Oxidative Stress–Induced Degradation of Thioredoxin-1 and Apoptosis Is Inhibited by Thioredoxin-1–Actin Interaction in Endothelial Cells
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Abstract
Objective—
Thioredoxin-1 (Trx-1), one important antioxidative enzyme in endothelial cells, is required for apoptosis inhibition. Apoptosis induction is dependent on cytoskeletal changes, which depend on actin rearrangements. Therefore, we wanted to elucidate whether a physical interaction exists between Trx-1 and actin and what the functional consequences are.
Methods and Results—
Combined immunoprecipitation/mass spectrometry identified actin as a new binding partner for Trx-1. A separate pool of Trx-1 forms a complex with apoptosis signaling kinase 1. Actin is required for stress fiber formation; thus, the interaction of actin with Trx-1 might interfere with this process. Stress fiber formation, which is directly linked to the phosphorylation of focal adhesion kinase (FAK), occurs as early as 1 hour after H2O2 treatment. It is inhibited by Trx-1 overexpression, treatment with exogenous Trx-1, or inhibition of FAK. Prolonged incubation with H2O2 induced stress fiber formation, reduced Trx-1 protein levels, and increased apoptosis. All these processes were inhibited by preincubation with the FAK inhibitor PF573228. On the contrary, incubation with PF573228 1 hour after H2O2 treatment did not block stress fiber formation, degradation of Trx-1, or apoptosis.
Conclusion—
These data demonstrate that the actin–Trx-1 complex protects Trx-1 from degradation and, thus, endothelial cells from apoptosis. Reciprocally, Trx-1 prevents stress fiber formation.
The thioredoxin system consists of 2 antioxidant oxidoreductase enzymes, thioredoxin-1 (Trx-1) and the thioredoxin reductase 1. Trx-1 is a small, 12-kDa, ubiquitous protein with 2 redox-active cysteine residues in an exposed active center, having the exact same amino acid sequence as
The aim of the present study was to identify new Trx-1 interaction partners, which could provide further insights into the protection of Trx-1 from degradation and, thus, into its antiapoptotic functions in endothelial cells. Here, we discovered actin as a new binding partner for Trx-1. The Trx-1–actin complex did not contain ASK-1. Thus, at least 2 different pools of Trx-1 exist, one binding to actin and the other one interacting with ASK-1. Both of them are required to protect endothelial cells from apoptosis. Moreover, interaction with Trx-1 inhibited H2O2-induced rigid bundle (so-called stress fiber) formation of actin, Trx-1 degradation, and thereby apoptosis induction in endothelial cells. Interestingly, once stress fibers have been formed, Trx-1 degradation and apoptosis induction cannot be blocked anymore. Therefore, the Trx-1–actin interaction seems to result in a mutual protection of the 2 proteins.
Materials and Methods
Cell Culture
Human primary endothelial cells (ECs) 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 (Lonza, Cologne, Germany). After detachment with trypsin, cells were grown for at least 18 hours as described previously.22,23
Plasmids and Transfection
Human Trx-1 was cloned out of endothelial cell–derived cDNA as described previously and inserted in pcDNA 4 (Invitrogen, Karlsruhe, Germany) or in the FLAG vector (Sigma-Aldrich, Munich, Germany).24 ECs were transiently transfected with Superfect (Qiagen, Hilden, Germany) as described previously.24
Immunoprecipitation and Immunoblotting
Lysates (500 μg) were immunoprecipitated with 5 μg of the respective antibody overnight at 4°C. After incubation with protein A and protein G–Sepharose (GE Healthcare, Munich, Germany) for 2 hours at 4°C, the resulting beads were washed and boiled in SDS-PAGE sample buffer, and proteins were resolved by SDS-PAGE. Immunoblotting was performed with antibodies directed against Trx-1 (1:500, overnight, 4°C, BD Biosciences, Karlsruhe, Germany), γ-actin (1:500), GAPDH (1:8000), ASK-1 (1:250) (overnight 4°C, Santa Cruz Biotechnology, Heidelberg, Germany), and phospho-focal adhesion kinase (phospho-FAK) (Tyr397) (1:250), FAK (1:1000) (both overnight 4°C, New England Biolabs, Frankfurt, Germany). Semiquantitative analyses were performed on scanned immunoblots using ImageJ 1.42q.25 Mass Spectrometry analysis was performed on gel isolated proteins by the Proteome Factory (Berlin, Germany).
