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Ref-1 suppressed protein kinase C-induced mitochondrial dysfunction in mouse endothelial cells
Mitochondrion 17 (2014) 42–49
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MitoMatters
Mitochondrial APE1/Ref-1 suppressed protein kinase C-induced mitochondrial dysfunction in mouse endothelial cells Hee Kyoung Joo a,b, Yu Ran Lee a,b, Myoung Soo Park a,b, Sunga Choi a,b, Kyoungsook Park c, Sang Ki Lee d, Cuk-Seong Kim a, Jin Bong Park a,b, Byeong Hwa Jeon a,b,⁎ a
Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea BioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea d Department of Sports Science, Chungnam National University, Daejeon 305-765, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 26 September 2013 Received in revised form 23 April 2014 Accepted 15 May 2014 Available online 23 May 2014 Keywords: APE1/Ref-1 Endothelial cell Mitochondria Phorbol 12-myristate 13-acetate Mitochondrial membrane potential
a b s t r a c t Protein kinase C (PKC) induces mitochondrial dysfunction, which is an important pathological factor in cardiovascular diseases. The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) on PKC-induced mitochondrial dysfunction has not been variously investigated. In this study, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced mitochondrial hyperpolarization and reactive oxygen species generation and also increased mitochondrial translocation of APE1/Ref-1. APE1/Ref-1 overexpression suppressed PMA-induced mitochondrial dysfunction. In contrast, gene silencing of APE1/Ref-1 increased the sensitivity of mitochondrial dysfunction. Moreover, mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 more effectively suppressed PMA-induced mitochondrial dysfunctions. These results suggest that mitochondrial APE1/Ref-1 is contributed to the protective role to protein kinase C-induced mitochondrial dysfunction in endothelial cells. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Disturbances in vascular function contribute to the development of cardiovascular diseases such as atherosclerosis. Because endothelial cells provide the frontline defense against disturbances that influence vascular function, endothelial dysfunction is a key early event in the pathogenesis of cardiovascular diseases. Specifically, endothelial mitochondria are critical targets of oxidative stress and play a crucial role in mediating cellular responses (Davidson and Duchen, 2007; Kluge et al., 2013). Mitochondrial membrane potentials drive the synthesis of ATP, and changes in the membrane potential indicate mitochondrial dysfunction (Junesch and Graber, 1991; Kadenbach, 2003). Sustained mitochondrial hyperpolarization increases the accidental transfer of electrons to oxygen to generate superoxide, or mitochondrial reactive oxygen species (ROS) (Andrews et al., 2005; Brownlee, 2001; Liu, 1997). Cardiovascular risk factors such as oxidized lowdensity lipoprotein, high glucose, and ischemic injury cause the
Abbreviations: APE1/Ref-1, apurinic/apyrimidinic endonuclease-1/redox factor-1; NLS, nuclear localization signal; MTS, mitochondrial targeting sequence; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; ROS, reactive oxygen species. ⁎ Corresponding author at: Department of Physiology, School of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea. Tel.: +82 42 580 8214; fax: +82 42 585 8440.
http://dx.doi.org/10.1016/j.mito.2014.05.006 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
mitochondrial hyperpolarization that precedes cellular dysfunction (Giovannini et al., 2002; Munusamy and MacMillan-Crow, 2009; Sommer et al., 2011). Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), disrupts mitochondrial membrane potential and increases the generation of mitochondrial ROS, leading to mitochondrial dysfunction (Majumder et al., 2000; Wang et al., 2006). Endothelial mitochondria are critical targets of oxidative stress and thus play a crucial role in the signaling that mediates cellular responses (Davidson and Duchen, 2007; Kluge et al., 2013). The activation of PKC regulates various vascular functions by modulating enzymatic activity and gene expression. PKC activation, especially PKCβ isoform, has been reported to induce the impairment of endothelium-dependent relaxations by increasing the phosphorylation of endothelial nitric oxide synthase at threonine 495 in endothelial cells (Chiasson et al., 2011). Overexpression of PKCβ2 increases p66shc phosphorylation which mediates ROS production in endothelial cells (Lee et al., 2011) and it also can exacerbate atherosclerosis in the aorta (Li et al., 2013). Also, PKC activation up-regulates adhesion molecule expression such as vascular cell adhesion molecule-1, suggesting the important role of PKC signaling on endothelial cell activation in vascular inflammatory disorders (Quagliaro et al., 2005). Apurinic apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is important for many physiological and pathological processes in a variety of cells (Bhakat et al., 2009). APE1/Ref-1 reduces the intracellular
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production of ROS and thus prevents oxidative stress (Angkeow et al., 2002; Guo et al., 2008; Ozaki et al., 2002). The activity of APE1/Ref-1 is highly dependent on its subcellular localization. The N terminus of APE1/Ref-1 contains a nuclear localization signal (NLS) and the residues 289–318 at the C terminus of APE1/Ref-1 contain its mitochondrial targeting sequence (MTS) (Jackson et al., 2005; Jeon et al., 2004; Perrino et al., 2010). Under basal conditions, APE1/Ref-1 localizes mainly in the nucleus. However, mitochondrial translocation of APE1/ Ref-1 occurs under conditions of oxidative stress or mitochondrial DNA damage (Perrino et al., 2010; Tell et al., 2005), and APE1/Ref-1 regulates mitochondrial function to protect cells after photodynamic therapy (Li et al., 2012). We hypothesized that PKC activation induces the mitochondrial translocation of APE1/Ref-1 and that mitochondrial APE1/Ref-1 protects PMA-induced mitochondrial dysfunction. To test this hypothesis, we investigated the effect of APE1/Ref-1 and MTS-tagged APE1/Ref-1 on PMA-induced changes in mitochondrial membrane potential and mitochondrial ROS generation in mouse endothelial cells. 2. Materials and methods 2.1. Reagents Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, was purchased from Sigma-Aldrich (St. Louis, MO). Anilinomonoindolylmaleimide (3-(1-(3-imidazol-1-ylpropyl)-1H-indol-3yl)-4-anilino-1H-pyrrole-2,5-dione), a PKCβ-specific inhibitor (PKCβi) (Tanaka et al., 2004), was purchased from Calbiochem. Triphenylphosphonium chloride (2-(2,2,6,6-Tetramethylpiperidin1-oxyl-4-ylamino)-2-oxoethyl) (Mito-TEMPO), a mitochondria specific antioxidant, was purchased from Enzo Life Sciences (Farmingdale, NY). Specific antibodies against APE1/Ref-1 (Abcam, Cambridge, MA), β-actin or FLAG (Sigma, MO), COX-IV (Cell Signaling, MA), and PARP (Santa Cruz Biotechnology, CA) were used in this study. 2.2. Plasmid construction Full-length human APE1/Ref-1 cDNA was subcloned into pCMVTag2 (Clontech, CA) to generate pCMV-hAPE1/Ref-1. The plasmid for mitochondrial targeting sequence (MTS)-tagged APE1/Ref-1 (MTS-APE1/Ref-1) was generated by addition of MTS cDNA (MLSRAVCGTSRQLAPVLGYLGSRQ) to a putative nuclear localization signal (NLS, N-terminus 1–28 amino acid)-deleted APE1/Ref-1 cDNA. EGFP-tagged MTS-APE1/Ref-1 was generated by inserting the MTS-APE1/Ref-1 cDNA into pEGFP-N1 (Clontech, CA). 2.3. Cell culture and transfection The mouse pancreatic endothelial cell line MS-1 (CRL-2279™) was purchased from American Type Culture Collection (Manassas, VA) and grown in DMEM supplemented with 5% fetal bovine serum (FBS) and antibiotics. Transient transfections of MS-1 cells were performed using Effectene transfection reagent (Qiagen, Valencia, CA), as recommended by the manufacturer. Typically, 1 × 105 cells were grown in 6-well plates and transiently transfected with 0.4 μg of plasmid encoding APE1/Ref-1. Transfected cells were washed 24 h later in phosphate-buffered saline (PBS), and harvested for analysis. Small interfering RNA (siRNA) oligonucleotides against mouse APE1/Ref-1 and a control scrambled siRNA were purchased from Bioneer Co. (Daejeon, South Korea); the siRNA targeting mouse APE1/ Ref-1 had the following gene-specific sense sequence: 5′-GUC UGG UAA GAC UGG AGU ACC-3′. Cells were transfected with 10 nM APE1/ Ref-1-specific or scrambled siRNA. The siRNAs and Lipofectamine 2000 (Invitrogen, San Diego, CA) were diluted separately in OptiMEM medium and incubated for 5 min at room temperature, and then
