Nuclear Redox-Signaling Is Essential for Apoptosis Inhibition in Endothelial Cells—Important Role for Nuclear Thioredoxin-1
Objective— The redox regulator thioredoxin-1 (Trx) is a potent antioxidative enzyme and exerts important cellular functions. Physiological concentrations of reactive oxygen species (ROS) and of nitric oxide (NO) act as second messengers. Previously, we demonstrated that ROS and NO reduced apoptosis in a Trx-dependent manner. The aim of this study was to determine the underlying mechanisms.
Methods and Results— First, we investigated the localization of Trx after H2O2 and NO. Both induced nuclear import of Trx, which required karyopherin-α. siRNA against karyopherin-α inhibited nuclear import of Trx. Analysis of the Trx amino acid sequence and subsequent immunoprecipitation studies revealed that Trx(K81/82E) is not imported into the nucleus under H2O2 treatment and Trx(K81/82/85E) was retained in the cytosol and induced cell death. Trx(K81/82E) abolished the antiapoptotic capacity of H2O2. Glutathione S-transferase P1 (GST-P1) was identified as one major target regulated by H2O2. siRNA against GST-P1 abolished the antiapoptotic effect of H2O2. Cysteine 69, but not cysteines 32 and 35, which are all required for the complete antiapoptotic function of Trx, is not imported into the nucleus.
Conclusion— H2O2-induced nuclear import of Trx depends on karyopherin-α and NO. Trx-dependent induction of GST-P1 expression is required for apoptosis inhibition in endothelial cells.
Oxygen and nitric oxide (NO) are physiologically relevant molecules. In physiological concentrations reactive oxygen species (ROS) and NO play important roles in the regulation of a variety of cellular functions including vascular tone, migration, apoptosis, and proliferation.1,2 One of these molecules, which is highly regulated by changes of the redox status in cells, is the oxidoreductase thioredoxin-1 (Trx). It is known that cells possess antioxidative enzymes, including superoxide dismutase, catalase, and Trx to maintain intracellular levels of ROS and reactive nitrogen species in physiological concentrations.3,4 The thioredoxin family includes 3 proteins, thioredoxin-1, thioredoxin-2, and Sp-thioredoxin.5–7 All of them contain a conserved -Cys-Gly-Pro-Cys- active site (cysteine 32 and cysteine 35 within thioredoxin-1), which is essential for the redox regulatory function of thioredoxins.8,9
Trx is a 12-kDa protein, which is ubiquitously expressed in mammalian cells8 and exerts its enzymatic activity as an oxidoreductase via cysteines 32 and 35 in the active site.8,9 This site is conserved among species from bacteria to humans.8,10 The active site cysteines are accessible on the surface of the protein and are oxidized to a disulfide upon reduction of a target protein.9,11,12 Trx itself is reduced by thioredoxin-reductase. These 2 oxidoreductases form the thioredoxin system in mammalian cells. Besides its well described function as an oxidoreductase, Trx exerts several other functions. By binding to different proteins, it modulates their function: Inhibition of binding to the apoptosis signaling kinase 1 and to the transcription factors AP1, Ref1, and NfκB modulates the ability of Trx to regulate cellular functions.13–16
Previously, we have demonstrated that physiological concentrations of H2O2 and NO inhibit apoptosis in endothelial cells in a Trx dependent manner.17,18 Although we were able to demonstrate that mutation of cysteine 69 in Trx reduced its antiapoptotic capacity,17 the underlying mechanisms are not entirely clear.
