 3 provides a schematic illustration of how the kinases and inhibitors studied are related to mitochondrial function and each other.


Figure 3
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  		Fig. 3. Diagrammatic illustration of the 3 independent pathways examined in this study that lead to BAD phosphorylation. BAD is a proapoptotic molecule, the function of which is regulated by phosphorylation at 1 of 3 sites (Ser112, Ser136, and Ser155). Phosphorylation at any of these three sites results in the loss of ability of BAD to heterodimerize with BCL-XL or BCL-2. Phosphorylated BAD binds to the 14-33-3 complex of proteins and is sequestered in the cytosol. Ser112 phosphorylation requires activation of the Ras-MEK-MAPK pathway and is prevented by the MEK inhibitor UO126. Ser136 phosphorylation results from activation of Akt. Ser155 (as well as Ser112) phosphorylation is induced by activation of PKA and prevented by the PKA inhibitors H-89 and KT5720. SL0101 is a highly specific ribosomal S6 kinase (RSK) inhibitor.

 
To understand the mechanism by which estradiol protection occurs, we investigated the signal transduction pathways that are activated in response to the nongenomic actions of estradiol. We tested whether E2 activates MAPK (via pERK1/2) and/or PI3K (via pAkt) signaling in quiescent HLE-B3 cultures by immunoblot analysis (Fig. 4A). We observed that E2 results in increased phosphorylation of ERK2 (p42 MAPK) in HLE-B3 cells and, to a lesser extent, increased phosphorylation of ERK1 (p44 MAPK; Fig. 4A). An increase in phosphorylation of Ser473 in Akt but not in Thr308 also occurred in response to 17β-E2. No change was observed in the total levels of MAPK or Akt. Phosphorylation generally peaked between 5 and 30 min and remained evident by 60 min. Serum stimulation produced a more rapid and robust phosphorylation over the same time span. Unlike E2, serum addition increased phosphorylation of both Ser473 and Thr308 in Akt. Alessi et al. (2) have previously reported that both Ser473 and Thr308 phosphorylations are required in order for Akt to be active. Collectively, these data demonstrate that E2 rapidly activates MAPK.


Figure 4
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  		Fig. 4. A: Western blot analyses of ERK1/2 and Akt phosphorylation in HLE-B3 subsequent to E2 or serum. Total cell lysates (20 µg protein/lane) were collected from quiescent HLE-B3 cultures that had been serum starved for ≥18 h before stimulation by either 1 µM E2 or 1% serum for 0, 5, 15, 30, and 60 min. B: Western blot analysis of estradiol-stimulated phosphorylation of BAD indicates that p112BAD is the predominant phosphorylated form of BAD in HLE-B3 cells. Total cell lysates (20 µg protein/lane) were collected after 0, 5, 15, 30, and 60 min of 1 µM E2 or 1% serum exposure and analyzed for Ser112-BAD, Ser136-BAD, and Ser155-BAD phosphorylation. Anti-VDAC was used as control to monitor equivalent lane loading. Experiment was run under serum-free conditions with subsequent bolus addition of estradiol (1 µM) or under serum-free conditions with subsequent bolus addition of 1% serum (as positive control). C: determination of pSer112-BAD after E2 treatment. Fast Activated Cell-Based ELISA was used to quantify the extent of pSer112-BAD stimulation with 1 µM E2 at 0, 5, 15, 30, 60, and 90 min using cultured HLE-B3 cells. The pSer112-BAD (left) and total BAD were quantified according to kit directions and graphed as means ± SE (n = 3). Statistical significance was determined using Student's t-test (2-tailed). The 0-min time point was tested against all later time points (*P < 0.05).

 
E2 and serum also stimulated the phosphorylation of Ser112 in BAD over a time course of 60 min. (Fig. 4B). These experiments were performed after 18 h of serum starvation to achieve basal levels of BAD phosphorylation before E2 addition. Total levels of BAD were not altered by E2 treatment. Of particular note, E2 failed to promote Ser136- and Ser155-BAD phosphorylation, indicating that these sites are not highly phosphorylated in HLE-B3 cells. These results were verified by using 1% serum as a positive control, which resulted in increased Ser112 phosphorylation but did not cause the phosphorylation of Ser136- or Ser155-BAD. Thus phosphorylation of Ser136 and Ser155 does not contribute to the estrogen protective effect.