Immunostaining
Cells were fixed in 4% paraformaldehyde and permeabilized using 0.3% Triton X-100 and 3% bovine serum albumin in PBS. For immunostaining, cells were incubated with an antibody against Trx-1 and Xpress (both 1:50, Invitrogen, Karlsruhe, Germany) overnight at 4°C and stained with anti-rabbit or anti-mouse Alexa Fluor 488–coupled antibodies (Invitrogen). Actin was stained with Alexa Fluor 568 phalloidin (1:500, Invitrogen) at room temperature for 20 minutes. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:2000, Invitrogen). Cells were visualized using an Axiovert 40 microscope from Zeiss (magnification, ×40, oil, Jena, Germany).
Detection of Cell Death by Fluorescence-Activated Cell Sorting
Detection of cell death was performed by fluorescence-activated cell sorting (FACS) analysis using annexin V–APC binding and 7 amino-actinomycin D (7-AAD) staining (BD Pharmingen, Heidelberg, Germany). In brief, cells were trypsinized of the dish and pelleted. After washing twice with annexin binding buffer cell pellets were resuspended in annexin binding buffer and incubated with 2.5 ng/mL annexin V–APC and 2.5 ng/mL 7-AAD for 20 minutes and analyzed using FACS.
Statistics
Statistical analyses were performed with the Student t test or Wilcoxon test using WinSTAT 2008.
Results
Actin Is a New Interaction Partner of Trx-1
We described previously that Trx-1 acts antioxidatively and is required to protect ECs from apoptosis.22,24,26 Oxidative stress–induced stress fiber formation has been demonstrated to induce apoptosis.27 Therefore, we hypothesized that a potential connection between Trx-1 and actin exists. Using Trx-1 immunoprecipitation combined with mass spectrometry, we identified actin as a new binding partner for Trx-1 (Figure 1A). It has been previously suggested that apoptosis of endothelial cells requires cytoskeletal rearrangements.28 Thus, an interaction between Trx-1 and actin could be involved in the antiapoptotic function of Trx-1. Using the reciprocal immunoprecipitation approach, we could identify Trx-1 in immunoprecipitates obtained with an anti-γ-actin antibody (Figure 1B). A known interaction partner of Trx-1 is ASK-1. The preservation of the Trx-1/ASK-1 complex is required to protect endothelial cells from apoptosis.16 Thus, we wanted to know whether ASK-1 is also part of the Trx-1–actin complex. Interestingly, we did not find ASK-1 associated with actin, demonstrating that at least 2 different pools of Trx-1 exist in endothelial cells (Figure 1B), one binding to actin and the other one to ASK-1. We also performed the reciprocal approach, but we did not find actin in a complex with ASK-1 after immunoprecipitation with an ASK-1 antibody (Figure 1C). In a second approach, we used immunofluorescence and found that Trx-1 colocalized with actin. Interestingly, costaining was observed predominantly with nonpolymerized actin (Figure 1D). To further strengthen this observation, we incubated endothelial cells with cytochalasin D, a known interrupter of actin fiber formation. Indeed, incubation of endothelial cells with cytochalasin D for 30 minutes increased the association of Trx-1 with actin, demonstrating that Trx-1 interacts predominantly with nonpolymerized actin (Figure 1E and Supplemental Figure I, available online at http://atvb.ahajournals.org).
Figure 1. Actin is a new interaction partner of Trx-1. A, Trx-1 was immunoprecipitated out of EC lysates. Immunoprecipitates were subjected to SDS-PAGE, and a dominant band of approximately 37 kDa was extracted. Mass spectrometry revealed 6 peptides completely matching human actin. B, The reciprocal immunoprecipitation was performed using an anti-γ-actin antibody. Trx-1 was coimmunoprecipitated with γ-actin, but ASK-1 did not form a complex with γ-actin. IgG served as a negative control. Top shows an immunoblot with an anti-actin antibody; middle, immunoblot with an anti-Trx-1 antibody; bottom, immunoblot with an anti-ASK-1 antibody. SN/IP indicates supernatant of immunoprecipitation. C, Cell lysates used in B were also subjected to an immunoprecipitation with an anti-ASK-1 antibody. ASK-1 did not form a complex with γ-actin. Top, immunoblot with an anti-ASK-1 antibody; bottom, immunoblot with an anti-actin antibody. IgG served as a negative control. D, Immunostainings were performed to visualize Trx-1 (green); actin was stained with Alexa Fluor 568 phalloidin (red) and nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The merge revealed that Trx-1 colocalizes with nonpolymerized actin (yellow). E, ECs were incubated with dimethyl sulfoxide (DMSO) as solvent or with 0.1 μmol/L cytochalasin D (CytD) for 30 minutes. Trx-1 was immunoprecipitated out of EC lysates. IgG served as a negative control. Lysate corresponds to untreated total endothelial cell lysates before immunoprecipitation. Top, immunoblot with an anti-actin antibody; middle and bottom, immunoblots with an anti-Trx-1 antibody (long exposure, middle blot; short exposure, lower blot).