Therefore, the aim of this study was to elucidate the mechanisms underlying the antiapoptotic properties of physiological concentrations of H2O2 and to identify the role of Trx. Here, we demonstrate that on exposure of cells to physiological concentrations of H2O2 and NO Trx is imported into the nucleus in a karyopherin-α–dependent manner. Nuclear import of Trx is required for the antiapoptotic properties of H2O2. Nuclear Trx binds to several transcription factors and increases transcription factor binding to antioxidant responsive elements (AREs). Glutathione S-transferase P1 (GST-P1) expression is increased. Genetic ablation of GST-P1 by siRNA completely abrogated H2O2-induced inhibition of apoptosis, demonstrating that increased expression of GST-P1 is 1 major downstream target for the Trx-dependent antiapoptotic properties of physiological concentrations of H2O2.
Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal medium supplemented with hydrocortisone (1 μg/mL), bovine brain extract (12 μg/mL), gentamicin (50 μg/mL), amphotericin B (50 ng/mL), epidermal growth factor (10 ng/mL), and 10% fetal calf serum. After detachment with trypsin, cells were grown for at least 18 hours. Human embryonic kidney cells (HEK293) were cultured in DMEM basal medium with 10% heat-inactivated fetal calf serum. Neonatal rat cardiac myocytes were isolated as described previously.19
Endothelial cell single strand cDNA was prepared out of endothelial cell RNA using RT-PCR. Trx was cloned out of endothelial cell cDNA using the following primers: 5′-GTGGTACCTTGGTGAAGCAGATCGAGAGC (sense) and 5′-CTCTAGACTTAGACTAATTCATTAATGGTGGC (antisense) incorporating KpnI and XbaI restriction sites. The amplified PCR product was subcloned into pcDNA4/His vector containing a Xpress-tag and an enterokinase (EK) recognition site (Invitrogen). TrxK85E, TrxK81E/K82E, TrxK81E/K82E/K85E, and TrxK94E/K96E were generated by site directed mutagenesis (Stratagene) out of Trx wt.
HUVECs were transfected with 3 μg plasmid and 25 μL Superfect with a transfection efficiency of 40%. For transfection of siRNA 6.6 μg double stranded DNA were transfected with JetSi (Eurogentec) according to the manufacturer‘s instructions.
cDNA Generation and PCR
cDNA was generated out of 5 μg total RNA using random hexamer primers according to the manufacturers instruction (Invitrogen). Primers to human karyopherin-α: 5′ATCCTGAGGCTTGGAGAACA-3′ and 5′-GGTCCCGAAGTAATGCTCAA-3′ to human glyceraldehyde-3-phosphate dehydrogenase, 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′.
Separation of Nuclear and Cytosolic Fractions
Nuclear and cytosolic fractions were separated as described previously.20 Purity of the nuclear and cytosolic fractions were assured by immunoblotting with topoisomerase 1 (nuclear) and HSP70 (cytosolic) as demonstrated previously.20
Lysates (500 μg) were immunoprecipitated with 5 μg Xpress-antibody overnight at 4°C. After incubation with G Sepharose (Amersham) for 2 hours at 4°C, resulting beads were washed, subjected to SDS-PAGE sample buffer, and resolved by a SDS-PAGE.
After stimulation for the indicated times, endothelial cells were scraped off the plates and lysed in RIPA buffer (50 mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, 1% Nonidet-P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate). After removing cell debris (15 minutes, 4°C, 20 000g), proteins were resolved on SDS-polyacrylamide gels and blotted onto polyvinylidene fluoride (PVDF) membranes. For detection of protein expression, membranes were incubated with antibodies against Trx (1:500, overnight, Pharmingen), karyopherin-α (1:250, overnight, Santa Cruz), topoisomerase I (1:250, overnight, Santa Cruz) or tubulin (1:1000, 2 hours, Neomarkers). After incubation for 2 hours with the corresponding secondary antibody tagged with horse radish peroxidase, signals were detected by the enhanced chemiluminescence system (Amersham).
