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. 2008 Jun;325(3):782-90.
doi: 10.1124/jpet.107.134072. Epub 2008 Mar 19.

Mitochondrial effects of estrogen are mediated by estrogen receptor alpha in brain endothelial cells

Ali Razmara  1 , Lorraine Sunday, Chris Stirone, Xiao Bo Wang, Diana N Krause, Sue P Duckles, Vincent Procaccio
Affiliations

Mitochondrial effects of estrogen are mediated by estrogen receptor alpha in brain endothelial cells

Ali Razmara et al. J Pharmacol Exp Ther. 2008 Jun.

Abstract

Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17beta-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17beta-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ERalpha, but not ERbeta. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17beta-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17beta-estradiol, but not 17alpha-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ERalpha, not ERbeta. The selective estrogen receptor modulators, raloxifene and 4-hydroxy-tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17beta-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ERalpha-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.

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Figures

Fig. 1
Fig. 1
Effect of estrogen on cytochrome c. After 24 h treatment with 17β-estradiol (10 nM) and vehicle control, cytochrome c mRNA and protein were measured. A. Quantitative real-time PCR measurement for cytochrome c mRNA. Data were normalized to beta-actin as an internal control and expressed relative to vehicle control. Values are means ± SEM; *Significantly different than vehicle; P≤ 0.05; n=4. B. Mean cytochrome c protein levels in estrogen-treated HBMEC and vehicle control and expressed relative to vehicle control. Values are means ± SEM; *Significantly different than vehicle; P≤ 0.05; n=4.
Fig. 2
Fig. 2
Effect of estrogen receptor RNA interference on levels of ERα and ERβ in HBMEC after 48 h transfection with either ERα RNAi, ERβ RNAi, a negative control RNAi construct or vehicle. Immunoblots show bands corresponding to the 66 kDa (A) and 45 kDa (B) forms of ERα and ERβ (C). Following densitometric analysis of each blot, all values were expressed relative to the vehicle-treated cells; means ± SEM are shown. *Significantly different than vehicle; P≤ 0.05; n=4.
Fig. 3
Fig. 3
Effect of estrogen treatment and ER RNA interference on levels of cytochrome c. After 48 h transfection with either ERα or ERβ RNAi construct or vehicle treatment, HBMEC were then treated with 10 nM 17β-estradiol for 24 h. Representative Western blot (A) and densitometric analysis (B) comparing cytochrome c protein levels in HBMEC treated with 17β-estradiol in the presence or absence of RNAi for ERα and expressed relative to vehicle control. Values are means ± SEM; *Significantly different than vehicle; P≤ 0.05; n=4. Representative immunoblot (C) and densitometric analysis (D) for levels of cytochrome c after treatment with 17β-estradiol in presence or absence of RNAi for ERβ and expressed relative to vehicle control. Values are means ± SEM; *Significantly different than vehicle; P≤ 0.05; n=4.
Fig. 4
Fig. 4
Effects of estrogen treatment on mitochondrial aconitase activity. Mitochondria were isolated from 17β-estradiol-treated and vehicle-treated control HBMEC. A. The ratio of activities of aconitase, inactivated by ROS, to fumarase, unaffected by ROS, was measured as a functional indicator of mitochondrial ROS production. The activity ratio relative to vehicle control is shown. Values are means ± SEM. *Significantly different from vehicle control; P ≤ 0.05; n=4. B. Aconitase activity measured in isolated HBMEC mitochondria before and after total enzyme reactivation with reducing reagents. Values are means ± SEM. *Significantly different from mitochondrial activity of vehicle control group; P ≤ 0.05; n=4. †Significantly different from activity before reactivation within each group; P ≤ 0.05; n=4.
Fig. 5
Fig. 5
Effects of estrogen on mitochondrial superoxide production. HBMEC were treated with 10 nM 17β-estradiol, 10 nM 17α-estradiol or vehicle control. Some HBMEC were pre-equilibrated with the estrogen receptor antagonist ICI-182,780 (1 μM) for 1 h and maintained during 17β-estradiol or vehicle treatment. Cells were exposed to these treatments for 24 h prior to measurement of superoxide production in live cells using the MitoSOX Red dye. A. MitoSOX Red mitochondrial superoxide indicator staining shown in red. Scale bar, 10 μm. B. Subunit I of complex IV, a mitochondrial DNA encoded-protein, staining shown in green. C. Immunofluorescence co-localization (yellow) of MitoSOX Red and subunit I of complex IV. D. Representative tracing of MitoSOX dye fluorescence intensity reflecting mitochondrial superoxide levels. Tracings for HBMEC pre-treated with 17β-estradiol or vehicle are shown. Pyruvate and malate, complex I substrates, both at 2 mM, were added to initiate the reaction. MnTBAP, a superoxide dismutase mimetic, was added after a plateau was reached. E. Mean values of mitochondrial superoxide production, corrected for sample protein concentration and expressed relative to vehicle control are shown. Values are means ± SEM. *Significantly different from other groups; P ≤ 0.05; n=8 for vehicle and 17β-estradiol groups; n=4 for other groups.
Fig. 6
Fig. 6
Effects of selective ER agonists and ER modulators on mitochondrial superoxide production. HBMEC were treated with 10 nM 17β-estradiol, 10 nM 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), 10 nM 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), 100 nM 4-hydroxy-tamoxifen, 100 nM raloxifene, or vehicle control. Some HBMEC were pre-equilibrated with 100 nM 4-hydroxy-tamoxifen for 1 h and maintained during 17β-estradiol treatment. A. The effects of the selective ER agonists, PPT and DPN, on the production of mitochondrial superoxide. B. The effects of the selective ER modulators, raloxifene and 4-hydroxy-tamoxifen (4-OH-tamoxifen), on mitochondrial superoxide production. Mean values of mitochondrial superoxide production, corrected for sample protein concentration and expressed relative to vehicle control are shown. Values are means ± SEM. *Significantly different from all groups without asterisk; P ≤ 0.05; n=8 for vehicle and 17β-estradiol groups; n=4 for all other groups.
Fig. 7
Fig. 7
Effects of estrogen on manganese superoxide dismutase (MnSOD). After 24 h treatment with estrogen and vehicle control, MnSOD mRNA, protein, and enzyme activity were measured. A. Quantitative real-time PCR measurement for MnSOD mRNA. Data were normalized to beta-actin as an internal control and expressed relative to vehicle control. Values are means ± SEM; P > 0.05; n=4. B. Mean MnSOD protein levels in mitochondria isolated from 17β-estradiol-treated and vehicle-treated control HBMEC and expressed relative to vehicle control. Values are means ± SEM; P > 0.05; n=4. C. Activity of MnSOD in mitochondria isolated from HBMEC treated with either 10 nM 17β-estradiol or vehicle control for 24 h expressed relative to vehicle control. Values are means ± SEM; P > 0.05; n=6.
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