Overexpression of Trx-1 Inhibits Stress Fiber Formation and FAK Phosphorylation
Because it is known that oxidative stress–induced bundle formation of actin leads to actin polymerization and so-called stress fibers, we next investigated whether Trx-1 can inhibit stress fiber formation. Incubation with H2O2 induced bundle formation of actin after only 1 hour, and overexpression of Trx-1 completely blocked these cytoskeletal changes (Figure 2A). Because polymerization of actin requires activation of FAK by phosphorylation,29 we incubated ECs with the FAK inhibitor PF573228 for 6 hours before 1 hour of H2O2 treatment. This preincubation inhibited stress fiber formation (Figure 2B). Interestingly, FAK inhibition in Trx-1 overexpressing ECs did not change the phenotype compared with PF573228 treatment (Figure 2B, lower left panel) or overexpression of Trx-1 (Figure 2A) alone. In line with this finding, treatment of ECs with H2O2 for 1 hour induced phosphorylation of FAK, which was completely blocked by preincubation with PF573228 for 6 hours (Figure 3A and 3B). Preincubation with exogenous human Trx-1 for 6 hours also inhibited phosphorylation of FAK, but no additive effects were observed when ECs were coincubated with PF573228 and Trx-1 (Figure 3A and 3B), suggesting a common pathway.
Figure 2. Inhibition of H2O2-induced stress fiber formation by Trx-1 and blockage of FAK. ECs were transfected with an expression vector for Xpress-tagged Trx-1 and treated with 200 μmol/L H2O2 for 1 hour (Trx-1+H2O2) or left untreated (Trx-1). A, Trx-1-overexpressing cells were immunostained with an anti-Xpress antibody (green), and actin was stained with Alexa Fluor 568 phalloidin (red). The enlarged pictures below show the sections marked with the corresponding numbers in the small photographs. Cells overexpressing Trx-1 do not exhibit stress fibers after H2O2 treatment. B, Incubations with 40 nmol/L FAK inhibitor PF573228 were done for 6 hours before 1 hour of H2O2 treatment (FAKi/H2O2). Actin was stained with Alexa Fluor 568 phalloidin (red). Cells transfected with a Trx-1-Xpress expression vector and treated with PF573228/H2O2 as before (Trx-1/FAKi/H2O2) were counterstained with an anti-Xpress antibody (green).
Figure 3. Preincubation with PF573228 and with exogenous human Trx-1 inhibits H2O2-induced phosphorylation of FAK. ECs were incubated with 40 nmol/L PF573228, 1 ng/mL recombinant human Trx-1 (Sigma-Aldrich), or both for 6 hours before treatment with 200 μmol/L H2O2 for 1 hour. A, FAK phosphorylation was assessed by Western blotting with an anti-phospho-FAK (Tyr397) antibody; total FAK amounts were determined with an anti–total FAK antibody. B, Total FAK and phospho-FAK were evaluated densitometrically, shown is the ratio between phospho-FAK (P-FAK) and total FAK (FAK) (n=3 to 6, *P<0.05 versus H2O2). n.s. indicates not significant; con, control; FAKi, FAK inhibitor.
Inhibition of FAK Prevents H2O2-Induced Trx-1 Degradation and Apoptosis in ECs
We next investigated whether actin bundle formation plays a role in endothelial cell apoptosis induction. H2O2 induced apoptosis in ECs (22,24 and Figure 4A). Preincubation with PF573228 for 6 hours significantly inhibited H2O2-induced stress fiber formation and apoptosis (Figure 4A and 4B). We have previously shown that H2O2 decreases Trx-1 protein levels and does not change Trx-1 mRNA levels.22,26 Therefore, we hypothesized that inhibition of nonphysiological, high activation of FAK might stabilize Trx-1. Indeed, preincubation with PF573228 for 6 hours reduced H2O2-induced Trx-1 degradation (Figure 4C and 4D). Thus, stress fiber formation seems to be a prerequisite for Trx-1 degradation and endothelial cell apoptosis induction.