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siRNA/Lipofectamine mixtures were incubated for 20 min before incubating them with the cells for 48 h. 2.4. Assessment of mitochondrial membrane potential Mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester perchlorate (TMRE), a lipophilic cationic probe (Molecular Probes, Eugene, OR). Briefly, cells were plated in 6-well plates and incubated with PMA for 1 h, and then exposed to 100 nM TMRE for 10 min at 37 °C in the dark. After washing with PBS, fluorescence intensity was measured at 590 nm using a plate reader. The data are presented as fold-changes in the mean intensity of TMRE fluorescence relative to controls. Fluorescence images were obtained by microscopy (Carl Zeiss Inc., Thornwood, NY). 2.5. Detection of mitochondrial ROS Mitochondrial superoxide production was assessed using MitoSox red (Molecular Probes), a mitochondrion-specific hydroethidinederivative fluorescent dye that undergoes oxidation to form the DNAbinding red fluorophore ethidium bromide. After 6 h of PMA exposure, cells were incubated with 5 μM MitoSox red for 10 min in a CO2 incubator. Fluorescence was measured at 590 nm. The data are presented as fold-changes in the mean intensity of MitoSox fluorescence relative to controls. Fluorescence images were obtained by microscopy (Carl Zeiss Inc.). In some experiments, mitochondrial ROS generation was also analyzed by subtracting the fluorescence intensity with MitoTEMPO, a specific antioxidant to mitochondria, from the total fluorescence intensity with 2′,7′-dichlorofluorescein diacetate (DCFDA, Molecular Probes). 2.6. Localization of APE1/Ref-1 Cells (5 × 104 cells) were cultured on glass coverslips in 12-well plates and then transiently transfected with 0.3 μg of EGFP-tagged APE1/Ref-1 plasmids as described in Section 2.3. At 24 h after transfection, the cells were exposed to 250 nM PMA for 6 h. The medium was removed and the cells were incubated in a Hank's Balanced Salt Solution (HBSS) buffer containing 250 nM MitoTracker Red CMXRos probe (Molecular Probes) for 15 min at 37 °C. After washing twice with PBS, the cells were fixed with 4% (w/v) paraformaldehyde (PFA) in PBS at room temperature for 15 min. After washing, the cells were stained with DAPI nuclear probe (Molecular Probes). The coverslips were mounted on microscope slides for examination under a fluorescence microscope. For immunocytochemistry, cells were cultured on glass coverslips in 12-well plates, incubated with MitoTracker Red, and fixed with 4% PFA. After blocking for 1 h with PBS containing 2% BSA and 5% horse serum, cells were incubated with APE1/Ref-1 antibody (1:500) overnight at 4 °C and then with Alexa488-conjugated secondary antibody (1:500) for 1 h in the dark at room temperature. Images were obtained by fluorescence microscopy. 2.7. Western blot analysis For western blotting, cells were harvested using 100 μL lysis buffer (20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, phosphatase-inhibitor cocktail, and protease-inhibitor cocktail). Cell homogenates (30 μg) were separated by 12% SDS-PAGE and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h at room temperature, blots were incubated overnight at 4 °C with specific primary antibodies (1:1000 anti-PARP, 1:1000 anti-COX-IV, 1:2000 anti-APE1/Ref-1, and 1:5000 anti-β-actin), which were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies. Blots
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were developed for visualization using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). Nuclear and mitochondrial fractions were prepared using a cell fractionation kit (Abcam), as recommended by the manufacturer. The purity of each fraction was confirmed using antibodies against the nuclear marker PARP and the mitochondrial marker COX-IV, and the fractions were examined for their APE1/Ref-1 content by western blotting. 2.8. Statistical analysis Statistical significance of differences in measured variables between control and PMA-treated groups was determined using one-way ANOVA followed by a Tukey's post-hoc test, and p b 0.05 was considered statistically significant. Values are expressed as means ± SEM. 3. Results 3.1. PMA induced mitochondrial hyperpolarization and mitochondrial ROS generation in mouse endothelial MS-1 cells PMA has been shown to disrupt mitochondrial membrane potential and induce mitochondrial ROS production, leading to mitochondrial dysfunction (Majumder et al., 2000; Wang et al., 2006). To assess the effects of PMA in mouse endothelial MS-1 cells, we first measured the changes in mitochondrial membrane potential and ROS generation after PMA treatment. PMA treatment induced a significant dosedependent increase in TMRE fluorescence intensity in mouse endothelial MS-1 cells, indicating mitochondrial hyperpolarization (Fig. 1A). Fluorescence intensity was elevated 1.98-fold in cells exposed to 250 nM PMA for 1 h relative to non-stimulated cells. The representative fluorescence images shown in Fig. 1A also suggest that PMA induced mitochondrial hyperpolarization in mouse endothelial MS-1 cells. The change in mitochondrial membrane potential also causes mitochondrial dysfunction by increasing the accidental transfer of
electrons to oxygen, thus generating superoxide or ROS (Andrews et al., 2005; Brownlee, 2001; Liu, 1997). To assess the effects of PMA on mitochondrial ROS generation in mouse endothelial MS-1 cells, we used MitoSox dye. PMA induced a dose-dependent increase in MitoSox fluorescence intensity, indicating mitochondrial ROS generation; the fluorescent signal obtained with 250 nM PMA was 1.87-fold higher than that obtained without PMA stimulation. The representative fluorescence photomicrographs shown in Fig. 1B indicate that PMA exposure induced mitochondrial ROS generation in mouse endothelial MS-1 cells. The above results together suggest that PMA induces mitochondrial dysfunction in mouse endothelial MS-1 cells. 3.2. Mitochondrial APE1/Ref-1 levels were elevated by PMA Mitochondrial APE1/Ref-1 levels increase in response to oxidative stress or mitochondrial DNA damage (Perrino et al., 2010). We investigated the localization of APE1/Ref-1 after exposing cells to PMA. Mouse endothelial MS-1 cells were treated with PMA and then nuclear and mitochondrial fractions of the cells were examined for APE1/Ref-1 through Western blotting. As shown in Fig. 2A, PMA increased mitochondrial APE1/Ref-1 expressions, in contrast, nuclear APE1/Ref-1 levels were decreased in response to PMA. Exposure to PMA for 1 h and 6 h elevated mitochondrial APE1/Ref-1 levels 1.6- and 1.8-fold over control levels (Fig. 2B). We confirmed the change in the subcellular distribution of APE1/ Ref-1 in PMA-stimulated cells by immunofluorescence staining; mitochondria and nuclei were visualized using MitoTracker (red) and DAPI (blue) staining, respectively. Consistent with our Western blotting results, APE1/Ref-1 intensity was increased in mitochondria after PMA treatment (Fig. 2C), suggesting that APE1/Ref-1 is translocated to mitochondria in endothelial cells in response to stimulation with PMA. Next, we investigated the effect of PKCβII inhibitor on PMA-induced mitochondrial APE1/Ref-1 translocation. Pretreatment of PKCβi (10 nM), a specific PKCβII inhibitor, inhibited PMA-induced mitochondrial APE1/Ref-1 translocation (Supplementary Fig. 1), suggesting that
Fig. 1. Dose-dependent changes in mitochondrial membrane potential and reactive oxygen species (ROS) generation in response to phorbol 12-myristate 13-acetate (PMA) treatment in mouse endothelial MS-1 cells. (A) TMRE fluorescence and (B) MitoSox Red fluorescence in PMA-treated cells. Fluorescence emission was measured at 590 nm with excitation at 530 nm. Values are expressed as mean fold-changes in arbitrary fluorescence values over basal value. Each bar shows mean ± S.E. (n = 4). * p b 0.05 vs. basal value.
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Fig. 2. Elevation of mitochondrial APE1/Ref-1 in response to PMA in mouse endothelial MS-1 cells. (A) Western blots of mitochondrial and nuclear fractions of PMA-treated cells harvested after 1 and 6 h treatment. Staining for APE1/Ref-1, COX-IV (loading control for mitochondria), and PARP (loading control for nuclei) are shown. (B) Densitometric analysis of Western blots. Data are expressed as fold-change values relative to basal levels of mitochondrial and nuclear APE1/Ref-1. Each bar represents mean ± S.E. (n = 4). * p b 0.05 vs. basal value. (C) After 6-h of treatment with 250 nM PMA, cells were stained for APE1/Ref-1 (green), mitochondria (red), and nuclei (blue). Magnification, 400 ×. Fluorescence photomicrographs of control and PMA-treated cells; results are representative of three independent experiments.