Protein-DNA Binding Array
Trx-Xpress was immunoprecipitated out of nuclear fractions after H2O2 incubation for 6 hours. Protein/DNA arrays were performed in the washed immunoprecipitates according to the manufacturer′s protocol (Panomics). Briefly, immunoprecipitate was incubated with the TranSignal Probe Mix. DNA/protein complexes were washed. Then, DNA was separated from protein and hybridized on the membranes at 42°C. Signal was detected using ECL-Hyperfilm. To semiquantitatively analyze the dot blots, spots were scanned and normalized to the internal controls on the membranes as suggested by the manufacturer‘s instructions. Scanned spots of Trxwt + H2O2 were set to 1 and intensity (=protein-DNA binding) of TrxK81E/K82E + H2O2 was calculated using scion image.
Detection of Cell Death by Fluorescence-Activated-Cell Sorter (FACS)
Detection of cell death was performed by FACS analysis using annexin V-PE binding and 7-Amino-actinomycin (7AAD)-fluorescein isothiocyanate (FITC) staining (Pharmingen). In brief, cells were trypsinized of the dish and pelleted. After washing twice with annexin binding buffer, cell pellets were resuspended in 50 μL of annexin binding buffer and incubated with 2.5 ng/mL annexin V-PE and 2.5 ng/mL 7AAD-FITC for 20 minutes and analyzed using FACS.
Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After permeabilization and blocking, cells were incubated with anti–Trx-antibody (1:50, BD Biosciences) or anti-Xpress antibody (1:50, Invitrogen) overnight at 4°C. After incubation with a Rhodamine Red X–conjugated anti-mouse antibody (1:300, Jackson ImmunoResearch), cells were incubated with RNase and nuclei were stained with Cytox-Blue (1:500, Invitrogen). For knockdown experiments against karyopherin-α, cells were first incubated with anti Trx-antibody (1:50, BD Biosciences) overnight at 4°C. After incubation with a Rhodamine Red X–conjugated anti-mouse antibody (1:300, Jackson ImmunoResearch), cells were incubated with karyopherin-α (1:25, Santa Cruz) antibody overnight at 4°C. After incubation with a FITC-conjugated anti-goat antibody (1:300, Molecular Probes), nuclei were stained with Tropro-3-iodide (1:1000, Invitrogen). Cells were visualized by confocal microscopy (Zeiss, LSM 510 META, Objective: Plan-Apochromat 63×, 1,4 oil).
Statistical analysis was performed with student t test or ANOVA followed by modified LSD (Bonferroni) test (SPSS-Software).
Trx Is Imported Into the Nucleus Under Physiological Concentrations of H2O2
Previously, we have demonstrated that low doses of H2O2 protect endothelial cells from apoptosis in a Trx-dependent manner. However, the underlying mechanism is completely unclear. It is known that Trx can bind to several proteins, including transcription factors, and thereby modulate their functions. Therefore, we first investigated the localization of Trx after treatment with 10 μmol/L H2O2 for 6 hours. H2O2 induced an increase in nuclear localization of Trx in endothelial cells (supplemental Figure I, available online at http://atvb.ahajournals.org; Figure 1A). This phenomenon was also seen in other cell types including cardiomyocytes (Figure 1B), suggesting a general mechanism for the regulation of Trx by low doses of reactive oxygen species.
Trx Is Imported Into the Nucleus via Karyopherins
To understand the nuclear import mechanism of Trx, we first analyzed the amino acid sequence of Trx and determined a potential nuclear import sequence for the import receptors, karyopherin-α and karyopherin-β, at lysines 81, 82, and 85 (supplemental Figure II). To verify whether Trx is indeed imported into the nucleus in a karyopherin-α– and karyopherin-β–dependent manner, we mutated the lysines to glutamates, to block nuclear import of Trx. We created several mutants: TrxK85E, TrxK81E/K82E, and TrxK81E/K82E/K85E. Overexpression of TrxK81E/K82E/K85E resulted in cell death of endothelial cells. Only after treatment with a pan-caspase inhibitor ZVAD and cell-permeable pepstatin A, we were able to detect endothelial cells overexpressing TrxK81E/K82E/K85E. TrxK81E/K82E/K85E is not detectable in the nucleus and shows a punctual staining (supplemental Figure III), demonstrating that lysines 81, 82, and 85 are required for nuclear import of Trx. Therefore, we did not pursue any further experiments with this mutant. We assured that Trxwt and TrxK81EK82E are overexpressed to a similar extent in endothelial cells (supplemental Figure II). Moreover, TrxK81E/K82E is in the nucleus under basal conditions, but H2O2 did not increase nuclear import of TrxK81E/K82E (supplemental Figure IV and Figure 2). In certain cases after H2O2 incubation, TrxK81E/K82E was only detected in the cytoplasm (supplemental Figure IV). In contrast, TrxK85E was imported under H2O2 treatment as efficiently as Trxwt (Figure 2).