The extent of stimulation of pSer112-BAD from total BAD by estrogen addition was determined by FACE ELISA (Fig. 4C). pSer112-BAD increased significantly 30 min post-E2 addition and remained elevated throughout the 90-min assay. No statistical change was observed in total BAD content in confirmation of the relatively small shift of total BAD to Ser112-BAD phosphorylation with E2 treatment observed by Western blot analysis (Fig. 4B).

If, indeed, BAD inactivation were a key component of the mitochondrial protection mechanism, then stimulation of ERK1/2, which likely results in Ser112-BAD phosphorylation, would have played a prominent role in mitochondrial defense.

RSK-independent Ser112-BAD phosphorylation. To determine whether ERK1/2 was responsible for the estrogen-induced increase in pSer112, we used the MEK inhibitor U0126 to indirectly inhibit MAPK activity. Pretreatment of HLE-B3 cells with UO126 prevented the estrogen-induced increase in phosphorylation of MAPK but did not effect phosphorylation of Ser112 in BAD (Fig. 5A).


Figure 5
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  		Fig. 5. A: Western blot analysis of UO126 inhibition of MAPK signaling indicates a MAPK-RSK-independent pathway of activation for pSer112-BAD. After a 1-h pretreatment with 10 µM UO126 (+UO126) or DMSO vehicle (–UO126), cells were treated with 1µM E2 for 30 min and total cell lysates (20 µg protein/lane) were collected and analyzed for pERK 1/2 and p112Bad. Each subset (E2 or UO+E2) represents triplicate samples from 3 individual cell populations. Note that UO126 treatment completely eradicates pERK but does not eliminate p112BAD levels, suggesting that a RSK-independent pathway to p112BAD activation exists in this virally-transformed lens epithelium. B: Western blot analysis using endogenous RSK-specific substrates indicates that SL0101 inhibits RSK activity in cultured human lens epithelial cells. HLE-B3 cells were pretreated with vehicle (DMSO) or 100 µM SL0101 for 2 h before addition of 500 nM PMA for 30 min before lysis. Protein concentration of lysates was measured, and lysates were electophoresed, transferred and immunoblotted. Equal loading of lysate is shown by the anti-Ran immunoblot. Antibodies against pS6 (#2211), S6 (#2212), peEF2 (32331), and eEF2 (#2332) were purchased from Cell Signaling Technology. The anti-Ran antibody (#610340) was purchased from BD Transduction Laboratories. C: Western blot analysis of SL0101 inhibition of RSK signaling supports a RSK-independent pathway of synthesis for p112BAD. After a 1-h pretreatment with 100 µM SL0101 (+SL0101) or DMSO vehicle (–SL0101), cells were treated with 1 µM E2 for 30 min and total cell lysates (20 µg protein/lane) were collected and analyzed for pERK1/2 and p112BAD. Controls were not exposed to SL0101 or E2. This experiment was repeated twice with similar results. Note the typical activation of pERK1/2 and p112BAD with E2 administration and that SL0101 treatment does not reduce pERK levels. Further note that SL0101 also does not attenuate p112BAD levels, confirming that a RSK-independent pathway for p112BAD activation exists in this virally-transformed lens epithelium. D: Western blot analysis of H-89 and KT5720 inhibition of PKA indicates that PKA is the RSK-independent source of BAD activation. After a 1-h pretreatment with 10 µM H-89 or 1 µM KT5720 (selective inhibitors of PKA) or DMSO vehicle, cells were treated with 1 µM E2 for 30 min and total cell lysates (20 µg protein/lane) were collected and analyzed for pERK1/2, p112BAD, and total BAD. VDAC was used to demonstrate consistency of lane loading. Controls were not exposed to metabolic inhibitors or E2. Note that both H-89 and KT5720 effectively reduce p112BAD levels, indicating that PKA is the RSK-independent source of BAD activation. Under these conditions, ERK1/2 levels were not diminished, proving that neither H-89 nor KT5720 influence upstream MAPK. This experiment was repeated twice with similar results.