Fig. 3. APE1/Ref-1 overexpression inhibited PMA-induced mitochondrial membrane hyperpolarization and mitochondrial ROS generation in mouse endothelial MS-1 cells. (A) FLAG-tagged APE1/Ref-1-transfected cells were exposed to 250 nM PMA for 1 h and then mitochondrial membrane potentials using TMRE fluorescence was measured. (B) FLAG-tagged APE1/Ref-1-transfected cells were exposed to 250 nM PMA for 6 h. Relative mitochondrial ROS generation was analyzed based on MitoSox fluorescence. Values are expressed as mean fold-change in arbitrary fluorescence values over basal value. Each bar shows mean ± S.E. (n = 5). * p b 0.05 vs. basal value, # p b 0.05 vs. PMA alone. FLAG-tagged APE1/Ref-1 overexpression was confirmed by anti-FLAG in Western blotting. β-actin was used as loading control (bottom).
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APE1/Ref-1 phosphorylation induced by PKCβII would be contributed to mitochondrial APE1/Ref-1 translocation in endothelial cells. 3.3. APE1/Ref-1 overexpression prevented PMA-induced mitochondrial hyperpolarization and ROS generation To gain insights into the functional significance of the increase in mitochondrial APE1/Ref-1 levels in response to PMA-induced stress, we examined how overexpressing FLAG-tagged APE1/Ref-1 in endothelial cells affected PMA-induced mitochondrial hyperpolarization and ROS generation. Interestingly, overexpression of APE1/Ref-1 prevented PMA-induced mitochondrial hyperpolarization (Fig. 3A). PMA treatment increased the mitochondrial membrane potential, but this increase in cells ectopically expressing APE1/Ref-1 was 39.3% lower than in control cells. Thus, APE1/Ref-1 rescued the PMA-induced disruption of mitochondrial membrane potential. Furthermore, overexpressing APE1/Ref-1 in endothelial cells inhibited mitochondrial ROS generation after PMA exposure (Fig. 3B). In APE1/Ref-1 overexpressing cells, mitochondrial ROS production was reduced by 55.6%, compared with that in PMA-treated control cells. The overexpression of FLAG-tagged APE1/ Ref-1 was confirmed with immunoreactivity of anti-FLAG in Western blotting (bottom of Fig. 3). 3.4. Downregulating APE1/Ref-1 increased PMA-induced mitochondrial hyperpolarization Having determined that APE1/Ref-1 overexpression inhibited PMA-induced mitochondrial dysfunction, we investigated whether downregulating APE1/Ref-1 affected PMA-dependent mitochondrial hyperpolarization. PMA-induced mitochondrial membrane potential was compared in mouse endothelial MS-1 cells transfected with an APE1/Ref-1-specific siRNA or a scrambled siRNA. As shown in Fig. 4, PMA (250 nM) significantly increased mitochondrial membrane potentials, but low dose of PMA (1–100 nM) did not significantly
increase the mitochondrial membrane potential in scramble siRNAtransfected cells. However, low dose of PMA (1–100 nM) significantly increased mitochondrial membrane potentials in APE1/Ref-1 siRNAtransfected cells, compared with each dose of scramble siRNA-treated cells. This data suggested that the gene silencing of APE1/Ref-1 increased the sensitivity of PMA-induced mitochondrial membrane potentials in endothelial cells. Western blot data showed that treatment for 48 h with 10 nM of APE1/Ref-1 siRNA, but not the scrambled siRNA, markedly reduced endogenous APE1/Ref-1 (bottom of Fig. 4). We also measured the mitochondrial ROS generation in response to PMA in endothelial cells transfected with the APE1/Ref-1-specific and scrambled siRNAs. To determine the contribution of mitochondrial ROS production, we used Mito-TEMPO, a mitochondria specific antioxidant (Dikalova et al., 2010). As results, gene silencing of APE1/ Ref-1 significantly increased basal or PMA-induced mitochondrial ROS generation (Supplementary Fig. 2). These results suggest that mitochondrial APE1/Ref-1 helps to maintain the homeostasis of the mitochondrial membrane potential and ROS. 3.5. Mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 was localized in mitochondria We next compared the effect of PMA on cells expressing mitochondrion-specific APE1/Ref-1 and wild-type APE1/Ref-1. First, we determined the subcellular localization of EGFP-tagged wild-type or MTS-APE1/Ref-1 in mouse endothelial MS-1 cells. Fluorescence imaging revealed that the green fluorescence of wild-type APE1/Ref-1 was mostly merged with blue DAPI fluorescence, indicating nuclear localization of the protein. By contrast, MTS-APE1/Ref-1 was localized in the mitochondria, producing a yellow-orange fluorescence (Fig. 5A). Western blot data against anti-FLAG showed subcellular localization of FLAG-tagged wild-type APE1/Ref-1 and MTS-APE1/Ref-1 expressed ectopically in mouse endothelial MS-1 cells. FLAG-tagged APE1/Ref-1 was detected mainly in the nuclear fraction, whereas FLAG-tagged MTS-APE1/Ref-1 was present in the mitochondrial fraction in endothelial cells (Fig. 5B). 3.6. Mitochondrial APE1/Ref-1 potently blocked PMA-induced disruption of mitochondrial function Finally, we investigated the effect of mitochondrion-specific MTSAPE1/Ref-1 on PMA-induced mitochondrial hyperpolarization and ROS generation. Surprisingly, MTS-APE1/Ref-1 suppressed PMAinduced mitochondrial hyperpolarization substantially more than wild-type APE1/Ref-1 (Fig. 6A): whereas wild-type APE1/Ref-1 produced 39.3% reduction, MTS-APE1/Ref-1 produced a 68.9% reduction in PMA-induced mitochondrial hyperpolarization. Furthermore, the PMA-induced increase in mitochondrial ROS generation was suppressed by 86.3% in cells expressing MTS-APE1/Ref-1 and by 55.6% in cells expressing wild-type APE1/Ref-1 (Fig. 6B). These results suggest that MTS-tagged APE1/Ref-1 rescues PMA-induced mitochondrial dysfunction in mouse endothelial MS-1 cells. 4. Discussion
Fig. 4. Effect of APE1/Ref-1 downregulation on PMA-induced mitochondrial hyperpolarization in mouse endothelial MS-1 cells. At 48 h after transfection with scrambled siRNA or APE1/Ref-1-specific siRNA (10 nM), cells were exposed to 1 or 250 nM PMA for 1 h. Relative mitochondrial membrane potential was analyzed using a fluorometer to measure TMRE fluorescence. Values are expressed as mean fold-change in arbitrary fluorescence values over basal values. Each bar shows mean ± S.E. (n = 4). * p b 0.05 vs. basal value, # p b 0.05 vs. scramble siRNA. Downregulation of APE1/Ref-1 using APE1/Ref-1-specific siRNA was confirmed by anti-APE1/Ref-1 in Western blotting. β-actin was used as loading control (bottom).
Our objective in this study was to determine the role of mitochondrial APE1/Ref-1 in PKC-induced mitochondrial dysfunction in endothelial cells. Our results showed that PKC activation induced mitochondrial translocation of APE1/Ref-1, mitochondrial APE1/Ref-1 suppressed PMA-induced mitochondrial hyperpolarization and ROS production in endothelial cells. PMA induces the changes of mitochondrial membrane potentials and mitochondrial ROS generation, leading to mitochondrial dysfunction (Majumder et al., 2000; Wang et al., 2006). Our data showed that PMA induced mitochondrial hyperpolarization in mouse endothelial MS-1 cells, which indicates that the protonic
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Fig. 5. Subcellular localization of mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 in mouse endothelial MS-1 cells. (A) Immunofluorescence images showed the subcellular localization of EGFP-tagged wild-type APE1/Ref-1 (WT) and MTS-APE1/Ref-1. MitoTracker (red) indicates mitochondria and DAPI (blue) indicates nuclei. Immunofluorescence images were obtained with fluorescence microscopy, magnification, 400×. (B) Western blot data showed subcellular localization of APE1/Ref-1 in nuclear (N) and mitochondrial (M) fractions of FLAG-tagged wild-type APE1/Ref-1- and MTS-APE1/Ref-1-transfected endothelial cells. Western blot was performed with anti-FLAG for FLAG-APE1/Ref-1 and FLAG-MTS-APE1/Ref-1. COX-IV and PARP served as loading controls for mitochondria and nuclei, respectively.