To verify that karyopherins are indeed the import receptors for Trx, we overexpressed Trxwt or TrxK81E/K82E in endothelial cells, treated the cells for 6 hours with 10 μmol/L H2O2, and immunoprecipitated Trxwt and the mutant with a Xpress antibody or for reverse Immunoprecipitation with a karyopherin-α antibody. As expected, Trx associated with karyopherin-α (Figure 3A and data not shown). The association increased after H2O2 treatment in Trxwt transfected cells, but not in cells overexpressing TrxK81E/K82E (Figure 3A). Next, we wanted to investigate a direct link between the association of Trx with karyopherin-α and the nuclear import of Trx. Therefore, we knocked down karyopherin-α by siRNA transfection in endothelial cells (Figure 3B and 3C). The maximal reduction of karyopherin-α, which did not kill the cells, was around 50% (Figure 3B and data not shown). Using this reduction in karyopherin-α, H2O2-induced nuclear import of Trx was strongly inhibited (Figure 3C).
Nuclear Import of Trx Is Required for the Antiapoptotic Properties of H2O2
To test whether nuclear import is indeed required for the antiapoptotic functions of low doses of H2O2, we overexpressed Trxwt and TrxK81E/K82E in endothelial cells. As a structural control, we also cloned TrxK94E/K96E, in which the putative second nuclear import motif is destroyed (see supplemental Figure II). As expected, Trxwt inhibited basal apoptosis17 (Figure 4A). Moreover, the antiapoptotic effect of low doses of H2O2 is completely abrogated in endothelial cells overexpressing TrxK81E/K82E (Figure 4A). In contrast, TrxK94E/K96E did not influence the antiapoptotic properties of low doses of H2O2 (Figure 4A). Serum-deprivation–induced apoptosis was also dependent on nuclear import of Trx. Overexpression of Trxwt, but not of TrxK81E/K82E, reduced apoptosis induction (Figure 4B) demonstrating that nuclear import of Trx is indeed required to protect from basal and stimulus-induced apoptosis.
Genes With an ARE Containing Promoter Are Necessary for Apoptosis Inhibition by Nuclear Trx
Trx has been demonstrated to regulate transcription factors, such as NF-κB, by protein binding. Thus, we hypothesized that nuclear import of Trx may influence the activity of transcription factors. To get insights in the underlying mechanisms, we investigated protein-DNA binding of 400 transcription factors. To only investigate the transcription factors bound to Trx, we first immunoprecipitated Trxwt and TrxK81E/K82E after stimulation with H2O2 out of nuclear extracts. The immunoprecipitates were then subjected to protein-DNA binding studies. The most pronounced differences were found by genes containing an ARE motif in their promoters (supplemental Figure V and Figure 4C). We also detected slight differences in AP1 and AP2, whereas activity of NfκB was unaltered (Figure 4C).