 
The p90 RSK family of serine/threonine protein kinases is a downstream effector of ERK1/2, which upon activation by growth factors leads to the phosphorylation of BAD at Ser112. To directly test whether RSK was responsible for the estradiol-induced phosphorylation of Ser112, we utilized the RSK-specific inhibitor SL0101 (37). SL0101 (100 µM) inhibits RSK activity in intact HLE-B3 cells as demonstrated in the studies of the RSK-mediated regulation of the endogenous lens epithelial cell RSK substrates, ribosomal protein S6, and eukaryotic elongation factor 2 (eEF2). S6 regulates glucose homeostasis and cell size, and phosphorylation of S6 modulates its activity. Interestingly, the basal level of S6 phosphorylation (pS6) is quite high in HLE-B3 cells, but as expected PMA further increased pS6 levels (Fig. 5B). SL0101 inhibited phosphorylation of S6 under both basal and PMA-stimulated conditions. Although phosphorylation of S6 decreases its electrophoretic mobility, the total level of S6 remained constant (the anti-S6 immunoblot; Fig. 5B). The second endogenous substrate eEF2 mediates the translocation step in mRNA translation. A highly specific kinase, EF2 kinase (EF2K), inactivates eEF2 by phosphorylation. RSK phosphorylates and inactivates EF2K in response to mitogenic stimulation, which leads to a decrease in phosphorylation of eEF2. Thus, when RSK activity is low, eEF2 is phosphorylated by the active EF2K. However, stimulation of RSK activity by mitogens results in reduced phosphorylation of eEF2 due to inactivation of EF2K by RSK. In basal conditions in HLE-B3 cells, there was no detectable peEF2 (Fig. 5B). These results are consistent with the data with pS6 and demonstrate that RSK activity is quite high even in the absence of exogenous mitogens in HLE-B3 cells. SL0101 dramatically increased peEF2 levels. The levels of total eEF2 were not altered by any of the treatments as shown by the anti-eEF2 immunoblot. These results demonstrate that SL0101 inhibits RSK activity in intact HLE-B3 cells.

Having established that SL0101 was effective in HLE-B3 cells, we pretreated cells with SL0101 or vehicle before addition of E2. As seen previously, we observed that E2 increased the levels of pERK1/2 and pSer112-BAD. SL0101 did not alter the level of E2-stimulated pERK1/2 or pSer112-BAD (Fig. 5C).

Together, our results suggest that the estrogen-induced phosphorylation of Ser112 is independent of the MAPK-RSK signal transduction pathway in HLE-B3 cells.

Protein kinase a-dependent Ser112-BAD phosphorylation. The inability of U0126 (Fig. 5A) and SL0101 (Fig. 5C) to block BAD phosphorylation at Ser112 demonstrates that a RSK-independent pathway contributes to the estrogen-induced increase in Ser112-BAD phosphorylation in HLE-B3 cells in response to estrogen. Harada et al. (19) previously described a mitochondria-anchored PKA that could phosphorylate BAD. To investigate whether PKA was responsible for the increase in Ser112 phosphorylation, we used two structurally different PKA inhibitors. Pretreatment with either H-89 or KT5720 blocked the E2-induced phosphorylation of Ser112-BAD while having no effect on E2-induced activation of MAPK (Fig. 5D). The failure to phoshorylate Ser112-BAD upon addition of the PKA inhibitors can be attributed to the specific blocking of PKA activity and not due to inhibitor-induced loss of total BAD protein (Fig. 5D). We concluded that PKA is responsible for the estrogen-induced phosphorylation of Ser112-BAD in HLE-B3.

We determined whether estrogen increased PKA activity using a PKA activity assay kit. Consistent with the pattern of increasing E2-induced phosphorylation of Ser112-BAD observed in Fig. 4, B and C, Table 1 shows that PKA activity was significantly elevated by 15 min postaddition of E2 and remained statistically higher than basal level up through 90 min.


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  		Table 1. Relative PKA activity

 
Potentials models of mitochondrial protection. Below we suggest several potential mechanisms of protection of mitochondria against oxidative stress, as it translates from estrogen-activated ERK2 to prevention of loss of mitochondrial membrane potential. Continued research is necessary to fully understand the molecular details of the interactions between signaling pathways and mitochondria involved in the protective mechanism.