potential across the mitochondrion was increased. However, how PMA increases mitochondrial membrane potential remains unclear. PMA treatment activates several PKCs belonging to both classical and non-conventional PKC families (Goel et al., 2007). Mitochondrial hyperpolarization triggered by PKCα and PKCε activation leads to mitochondrial dysfunction in renal proximal tubular cells. The mitochondrial membrane potential depends on the activity of F0F1ATPase, which allows protons to return to the mitochondrial matrix, and the suppression of F0 F1 -ATPase activity in response to the
activation of PKCα and PKCε results in mitochondrial hyperpolarization (Nowak, 2002; Nowak et al., 2011). Moreover, activated PKCε phosphorylates voltage-dependent anion channel-1 (VDAC-1), which locks the channel in the closed conformation and leads to mitochondrial hyperpolarization (Baines et al., 2003). Thus, PKC activation by PMA could induce VDAC-1 closure or reduce F0F1-ATPase activity to cause mitochondrial hyperpolarization. A substantial increase in mitochondrial membrane potential has been reported to enhance mitochondrial production of ROS (Andrews et al., 2005;
Fig. 6. Effect of mitochondrial APE1/Ref-1 on mitochondrial membrane potential and mitochondrial ROS generation in PMA-stimulated mouse endothelial MS-1 cells. Cells transfected with FLAG-tagged wild-type APE1/Ref-1 and MTS-APE1/Ref-1 were exposed to 250 nM PMA for 1 h. Relative mitochondrial membrane potential was analyzed by measuring TMRE fluorescence. (B) FLAG-tagged APE1/Ref-1- and MTS-APE1/Ref-1-transfected cells were exposed to 250 nM PMA for 6 h. Relative mitochondrial ROS generation was analyzed by measuring MitoSox fluorescence. Values are expressed as mean fold-change in arbitrary fluorescence values over basal values. Each bar shows mean ± S.E. (n = 5). * p b 0.05 vs. basal value, # p b 0.05 vs. PMA alone, ## p b 0.05 vs. PMA with wild-type APE1/Ref-1.
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Brownlee, 2001; Korshunov et al., 1997). In this study, we confirmed that PMA induced mitochondrial hyperpolarization and mitochondrial ROS generation, leading to mitochondrial dysfunction in mouse endothelial cells. Interestingly, our data showed that overexpressing APE1/Ref-1 in endothelial cells significantly inhibited PMA-induced mitochondrial hyperpolarization and ROS generation. APE1/Ref-1 is dynamically regulated at both transcriptional and post-translational levels through the control of its subcellular localization and posttranslational modification. The role of APE1/Ref-1 in various subcellular and nuclear functions has been reported. For example, mitochondrial APE1/Ref-1 has been proposed recently to protect cells by regulating mitochondrial function after photodynamic therapy (Li et al., 2012). In this study, the expression of mitochondrionspecific MTS-APE1/Ref-1 suppressed PMA-induced mitochondrial hyperpolarization and ROS generation considerably more potently than the expression of wild-type APE1/Ref-1. However, the mechanism by which mitochondrial APE1/Ref-1 inhibits PMA-induced mitochondrial hyperpolarization and ROS generation is unclear. APE1/Ref-1 inhibition of mitochondrial hyperpolarization could also be related with the redox function to the mitochondrial uncoupling system. Uncoupling proteins such as UCP-2 and ANT-1 dissipate the mitochondrial proton gradient and stabilize the inner mitochondrial membrane potential and thereby reduce the formation of ROS (Haines and Li, 2012). APE1/Ref-1 may regulate the activity of uncoupling proteins and thus normalize mitochondrial hyperpolarization. Another possibility of the redox function of APE1/Ref-1 is that APE1/Ref-1 governed PKC-induced mitochondrial p66shc phosphorylation which mediated ROS production in endothelial cells (Lee et al., 2011). Mitochondrial DNA is a major site of oxidative stress damage and increased oxidative stress is closely linked with mitochondrial dysfunctions. Moreover, APE1/Ref-1 is localized to the mitochondria in response to oxidative stress (Perrino et al., 2010; Tell et al., 2005). Therefore, in addition of the redox function of APE1/Ref-1, DNA repair function would contribute in protecting PMA-induced mitochondrial dysfunctions. In this study, APE1/Ref-1 expression in mitochondrial fractions was increased within 1 h after PMA treatment, suggesting the mitochondrial translocation of APE1/Ref-1. Most mitochondrial proteins are encoded by nuclear genes and then translocated into the mitochondria. Recently, the MTS of APE1/Ref-1 was identified within residues 289–318 at the C terminus, which is normally masked by the nuclear localization signal (Perrino et al., 2010). This existence of an MTS in APE1/Ref-1 may explain the conditional mitochondrial localization of the protein in response to oxidative stress. How APE1/Ref-1 is translocated into the mitochondria is not fully established, although Tom 20 protein, a translocase of the mitochondrial outer membrane, may play a key role in the mitochondrial translocation of APE1/Ref-1. In the present study, PKC activation induced mitochondrial translocation of APE1/Ref-1 which was inhibited by PKCβΙΙ inhibitor, an anilinomonoindolylmaleimide compound, as a potent, cell-permeable, reversible, and ATP-competitive inhibitor of PKCβ isozymes (IC50 = 5 nM and 21 nM for human PKCβΙΙ and βΙ, respectively (Tanaka et al., 2004). It suggested that the phosphorylation of APE1/ Ref-1 by PKCβΙΙ would be contributed to mitochondrial translocation of APE1/Ref-1 in endothelial cells (Supplementary Fig. 1). Another possibility for mitochondrial translocation of APE1/Ref-1 is that the phosphorylation of nuclear localization signal of Nterminus of APE1/Ref-1 by PKC activation might unmask MTS signals, then phosphorylated APE1/Ref-1 can be translocated into the mitochondria. Our findings suggest that mitochondrial translocation of APE1/Ref-1 reverses the PMA-induced increase in mitochondrial membrane potential and ROS production. Moreover, we have demonstrated this effect of APE1/Ref-1 on endothelial mitochondrial function, which is a critical factor in several vascular disorders. Thus, our results suggest that
mitochondrial regulation by APE1/Ref-1 may play a therapeutic role in vascular diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2014.05.006. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (20110016797, 2007-0054932). The English grammar has been checked by a professional editor who is a native speaker of English. References Andrews, Z.B., Diano, S., Horvath, T.L., 2005. Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Nat. Rev. Neurosci. 6, 829–840. Angkeow, P., Deshpande, S.S., Qi, B., Liu, Y.X., Park, Y.C., Jeon, B.H., Ozaki, M., Irani, K., 2002. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death Differ. 9, 717–725. Baines, C.P., Song, C.X., Zheng, Y.T., Wang, G.W., Zhang, J., Wang, O.L., Guo, Y., Bolli, R., Cardwell, E.M., Ping, P., 2003. Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circ. Res. 92, 873–880. Bhakat, K.K., Mantha, A.K., Mitra, S., 2009. Transcriptional regulatory functions of mammalian AP-endonuclease (APE1/Ref-1), an essential multifunctional protein. Antioxid. Redox Signal. 11, 621–638. Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820. Chiasson, V.L., Quinn, M.A., Young, K.J., Mitchell, B.M., 2011. Protein kinase CbetaIImediated phosphorylation of endothelial nitric oxide synthase threonine 495 mediates the endothelial dysfunction induced by FK506 (tacrolimus). J. Pharmacol. Exp. Ther. 337, 718–723. Davidson, S.M., Duchen, M.R., 2007. Endothelial mitochondria: contributing to vascular function and disease. Circ. Res. 100, 1128–1141. Dikalova, A.E., Bikineyeva, A.T., Budzyn, K., Nazarewicz, R.R., McCann, L., Lewis, W., Harrison, D.G., Dikalov, S.I., 2010. 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FEBS Lett. 416, 15–18. Lee, S.K., Chung, J.I., Park, M.S., Joo, H.K., Lee, E.J., Cho, E.J., Park, J.B., Ryoo, S., Irani, K., Jeon, B.H., 2011. Apurinic/apyrimidinic endonuclease 1 inhibits protein kinase C-mediated p66shc phosphorylation and vasoconstriction. Cardiovasc. Res. 91, 502–509. Li, M.X., Shan, J.L., Wang, D., He, Y., Zhou, Q., Xia, L., Zeng, L.L., Li, Z.P., Wang, G., Yang, Z.Z., 2012. Human apurinic/apyrimidinic endonuclease 1 translocalizes to mitochondria after photodynamic therapy and protects cells from apoptosis. Cancer Sci. 103, 882–888. Li, Q., Park, K., Li, C., Rask-Madsen, C., Mima, A., Qi, W., Mizutani, K., Huang, P., King, G.L., 2013. Induction of vascular insulin resistance and endothelin-1 expression and acceleration of atherosclerosis by the overexpression of protein kinase C-beta isoform in the endothelium. Circ. Res. 113, 418–427. Liu, S.S., 1997. Generating, partitioning, targeting and functioning of superoxide in mitochondria. Biosci. Rep. 17, 259–272. 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human umbilical vein endothelial cells in culture: the distinct role of protein kinase C and mitochondrial superoxide production. Atherosclerosis 183, 259–267. Sommer, S.P., Sommer, S., Sinha, B., Wiedemann, J., Otto, C., Aleksic, I., Schimmer, C., Leyh, R.G., 2011. Ischemia–reperfusion injury-induced pulmonary mitochondrial damage. J. Heart Lung Transplant. 30, 811–818. Tanaka, M., Sagawa, S., Hoshi, J., Shimoma, F., Matsuda, I., Sakoda, K., Sasase, T., Shindo, M., Inaba, T., 2004. Synthesis of anilino-monoindolylmaleimides as potent and selective PKCbeta inhibitors. Bioorg. Med. Chem. Lett. 14, 5171–5174. Tell, G., Damante, G., Caldwell, D., Kelley, M.R., 2005. The intracellular localization of APE1/Ref-1: more than a passive phenomenon? Antioxid. Redox Signal. 7, 367–384. Wang, Y., Biswas, G., Prabu, S.K., Avadhani, N.G., 2006. Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Calpha in C2C12 myocytes. Biochem. Pharmacol. 72, 881–892.