Glutathione S-Transferase P1 (GST-P1) Is Required for the Antiapoptotic Effects of Low Doses of H2O2
One important protein which contributes to the redox status of cells is GST-P1. GST-P1 contains 4 ARE motifs in its promoter. Therefore, we next investigated the regulation of GST-P1 protein expression by low doses of H2O2 in endothelial cells. Low doses of H2O2 increased protein expression of GST-P1 in cells expressing Trxwt or TrxK85E (Figure 5A). Interestingly, in endothelial cells overexpressing TrxK81E/K82E, H2O2 was unable to upregulate GST-P1 protein expression. To elucidate whether the protein expression of GST-P1 is indeed required as a downstream target of nuclear Trx for the antiapoptotic effects of H2O2, we genetically ablated GST-P1 expression by siRNA (Figure 5B). Genetic knock down of GST-P1 protein expression inversed the antiapoptotic effects of low doses of H2O2 (Figure 5C), demonstrating that GST-P1 is one of the targets regulated by nuclear Trx which plays an important role for the antiapoptotic effects of low doses of H2O2.
Posttranslational Modification by Nitric Oxide Is Required for Nuclear Import of Thioredoxin
Finally, we investigated the signals responsible to import Trx into the nucleus. Trx has been described to be posttranslationally modified by oxidation, nitros(yl)ation, or glutathionylation (for review see21). Therefore, we determined whether one of these posttranslational modifications has an impact on nuclear import of Trx. Oxidation of Trx occurs on the active site cysteines 32 and 35. Therefore, we isolated nuclear extracts from mouse hearts overexpressing either Trxwt or TrxC32S/C35S.22 We did not detect any difference between nuclear localization of Trxwt and TrxC32S/C35S in mouse hearts as well as in endothelial cells (Figure 6A and data not shown). This is in accordance to findings by Hirota et al who excluded modifications on cysteine 32 and cysteine 35 for nuclear import of Trx in their cell system.23 It has been demonstrated in cells and in a mouse model that S-nitros(yl)ation of Trx enhanced its activity, its antiapoptotic capacity, and its cardioprotective properties.17,24 Thus, it is tempting to speculate that NO influences the nuclear import of Trx. Therefore, we stimulated endothelial cells with different NO-donors and measured nuclear Trx protein. Trx protein levels are increased in the nucleus after stimulation with NO (Figure 6B). Moreover, TrxC69S, a Trx mutant which cannot be S-nitros(yl)ated in endothlelial cells, was barely detected in the nucleus, demonstrating that posttranslational modification by NO is required for nuclear import of Trx (Figure 6C).
The findings of our present study demonstrate that nuclear Trx is required for the antiapoptotic effects of low doses of H2O2. We determined for the first time that (1) Trx is imported into the nucleus by karyopherin alpha, (2) GST-P1 is one important downstream target activated by nuclear Trx, and (3) posttranslational modification of Trx by NO is necessary for nuclear import of Trx.
ROS are formed and degraded by all aerobic organisms, leading to either physiological concentrations required for normal cell function, or excessive quantities resulting in oxidative stress. Ideally, a metabolically active cell should strike a balance between ROS production and the cellular antioxidant defense system. Different studies, predominantly in smooth muscle cells, show that on stimulation with EGF or PDGF, proliferation is induced in a ROS-dependent manner.25,26 Furthermore, ROS can inhibit apoptosis and induce angiogenesis in endothelial cells and other cell types.18,27,28 Therefore, intracellular ROS can be seen as a signal transduction messenger involved in several intracellular mechanisms. Here, we extend these findings and demonstrate that the antiapoptotic effects of H2O2 depend on nuclear Trx. Nuclear Trx regulates ARE containing promoters of genes and thereby acts antiapoptotic. It has to be noted that the promoter of Trx contains ARE binding sites and that expression of the Trx gene itself can be induced through ARE.29 This activation is dependent on the NF-E2–related factor (Nrf2). Kim et al postulated that Trx served as a factor facilitating DNA binding of Nrf2 to the ARE. Thereby, the ARE-triggered Trx gene activation may enhance the ARE-mediated induction of ARE-controlled enzymes. Indeed, we demonstrate here that nuclear Trx is required to induce GST-P1, which contains AREs in its promoter. This suggested that a feedback loop exists for balancing oxidant and antioxidant systems, which is essential for a cell to survive, because a gain of ROS product formation or a loss in antioxidative capacity can disturb the equilibrium and subsequently lead to cellular destruction.