Wild-type estrogen receptor (ER)-β resides within mitochondria (28, 29). Using immunohistochemistry with confocal microscopy and immunogold electron microscopy, Chen et al. (9) also demonstrated that ERβ (and ER{alpha}) are located within the human breast adenocarcinoma cell (MCF-7) mitochondrial matrix. There are estrogen-binding sites in the mitochondria (47), suggesting, but not definitively proving, that relatively high levels of the hormone may localize to this organelle. pERK (48) resides in mitochondria. E2 prevents H2O2-induced injury to several oxidant-susceptible components of the cellular ATP-generating machinery, as well as loss of mitochondrial membrane potential, thereby preserving the driving force for ATP synthesis (29). It is entirely plausible that estrogen may oppose or protect against the toxic action of oxidative stress via activation of ERK (26, 28). Estrogen (and pERK) integration into mitochondria may prevent lipid peroxidation in the face of oxidative stress (13). E2 has recently been shown to effectively reduce lipid peroxidation induced by H2O2 exposure (43). It is not currently understood how estrogen and pERK might cooperatively interact to provide the stabilization of mitochondrial membrane potential against oxidative stress. Cardiolipin has recently been shown to decrease with oxidative stress (hyperoxic exposure; Ref. 22). Prevention of the loss of cardiolipin prevents the decrease of impermeability of the inner mitochondrial membrane or, put another way, by preventing the loss of cardiolipin, mitochondrial membrane potential might be preserved. Stabilizing against the loss of mitochondrial membrane potential prevents the otherwise deleterious downstream effects (release of cytochrome c, mitochondrial swelling, and DNA laddering) leading to cell death.

Are the protective effects of estradiol against oxidative stress ER dependent or independent? We previously determined the level of expression and intracellular localization of the ER subtypes, ER{alpha} and ERβ, in cultured human lens epithelial cells (HLE-B3; Ref. 6). ER{alpha} and ERβ mRNA expression was evaluated by coupled RT-PCR and Southern blot analysis. Subcellular localization of ER{alpha} and ERβ was determined on formaldehyde-fixed, saponin-permeabilized cells using conventional immunofluorescence techniques, as well as immunodetection of differential cellular components after the cultured cells were subjected to fractionation by sucrose gradient centrifugation. With the use of 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 (ER{alpha}) and 167-bp (ERβ) PCR products were verified by sequence analysis. Southern blot analysis using internal oligonucleotides directed to specific primer pairs for ER{alpha} and ERβ, respectively, further confirmed the authenticity of the PCR products. HLE-B3 cells expressed ER{alpha} and ERβ in association with the nucleus and ERβ in the mitochondria. That the mitochondrial-enriched subfraction correlated with the presence of the ERβ subtype was confirmed by Western blot analysis. The differential subcellular partitioning of ER{alpha} and ERβ subtypes suggested to us a new aspect to the estrogen-signaling system wherein mitochondrial stabilization 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 ER{alpha} and ERβ raises the interesting probability that the two receptors, dependent on subcellular localization, may have differential cytoprotective potential and, by inference, disparity in their mechanisms of action. We have also reported the fact that two nonfeminizing estrogens, 17{alpha}-estradiol and ent-estradiol, both of which do not bind to either ER{alpha} or ERβ, were as effective as E2 in the recovery of cell viability after acute oxidative stress (42). The estrogen receptor antagonist ICI 182,780 also did not block protection by E2. Both E2 and 17{alpha}-estradiol moderated the collapse of mitochondrial membrane potential in response to either H2O2 or excessive Ca2+ loading. We concluded that both 17{alpha}-estradiol and E2 can preserve mitochondrial function, cell viability, and ATP levels in human lens cells during oxidative stress. Although the precise mechanism responsible for protection by the estradiols against oxidative stress remains to be determined, the ability of nonfeminizing estrogens, which do not bind to estrogen receptors, to protect against H2O2 toxicity indicates that this conservation is not likely to be mediated through classic estrogen receptors.