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MitoMatters
Mitochondrial APE1/Ref-1 suppressed protein kinase C-induced mitochondrial dysfunction in mouse endothelial cells Hee Kyoung Joo a,b, Yu Ran Lee a,b, Myoung Soo Park a,b, Sunga Choi a,b, Kyoungsook Park c, Sang Ki Lee d, Cuk-Seong Kim a, Jin Bong Park a,b, Byeong Hwa Jeon a,b,⁎ a
Department of Physiology, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea BioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea d Department of Sports Science, Chungnam National University, Daejeon 305-765, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 26 September 2013 Received in revised form 23 April 2014 Accepted 15 May 2014 Available online 23 May 2014 Keywords: APE1/Ref-1 Endothelial cell Mitochondria Phorbol 12-myristate 13-acetate Mitochondrial membrane potential
a b s t r a c t Protein kinase C (PKC) induces mitochondrial dysfunction, which is an important pathological factor in cardiovascular diseases. The role of apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) on PKC-induced mitochondrial dysfunction has not been variously investigated. In this study, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, induced mitochondrial hyperpolarization and reactive oxygen species generation and also increased mitochondrial translocation of APE1/Ref-1. APE1/Ref-1 overexpression suppressed PMA-induced mitochondrial dysfunction. In contrast, gene silencing of APE1/Ref-1 increased the sensitivity of mitochondrial dysfunction. Moreover, mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 more effectively suppressed PMA-induced mitochondrial dysfunctions. These results suggest that mitochondrial APE1/Ref-1 is contributed to the protective role to protein kinase C-induced mitochondrial dysfunction in endothelial cells. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Disturbances in vascular function contribute to the development of cardiovascular diseases such as atherosclerosis. Because endothelial cells provide the frontline defense against disturbances that influence vascular function, endothelial dysfunction is a key early event in the pathogenesis of cardiovascular diseases. Specifically, endothelial mitochondria are critical targets of oxidative stress and play a crucial role in mediating cellular responses (Davidson and Duchen, 2007; Kluge et al., 2013). Mitochondrial membrane potentials drive the synthesis of ATP, and changes in the membrane potential indicate mitochondrial dysfunction (Junesch and Graber, 1991; Kadenbach, 2003). Sustained mitochondrial hyperpolarization increases the accidental transfer of electrons to oxygen to generate superoxide, or mitochondrial reactive oxygen species (ROS) (Andrews et al., 2005; Brownlee, 2001; Liu, 1997). Cardiovascular risk factors such as oxidized lowdensity lipoprotein, high glucose, and ischemic injury cause the
Abbreviations: APE1/Ref-1, apurinic/apyrimidinic endonuclease-1/redox factor-1; NLS, nuclear localization signal; MTS, mitochondrial targeting sequence; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; ROS, reactive oxygen species. ⁎ Corresponding author at: Department of Physiology, School of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea. Tel.: +82 42 580 8214; fax: +82 42 585 8440.
http://dx.doi.org/10.1016/j.mito.2014.05.006 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
mitochondrial hyperpolarization that precedes cellular dysfunction (Giovannini et al., 2002; Munusamy and MacMillan-Crow, 2009; Sommer et al., 2011). Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), disrupts mitochondrial membrane potential and increases the generation of mitochondrial ROS, leading to mitochondrial dysfunction (Majumder et al., 2000; Wang et al., 2006). Endothelial mitochondria are critical targets of oxidative stress and thus play a crucial role in the signaling that mediates cellular responses (Davidson and Duchen, 2007; Kluge et al., 2013). The activation of PKC regulates various vascular functions by modulating enzymatic activity and gene expression. PKC activation, especially PKCβ isoform, has been reported to induce the impairment of endothelium-dependent relaxations by increasing the phosphorylation of endothelial nitric oxide synthase at threonine 495 in endothelial cells (Chiasson et al., 2011). Overexpression of PKCβ2 increases p66shc phosphorylation which mediates ROS production in endothelial cells (Lee et al., 2011) and it also can exacerbate atherosclerosis in the aorta (Li et al., 2013). Also, PKC activation up-regulates adhesion molecule expression such as vascular cell adhesion molecule-1, suggesting the important role of PKC signaling on endothelial cell activation in vascular inflammatory disorders (Quagliaro et al., 2005). Apurinic apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is important for many physiological and pathological processes in a variety of cells (Bhakat et al., 2009). APE1/Ref-1 reduces the intracellular
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production of ROS and thus prevents oxidative stress (Angkeow et al., 2002; Guo et al., 2008; Ozaki et al., 2002). The activity of APE1/Ref-1 is highly dependent on its subcellular localization. The N terminus of APE1/Ref-1 contains a nuclear localization signal (NLS) and the residues 289–318 at the C terminus of APE1/Ref-1 contain its mitochondrial targeting sequence (MTS) (Jackson et al., 2005; Jeon et al., 2004; Perrino et al., 2010). Under basal conditions, APE1/Ref-1 localizes mainly in the nucleus. However, mitochondrial translocation of APE1/ Ref-1 occurs under conditions of oxidative stress or mitochondrial DNA damage (Perrino et al., 2010; Tell et al., 2005), and APE1/Ref-1 regulates mitochondrial function to protect cells after photodynamic therapy (Li et al., 2012). We hypothesized that PKC activation induces the mitochondrial translocation of APE1/Ref-1 and that mitochondrial APE1/Ref-1 protects PMA-induced mitochondrial dysfunction. To test this hypothesis, we investigated the effect of APE1/Ref-1 and MTS-tagged APE1/Ref-1 on PMA-induced changes in mitochondrial membrane potential and mitochondrial ROS generation in mouse endothelial cells. 2. Materials and methods 2.1. Reagents Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, was purchased from Sigma-Aldrich (St. Louis, MO). Anilinomonoindolylmaleimide (3-(1-(3-imidazol-1-ylpropyl)-1H-indol-3yl)-4-anilino-1H-pyrrole-2,5-dione), a PKCβ-specific inhibitor (PKCβi) (Tanaka et al., 2004), was purchased from Calbiochem. Triphenylphosphonium chloride (2-(2,2,6,6-Tetramethylpiperidin1-oxyl-4-ylamino)-2-oxoethyl) (Mito-TEMPO), a mitochondria specific antioxidant, was purchased from Enzo Life Sciences (Farmingdale, NY). Specific antibodies against APE1/Ref-1 (Abcam, Cambridge, MA), β-actin or FLAG (Sigma, MO), COX-IV (Cell Signaling, MA), and PARP (Santa Cruz Biotechnology, CA) were used in this study. 2.2. Plasmid construction Full-length human APE1/Ref-1 cDNA was subcloned into pCMVTag2 (Clontech, CA) to generate pCMV-hAPE1/Ref-1. The plasmid for mitochondrial targeting sequence (MTS)-tagged APE1/Ref-1 (MTS-APE1/Ref-1) was generated by addition of MTS cDNA (MLSRAVCGTSRQLAPVLGYLGSRQ) to a putative nuclear localization signal (NLS, N-terminus 1–28 amino acid)-deleted APE1/Ref-1 cDNA. EGFP-tagged MTS-APE1/Ref-1 was generated by inserting the MTS-APE1/Ref-1 cDNA into pEGFP-N1 (Clontech, CA). 2.3. Cell culture and transfection The mouse pancreatic endothelial cell line MS-1 (CRL-2279™) was purchased from American Type Culture Collection (Manassas, VA) and grown in DMEM supplemented with 5% fetal bovine serum (FBS) and antibiotics. Transient transfections of MS-1 cells were performed using Effectene transfection reagent (Qiagen, Valencia, CA), as recommended by the manufacturer. Typically, 1 × 105 cells were grown in 6-well plates and transiently transfected with 0.4 μg of plasmid encoding APE1/Ref-1. Transfected cells were washed 24 h later in phosphate-buffered saline (PBS), and harvested for analysis. Small interfering RNA (siRNA) oligonucleotides against mouse APE1/Ref-1 and a control scrambled siRNA were purchased from Bioneer Co. (Daejeon, South Korea); the siRNA targeting mouse APE1/ Ref-1 had the following gene-specific sense sequence: 5′-GUC UGG UAA GAC UGG AGU ACC-3′. Cells were transfected with 10 nM APE1/ Ref-1-specific or scrambled siRNA. The siRNAs and Lipofectamine 2000 (Invitrogen, San Diego, CA) were diluted separately in OptiMEM medium and incubated for 5 min at room temperature, and then