In this study, we identified the nuclear import mechanism of Trx. The nuclear import receptors karyopherin-α and -β have been described for nuclear import of several targets.30 Proteins, which are smaller than 40 kDa have been proposed to freely diffuse through the nuclear pores. Here, we provide evidence that the 12-kDa protein Trx is imported into the nucleus by karyopherin-α under certain conditions in endothelial cells. Thus, one may speculate that depending on the cellular conditions, a regulated nuclear import of small proteins exists.
In this study we identified GST-P1 as one major downstream target for nuclear Trx signaling. GST-P1 is an antioxidative enzyme, which was recently been implicated to protect from oxidative stress–induced cardiac injury.31 The underlying mechanisms are not well understood. Recently, Zhao et al demonstrated that GST-P1 inhibited etoposide-induced apoptosis by inhibiting MEKK-1 activity, pro–caspase-3 activation, and PARP cleavage.32 Here, we demonstrate an upstream mediator for GST-P1, namely nuclear Trx. Our findings provide evidence for the underlying mechanism, because induction of nuclear Trx did not only occur in endothelial cells but also in cardiac myocytes. Therefore, it is tempting to speculate that nuclear Trx is essential to inhibit oxidative stress induced cardiac injury. Indeed, it has been shown that specific overexpression of the catalytically inactive TrxC32S/C35S in the heart led to hypertrophy and oxidative stress–induced DNA damage in vivo.22 Our data clearly demonstrate that TrxC32S/C35S can still be imported into the nucleus. However, cysteines 32 and 35 are required for the ability of Trx to bind to transcription factors and thereby modulate their protein functions.
Interestingly, nuclear import of Trx depends on its posttranslational modification by NO in endothelial cells. We have demonstrated that binding of NO by Trx increased its antiapoptotic function.17 NO is a crucial factor for the integrity and function of the endothelium. Besides its role in blood pressure regulation, NO acts antithrombotically and antiapoptotically. Moreover, the cardioprotective and antiapoptotic effects of Trx against ischemia-reperfusion injury were more pronounced when S-nitros(yl)ated Trx was infused into mice.24 Thus, Trx in concert with NO seems to be crucial for protection in endothelial cells and cardiac myocytes. Moreover, it seems that a balance between antioxidative and oxidative systems also exists in the nucleus, because we demonstrate here that Trx localized in the nucleus. Another important question is where is Trx S-nitros(yl)ated in the cell? Because the nuclear import of Trx is greatly impaired when cysteine 69 is mutated to serine, one may speculate that modification of Trx has to occur in the cytoplasm to allow Trx to be imported into the nucleus. However, further studies are needed to determine the compartment of S-nitros(yl)ation of Trx.
In conclusion, our data provide compelling evidence that nuclear Trx is one important signaling molecule, which modulates transcription factor activities. Trx is imported into the nucleus in a karyopherin-α–dependent manner, and this nuclear import of Trx is required for apoptosis inhibition. Moreover, one major downstream target, which is activated by nuclear Trx, is GST-P1. GST-P1 also plays an important role as an antioxidant. Strikingly, the nuclear import of Trx depends mainly on its S-nitros(yl)ation. Thus, S-nitros(yl)ated Trx could be one important strategy to enhance nuclear Trx and to protect from cardiac disorders.
We thank Diane Schmiegelt for expert technical assistance.
Sources of Funding
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to J.H. (HA2868/3-2).
P.S. and R.P. contributed equally to this study.
Original received June 6, 2007; final version accepted August 28, 2007.
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