Data presented in this study clearly establish a role for MAPK and, more specifically, the phosphorylated component pERK2, insofar as the role it plays in the estrogen-mediated mitochondrial protection mechanism against acute oxidative stress. Nevertheless, our data are not meant to imply that pERK, in and of itself, is the sole constituent involved in the protection mechanism that prevents loss of mitochondrial membrane potential. This fact was evident to us in the early stage of these studies, wherein it was noted that the treatment of HLE-B3 cells with UO126, while exacerbating mitochondrial membrane depolarization (because of the inhibition of ERK phosphorylation), still showed a degree of protection against depolarization 4 h postperoxide insult when estrogen was present (refer to Fig. 7 in Ref. 28); that is, a substantial fraction of mitochondrial protection against depolarization could be ascribed to pERK. However, had pERK alone been the sole component driving the protection mechanism, one might reasonably have expected to observe equal levels of mitochondrial membrane potential loss with the application of UO126 as with UO126 plus estrogen, and estrogen should have proved to be ineffective in preventing depolarization. The data obtained with ERK suppression by specific siRNA 50 min postoxidative stress but in the presence of estrogen confirm that estrogen protects against mitochondrial membrane depolarization via an ERK-independent mechanism (Fig. 2D). It should therefore be stressed that the protective mechanism(s) activated by E2 will likely prove to be complex and multifactorial. In that respect, Baines et al. (4) previously reported that PKC-{varepsilon} forms subcellular-targeted signaling modules with ERKs and that activated PKC-{varepsilon} increased phosphorylation of mitochondrial ERKs. Baines has postulated that PKC-{varepsilon}-ERK plays a role in PKC-{varepsilon}-mediated cardioprotection. Future studies aimed at discovering the means by which phosphorylated ERK2 prevents mitochondrial membrane permeability transition, as well as the means by which estrogen might directly associate with elements of the mitochondrial transition pore or indirectly activate/promote phosphorylation of components opposing the cell death machinery will undeniably be of great consequence to understanding the estrogen-mediated prevention of mitochondrial membrane permeability transition.

Other explanations for the protective mechanism(s) activated by E2 might include the restraint of {Delta}{Psi}m collapse by a repression of Ca2+ uptake into the mitochondria, increased tolerance to mitochondrial calcium sequestration, increased Ca2+ efflux from the mitochondria, increased resorption of Ca2+ into endoplasmic reticulum, and/or increased efflux of Ca2+ via the plasma membrane. With respect to Ca2+ mobilization, it seems a possibility that estradiol might indirectly activate the calcium pump by virtue of the fact that PKA activates Ca-ATPase (38). Activation of Ca-ATPase might then lower intracellular calcium, in turn repressing calcium uptake into the mitochondria, thereby preventing initiation of the mitochondrial cell death pathway. One cannot dismiss the compelling observation that estrogen, itself, may be acting as an antioxidant. Moosmann and Behl (30) reported that the antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties. An estrogen redox cycle has been proposed (32), which may control glutathione and NAD(P)H flux, and such action may also contribute as a defense mechanism against reactive oxygen species. In other words, estrogen, due to its steroidal phenol moiety, may intercalate into mitochondrial membranes where it blocks lipid peroxidation reactions and in turn is recycled via glutathione. We support the notion that the estrogen-driven nongenomic responses driving mitochondrial protection against membrane potential loss are likely to prove to be dynamic and multifaceted in that the protective stabilization of mitochondrial membrane potential by estrogens may be attributed to consolidation of several mechanisms of action working in concert.

Targeting mitochondrial function during periods of oxidative stress, using estrogen as a site-selective bioactive compound, preventing loss of mitochondrial membrane potential, characterizes a fresh conceptual approach that will contribute to novel innovative regimens for prevention or treatment of oxidative stress-related mitochondrial pathology associated with neurodegenerative diseases, obesity, diabetes, cardiovascular disease, and cataractogenesis.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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REFERENCES
 
This work was supported by Public Health National Science Award EY05570 (to P. R. Cammarata) and the Virginia Kincaid Cancer Research Fund (to D. A. Lannigan).


   ACKNOWLEDGMENTS
 
This project is taken in part from a dissertation submitted to the University of North Texas Health Science Center in partial fulfillment of the requirements for J. M. Flynn's Ph.D.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. R. Cammarata, Dept. of Cell Biology and Genetics, Univ. of North Texas Health Science Center, 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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 RESULTS AND DISCUSSION
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J. M. Flynn, S. D. Dimitrijevich, M. Younes, G. Skliris, L. C. Murphy, and P. R. Cammarata
Role of wild-type estrogen receptor-{beta} in mitochondrial cytoprotection of cultured normal male and female human lens epithelial cells
Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E637 - E647.
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