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siRNA/Lipofectamine mixtures were incubated for 20 min before incubating them with the cells for 48 h. 2.4. Assessment of mitochondrial membrane potential Mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester perchlorate (TMRE), a lipophilic cationic probe (Molecular Probes, Eugene, OR). Briefly, cells were plated in 6-well plates and incubated with PMA for 1 h, and then exposed to 100 nM TMRE for 10 min at 37 °C in the dark. After washing with PBS, fluorescence intensity was measured at 590 nm using a plate reader. The data are presented as fold-changes in the mean intensity of TMRE fluorescence relative to controls. Fluorescence images were obtained by microscopy (Carl Zeiss Inc., Thornwood, NY). 2.5. Detection of mitochondrial ROS Mitochondrial superoxide production was assessed using MitoSox red (Molecular Probes), a mitochondrion-specific hydroethidinederivative fluorescent dye that undergoes oxidation to form the DNAbinding red fluorophore ethidium bromide. After 6 h of PMA exposure, cells were incubated with 5 μM MitoSox red for 10 min in a CO2 incubator. Fluorescence was measured at 590 nm. The data are presented as fold-changes in the mean intensity of MitoSox fluorescence relative to controls. Fluorescence images were obtained by microscopy (Carl Zeiss Inc.). In some experiments, mitochondrial ROS generation was also analyzed by subtracting the fluorescence intensity with MitoTEMPO, a specific antioxidant to mitochondria, from the total fluorescence intensity with 2′,7′-dichlorofluorescein diacetate (DCFDA, Molecular Probes). 2.6. Localization of APE1/Ref-1 Cells (5 × 104 cells) were cultured on glass coverslips in 12-well plates and then transiently transfected with 0.3 μg of EGFP-tagged APE1/Ref-1 plasmids as described in Section 2.3. At 24 h after transfection, the cells were exposed to 250 nM PMA for 6 h. The medium was removed and the cells were incubated in a Hank's Balanced Salt Solution (HBSS) buffer containing 250 nM MitoTracker Red CMXRos probe (Molecular Probes) for 15 min at 37 °C. After washing twice with PBS, the cells were fixed with 4% (w/v) paraformaldehyde (PFA) in PBS at room temperature for 15 min. After washing, the cells were stained with DAPI nuclear probe (Molecular Probes). The coverslips were mounted on microscope slides for examination under a fluorescence microscope. For immunocytochemistry, cells were cultured on glass coverslips in 12-well plates, incubated with MitoTracker Red, and fixed with 4% PFA. After blocking for 1 h with PBS containing 2% BSA and 5% horse serum, cells were incubated with APE1/Ref-1 antibody (1:500) overnight at 4 °C and then with Alexa488-conjugated secondary antibody (1:500) for 1 h in the dark at room temperature. Images were obtained by fluorescence microscopy. 2.7. Western blot analysis For western blotting, cells were harvested using 100 μL lysis buffer (20 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, phosphatase-inhibitor cocktail, and protease-inhibitor cocktail). Cell homogenates (30 μg) were separated by 12% SDS-PAGE and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h at room temperature, blots were incubated overnight at 4 °C with specific primary antibodies (1:1000 anti-PARP, 1:1000 anti-COX-IV, 1:2000 anti-APE1/Ref-1, and 1:5000 anti-β-actin), which were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies. Blots
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were developed for visualization using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). Nuclear and mitochondrial fractions were prepared using a cell fractionation kit (Abcam), as recommended by the manufacturer. The purity of each fraction was confirmed using antibodies against the nuclear marker PARP and the mitochondrial marker COX-IV, and the fractions were examined for their APE1/Ref-1 content by western blotting. 2.8. Statistical analysis Statistical significance of differences in measured variables between control and PMA-treated groups was determined using one-way ANOVA followed by a Tukey's post-hoc test, and p b 0.05 was considered statistically significant. Values are expressed as means ± SEM. 3. Results 3.1. PMA induced mitochondrial hyperpolarization and mitochondrial ROS generation in mouse endothelial MS-1 cells PMA has been shown to disrupt mitochondrial membrane potential and induce mitochondrial ROS production, leading to mitochondrial dysfunction (Majumder et al., 2000; Wang et al., 2006). To assess the effects of PMA in mouse endothelial MS-1 cells, we first measured the changes in mitochondrial membrane potential and ROS generation after PMA treatment. PMA treatment induced a significant dosedependent increase in TMRE fluorescence intensity in mouse endothelial MS-1 cells, indicating mitochondrial hyperpolarization (Fig. 1A). Fluorescence intensity was elevated 1.98-fold in cells exposed to 250 nM PMA for 1 h relative to non-stimulated cells. The representative fluorescence images shown in Fig. 1A also suggest that PMA induced mitochondrial hyperpolarization in mouse endothelial MS-1 cells. The change in mitochondrial membrane potential also causes mitochondrial dysfunction by increasing the accidental transfer of
electrons to oxygen, thus generating superoxide or ROS (Andrews et al., 2005; Brownlee, 2001; Liu, 1997). To assess the effects of PMA on mitochondrial ROS generation in mouse endothelial MS-1 cells, we used MitoSox dye. PMA induced a dose-dependent increase in MitoSox fluorescence intensity, indicating mitochondrial ROS generation; the fluorescent signal obtained with 250 nM PMA was 1.87-fold higher than that obtained without PMA stimulation. The representative fluorescence photomicrographs shown in Fig. 1B indicate that PMA exposure induced mitochondrial ROS generation in mouse endothelial MS-1 cells. The above results together suggest that PMA induces mitochondrial dysfunction in mouse endothelial MS-1 cells. 3.2. Mitochondrial APE1/Ref-1 levels were elevated by PMA Mitochondrial APE1/Ref-1 levels increase in response to oxidative stress or mitochondrial DNA damage (Perrino et al., 2010). We investigated the localization of APE1/Ref-1 after exposing cells to PMA. Mouse endothelial MS-1 cells were treated with PMA and then nuclear and mitochondrial fractions of the cells were examined for APE1/Ref-1 through Western blotting. As shown in Fig. 2A, PMA increased mitochondrial APE1/Ref-1 expressions, in contrast, nuclear APE1/Ref-1 levels were decreased in response to PMA. Exposure to PMA for 1 h and 6 h elevated mitochondrial APE1/Ref-1 levels 1.6- and 1.8-fold over control levels (Fig. 2B). We confirmed the change in the subcellular distribution of APE1/ Ref-1 in PMA-stimulated cells by immunofluorescence staining; mitochondria and nuclei were visualized using MitoTracker (red) and DAPI (blue) staining, respectively. Consistent with our Western blotting results, APE1/Ref-1 intensity was increased in mitochondria after PMA treatment (Fig. 2C), suggesting that APE1/Ref-1 is translocated to mitochondria in endothelial cells in response to stimulation with PMA. Next, we investigated the effect of PKCβII inhibitor on PMA-induced mitochondrial APE1/Ref-1 translocation. Pretreatment of PKCβi (10 nM), a specific PKCβII inhibitor, inhibited PMA-induced mitochondrial APE1/Ref-1 translocation (Supplementary Fig. 1), suggesting that
Fig. 1. Dose-dependent changes in mitochondrial membrane potential and reactive oxygen species (ROS) generation in response to phorbol 12-myristate 13-acetate (PMA) treatment in mouse endothelial MS-1 cells. (A) TMRE fluorescence and (B) MitoSox Red fluorescence in PMA-treated cells. Fluorescence emission was measured at 590 nm with excitation at 530 nm. Values are expressed as mean fold-changes in arbitrary fluorescence values over basal value. Each bar shows mean ± S.E. (n = 4). * p b 0.05 vs. basal value.
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Fig. 2. Elevation of mitochondrial APE1/Ref-1 in response to PMA in mouse endothelial MS-1 cells. (A) Western blots of mitochondrial and nuclear fractions of PMA-treated cells harvested after 1 and 6 h treatment. Staining for APE1/Ref-1, COX-IV (loading control for mitochondria), and PARP (loading control for nuclei) are shown. (B) Densitometric analysis of Western blots. Data are expressed as fold-change values relative to basal levels of mitochondrial and nuclear APE1/Ref-1. Each bar represents mean ± S.E. (n = 4). * p b 0.05 vs. basal value. (C) After 6-h of treatment with 250 nM PMA, cells were stained for APE1/Ref-1 (green), mitochondria (red), and nuclei (blue). Magnification, 400 ×. Fluorescence photomicrographs of control and PMA-treated cells; results are representative of three independent experiments.
Fig. 3. APE1/Ref-1 overexpression inhibited PMA-induced mitochondrial membrane hyperpolarization and mitochondrial ROS generation in mouse endothelial MS-1 cells. (A) FLAG-tagged APE1/Ref-1-transfected cells were exposed to 250 nM PMA for 1 h and then mitochondrial membrane potentials using TMRE fluorescence was measured. (B) FLAG-tagged APE1/Ref-1-transfected cells were exposed to 250 nM PMA for 6 h. Relative mitochondrial ROS generation was analyzed based on MitoSox fluorescence. Values are expressed as mean fold-change in arbitrary fluorescence values over basal value. Each bar shows mean ± S.E. (n = 5). * p b 0.05 vs. basal value, # p b 0.05 vs. PMA alone. FLAG-tagged APE1/Ref-1 overexpression was confirmed by anti-FLAG in Western blotting. β-actin was used as loading control (bottom).
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APE1/Ref-1 phosphorylation induced by PKCβII would be contributed to mitochondrial APE1/Ref-1 translocation in endothelial cells. 3.3. APE1/Ref-1 overexpression prevented PMA-induced mitochondrial hyperpolarization and ROS generation To gain insights into the functional significance of the increase in mitochondrial APE1/Ref-1 levels in response to PMA-induced stress, we examined how overexpressing FLAG-tagged APE1/Ref-1 in endothelial cells affected PMA-induced mitochondrial hyperpolarization and ROS generation. Interestingly, overexpression of APE1/Ref-1 prevented PMA-induced mitochondrial hyperpolarization (Fig. 3A). PMA treatment increased the mitochondrial membrane potential, but this increase in cells ectopically expressing APE1/Ref-1 was 39.3% lower than in control cells. Thus, APE1/Ref-1 rescued the PMA-induced disruption of mitochondrial membrane potential. Furthermore, overexpressing APE1/Ref-1 in endothelial cells inhibited mitochondrial ROS generation after PMA exposure (Fig. 3B). In APE1/Ref-1 overexpressing cells, mitochondrial ROS production was reduced by 55.6%, compared with that in PMA-treated control cells. The overexpression of FLAG-tagged APE1/ Ref-1 was confirmed with immunoreactivity of anti-FLAG in Western blotting (bottom of Fig. 3). 3.4. Downregulating APE1/Ref-1 increased PMA-induced mitochondrial hyperpolarization Having determined that APE1/Ref-1 overexpression inhibited PMA-induced mitochondrial dysfunction, we investigated whether downregulating APE1/Ref-1 affected PMA-dependent mitochondrial hyperpolarization. PMA-induced mitochondrial membrane potential was compared in mouse endothelial MS-1 cells transfected with an APE1/Ref-1-specific siRNA or a scrambled siRNA. As shown in Fig. 4, PMA (250 nM) significantly increased mitochondrial membrane potentials, but low dose of PMA (1–100 nM) did not significantly
increase the mitochondrial membrane potential in scramble siRNAtransfected cells. However, low dose of PMA (1–100 nM) significantly increased mitochondrial membrane potentials in APE1/Ref-1 siRNAtransfected cells, compared with each dose of scramble siRNA-treated cells. This data suggested that the gene silencing of APE1/Ref-1 increased the sensitivity of PMA-induced mitochondrial membrane potentials in endothelial cells. Western blot data showed that treatment for 48 h with 10 nM of APE1/Ref-1 siRNA, but not the scrambled siRNA, markedly reduced endogenous APE1/Ref-1 (bottom of Fig. 4). We also measured the mitochondrial ROS generation in response to PMA in endothelial cells transfected with the APE1/Ref-1-specific and scrambled siRNAs. To determine the contribution of mitochondrial ROS production, we used Mito-TEMPO, a mitochondria specific antioxidant (Dikalova et al., 2010). As results, gene silencing of APE1/ Ref-1 significantly increased basal or PMA-induced mitochondrial ROS generation (Supplementary Fig. 2). These results suggest that mitochondrial APE1/Ref-1 helps to maintain the homeostasis of the mitochondrial membrane potential and ROS. 3.5. Mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 was localized in mitochondria We next compared the effect of PMA on cells expressing mitochondrion-specific APE1/Ref-1 and wild-type APE1/Ref-1. First, we determined the subcellular localization of EGFP-tagged wild-type or MTS-APE1/Ref-1 in mouse endothelial MS-1 cells. Fluorescence imaging revealed that the green fluorescence of wild-type APE1/Ref-1 was mostly merged with blue DAPI fluorescence, indicating nuclear localization of the protein. By contrast, MTS-APE1/Ref-1 was localized in the mitochondria, producing a yellow-orange fluorescence (Fig. 5A). Western blot data against anti-FLAG showed subcellular localization of FLAG-tagged wild-type APE1/Ref-1 and MTS-APE1/Ref-1 expressed ectopically in mouse endothelial MS-1 cells. FLAG-tagged APE1/Ref-1 was detected mainly in the nuclear fraction, whereas FLAG-tagged MTS-APE1/Ref-1 was present in the mitochondrial fraction in endothelial cells (Fig. 5B). 3.6. Mitochondrial APE1/Ref-1 potently blocked PMA-induced disruption of mitochondrial function Finally, we investigated the effect of mitochondrion-specific MTSAPE1/Ref-1 on PMA-induced mitochondrial hyperpolarization and ROS generation. Surprisingly, MTS-APE1/Ref-1 suppressed PMAinduced mitochondrial hyperpolarization substantially more than wild-type APE1/Ref-1 (Fig. 6A): whereas wild-type APE1/Ref-1 produced 39.3% reduction, MTS-APE1/Ref-1 produced a 68.9% reduction in PMA-induced mitochondrial hyperpolarization. Furthermore, the PMA-induced increase in mitochondrial ROS generation was suppressed by 86.3% in cells expressing MTS-APE1/Ref-1 and by 55.6% in cells expressing wild-type APE1/Ref-1 (Fig. 6B). These results suggest that MTS-tagged APE1/Ref-1 rescues PMA-induced mitochondrial dysfunction in mouse endothelial MS-1 cells. 4. Discussion
Fig. 4. Effect of APE1/Ref-1 downregulation on PMA-induced mitochondrial hyperpolarization in mouse endothelial MS-1 cells. At 48 h after transfection with scrambled siRNA or APE1/Ref-1-specific siRNA (10 nM), cells were exposed to 1 or 250 nM PMA for 1 h. Relative mitochondrial membrane potential was analyzed using a fluorometer to measure TMRE fluorescence. Values are expressed as mean fold-change in arbitrary fluorescence values over basal values. Each bar shows mean ± S.E. (n = 4). * p b 0.05 vs. basal value, # p b 0.05 vs. scramble siRNA. Downregulation of APE1/Ref-1 using APE1/Ref-1-specific siRNA was confirmed by anti-APE1/Ref-1 in Western blotting. β-actin was used as loading control (bottom).
Our objective in this study was to determine the role of mitochondrial APE1/Ref-1 in PKC-induced mitochondrial dysfunction in endothelial cells. Our results showed that PKC activation induced mitochondrial translocation of APE1/Ref-1, mitochondrial APE1/Ref-1 suppressed PMA-induced mitochondrial hyperpolarization and ROS production in endothelial cells. PMA induces the changes of mitochondrial membrane potentials and mitochondrial ROS generation, leading to mitochondrial dysfunction (Majumder et al., 2000; Wang et al., 2006). Our data showed that PMA induced mitochondrial hyperpolarization in mouse endothelial MS-1 cells, which indicates that the protonic
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Fig. 5. Subcellular localization of mitochondrial targeting sequence (MTS)-fused APE1/Ref-1 in mouse endothelial MS-1 cells. (A) Immunofluorescence images showed the subcellular localization of EGFP-tagged wild-type APE1/Ref-1 (WT) and MTS-APE1/Ref-1. MitoTracker (red) indicates mitochondria and DAPI (blue) indicates nuclei. Immunofluorescence images were obtained with fluorescence microscopy, magnification, 400×. (B) Western blot data showed subcellular localization of APE1/Ref-1 in nuclear (N) and mitochondrial (M) fractions of FLAG-tagged wild-type APE1/Ref-1- and MTS-APE1/Ref-1-transfected endothelial cells. Western blot was performed with anti-FLAG for FLAG-APE1/Ref-1 and FLAG-MTS-APE1/Ref-1. COX-IV and PARP served as loading controls for mitochondria and nuclei, respectively.
potential across the mitochondrion was increased. However, how PMA increases mitochondrial membrane potential remains unclear. PMA treatment activates several PKCs belonging to both classical and non-conventional PKC families (Goel et al., 2007). Mitochondrial hyperpolarization triggered by PKCα and PKCε activation leads to mitochondrial dysfunction in renal proximal tubular cells. The mitochondrial membrane potential depends on the activity of F0F1ATPase, which allows protons to return to the mitochondrial matrix, and the suppression of F0 F1 -ATPase activity in response to the
activation of PKCα and PKCε results in mitochondrial hyperpolarization (Nowak, 2002; Nowak et al., 2011). Moreover, activated PKCε phosphorylates voltage-dependent anion channel-1 (VDAC-1), which locks the channel in the closed conformation and leads to mitochondrial hyperpolarization (Baines et al., 2003). Thus, PKC activation by PMA could induce VDAC-1 closure or reduce F0F1-ATPase activity to cause mitochondrial hyperpolarization. A substantial increase in mitochondrial membrane potential has been reported to enhance mitochondrial production of ROS (Andrews et al., 2005;
Fig. 6. Effect of mitochondrial APE1/Ref-1 on mitochondrial membrane potential and mitochondrial ROS generation in PMA-stimulated mouse endothelial MS-1 cells. Cells transfected with FLAG-tagged wild-type APE1/Ref-1 and MTS-APE1/Ref-1 were exposed to 250 nM PMA for 1 h. Relative mitochondrial membrane potential was analyzed by measuring TMRE fluorescence. (B) FLAG-tagged APE1/Ref-1- and MTS-APE1/Ref-1-transfected cells were exposed to 250 nM PMA for 6 h. Relative mitochondrial ROS generation was analyzed by measuring MitoSox fluorescence. Values are expressed as mean fold-change in arbitrary fluorescence values over basal values. Each bar shows mean ± S.E. (n = 5). * p b 0.05 vs. basal value, # p b 0.05 vs. PMA alone, ## p b 0.05 vs. PMA with wild-type APE1/Ref-1.
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Brownlee, 2001; Korshunov et al., 1997). In this study, we confirmed that PMA induced mitochondrial hyperpolarization and mitochondrial ROS generation, leading to mitochondrial dysfunction in mouse endothelial cells. Interestingly, our data showed that overexpressing APE1/Ref-1 in endothelial cells significantly inhibited PMA-induced mitochondrial hyperpolarization and ROS generation. APE1/Ref-1 is dynamically regulated at both transcriptional and post-translational levels through the control of its subcellular localization and posttranslational modification. The role of APE1/Ref-1 in various subcellular and nuclear functions has been reported. For example, mitochondrial APE1/Ref-1 has been proposed recently to protect cells by regulating mitochondrial function after photodynamic therapy (Li et al., 2012). In this study, the expression of mitochondrionspecific MTS-APE1/Ref-1 suppressed PMA-induced mitochondrial hyperpolarization and ROS generation considerably more potently than the expression of wild-type APE1/Ref-1. However, the mechanism by which mitochondrial APE1/Ref-1 inhibits PMA-induced mitochondrial hyperpolarization and ROS generation is unclear. APE1/Ref-1 inhibition of mitochondrial hyperpolarization could also be related with the redox function to the mitochondrial uncoupling system. Uncoupling proteins such as UCP-2 and ANT-1 dissipate the mitochondrial proton gradient and stabilize the inner mitochondrial membrane potential and thereby reduce the formation of ROS (Haines and Li, 2012). APE1/Ref-1 may regulate the activity of uncoupling proteins and thus normalize mitochondrial hyperpolarization. Another possibility of the redox function of APE1/Ref-1 is that APE1/Ref-1 governed PKC-induced mitochondrial p66shc phosphorylation which mediated ROS production in endothelial cells (Lee et al., 2011). Mitochondrial DNA is a major site of oxidative stress damage and increased oxidative stress is closely linked with mitochondrial dysfunctions. Moreover, APE1/Ref-1 is localized to the mitochondria in response to oxidative stress (Perrino et al., 2010; Tell et al., 2005). Therefore, in addition of the redox function of APE1/Ref-1, DNA repair function would contribute in protecting PMA-induced mitochondrial dysfunctions. In this study, APE1/Ref-1 expression in mitochondrial fractions was increased within 1 h after PMA treatment, suggesting the mitochondrial translocation of APE1/Ref-1. Most mitochondrial proteins are encoded by nuclear genes and then translocated into the mitochondria. Recently, the MTS of APE1/Ref-1 was identified within residues 289–318 at the C terminus, which is normally masked by the nuclear localization signal (Perrino et al., 2010). This existence of an MTS in APE1/Ref-1 may explain the conditional mitochondrial localization of the protein in response to oxidative stress. How APE1/Ref-1 is translocated into the mitochondria is not fully established, although Tom 20 protein, a translocase of the mitochondrial outer membrane, may play a key role in the mitochondrial translocation of APE1/Ref-1. In the present study, PKC activation induced mitochondrial translocation of APE1/Ref-1 which was inhibited by PKCβΙΙ inhibitor, an anilinomonoindolylmaleimide compound, as a potent, cell-permeable, reversible, and ATP-competitive inhibitor of PKCβ isozymes (IC50 = 5 nM and 21 nM for human PKCβΙΙ and βΙ, respectively (Tanaka et al., 2004). It suggested that the phosphorylation of APE1/ Ref-1 by PKCβΙΙ would be contributed to mitochondrial translocation of APE1/Ref-1 in endothelial cells (Supplementary Fig. 1). Another possibility for mitochondrial translocation of APE1/Ref-1 is that the phosphorylation of nuclear localization signal of Nterminus of APE1/Ref-1 by PKC activation might unmask MTS signals, then phosphorylated APE1/Ref-1 can be translocated into the mitochondria. Our findings suggest that mitochondrial translocation of APE1/Ref-1 reverses the PMA-induced increase in mitochondrial membrane potential and ROS production. Moreover, we have demonstrated this effect of APE1/Ref-1 on endothelial mitochondrial function, which is a critical factor in several vascular disorders. Thus, our results suggest that
mitochondrial regulation by APE1/Ref-1 may play a therapeutic role in vascular diseases. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2014.05.006. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (20110016797, 2007-0054932). The English grammar has been checked by a professional editor who is a native speaker of English. References Andrews, Z.B., Diano, S., Horvath, T.L., 2005. Mitochondrial uncoupling proteins in the CNS: in support of function and survival. Nat. Rev. Neurosci. 6, 829–840. Angkeow, P., Deshpande, S.S., Qi, B., Liu, Y.X., Park, Y.C., Jeon, B.H., Ozaki, M., Irani, K., 2002. Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis. Cell Death Differ. 9, 717–725. Baines, C.P., Song, C.X., Zheng, Y.T., Wang, G.W., Zhang, J., Wang, O.L., Guo, Y., Bolli, R., Cardwell, E.M., Ping, P., 2003. 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