Figure 4. Blocking FAK phosphorylation before H2O2 treatment inhibits apoptosis induction, stress fiber formation, and Trx-1 degradation. ECs were preincubated with or without 40 nmol/L PF573228 for 6 hours and incubation was continued in the presence (FAKi/H2O2) or absence (FAKi) of 200 μmol/L H2O2 for another 18 hours. A, Apoptosis rates were determined by FACS analysis using annexin V–APC binding and 7-amino-actinomycin (7AAD) staining. (n=3, *P<0.05 versus H2O2). con indicates control. B, Actin was stained with Alexa Fluor 568 phalloidin (red), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). C, Trx-1 and GAPDH levels were assessed by Western blotting. D, Trx-1 and GAPDH were evaluated densitometrically. Shown is the ratio between Trx-1 and GAPDH (n=4, *P<0.05 versus control, **P<0.05 versus H2O2).
H2O2-Induced Stress Fiber Formation Is a Prerequisite for Trx-1 Degradation and Endothelial Cell Apoptosis Induction
To analyze the sequence of events during apoptosis induction, we set up experiments in which stress fiber formation was induced before FAK inhibition. Therefore, we pretreated ECs for 1 hour with H2O2, continued incubation for 17 hours in the presence or absence of PF573228, and analyzed stress fiber formation, Trx-1 protein levels, and apoptosis. Interestingly, this postincubation with PF573228 did not inhibit H2O2-induced apoptosis (Figure 5A). This was because incubation with PF573228 after H2O2 treatment inhibited neither stress fiber formation (Figure 5B) nor degradation of Trx-1 protein (Figure 5C and 5D). Based on our observations that Trx-1 interacts predominantly with nonpolymerized actin (Figure 1D and 1E and Supplemental Figure I), these data suggest that disruption of Trx-1–actin interactions by induction of actin polymerization with H2O2 exposes Trx-1 and, thus, enhances its degradation.
Figure 5. Blocking FAK phosphorylation after H2O2 treatment does not inhibit apoptosis induction, stress fiber formation, or Trx-1 degradation. ECs were preincubated with or without 200 μmol/L H2O2 for 1 hour, and incubation was continued in the presence or absence of 40 nmol/L PF573228 for another 17 hours. A, Apoptosis rates were determined by FACS analysis using annexin V–APC binding and 7-amino-actinomycin (7AAD) staining (n=3). n.s. indicates not significant; con, control. B, Actin was stained with Alexa Fluor 568 phalloidin (red), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). C, Trx-1 and GAPDH levels were assessed by Western blotting. D, Trx-1 and GAPDH were evaluated densitometrically. Shown is the ratio between Trx-1 and GAPDH (n=4, *P<0.05 versus control).
Discussion
The data from our present study reveal actin as a new binding partner for Trx-1. Interaction of Trx-1 predominantly occurs with nonpolymerized actin. Inhibition of nonphysiological, high FAK phosphorylation and thus activation before H2O2 treatment inhibited stress fiber formation, Trx-1 degradation, and apoptosis induction. In contrast, blockade of FAK activity after preincubation with H2O2 did not prevent stress fiber formation, Trx-1 degradation, and apoptosis induction, demonstrating that the interaction with actin protects Trx-1 from degradation and thereby ECs from apoptosis induction.
Trx-1 is a multifunctional protein that has been demonstrated to induce transcriptome changes by interacting with several transcription factors and thereby modulating their functions.30 With respect to apoptosis induction in vascular cells in vitro and in vivo, it has been shown that Trx-1 interacts with ASK-1 and thereby inhibits its proapoptotic action.15–17,31 Another binding partner for Trx-1, which has antiproliferative and proinflammatory properties, is TXNIP.14,18,20,21,31 Inhibition of TXNIP–Trx-1 interaction or reducing TXNIP expression in vascular cells, including endothelial and vascular smooth muscle cells, increases Trx-1 activity and also reduces ASK-1 activation, probably by binding of Trx-1 to ASK-1.31 One of the recently described pathways to reduce TXNIP expression is steady laminar flow, the most potent antiinflammatory and antiapoptotic stimulus for endothelial cells, which seems to make more reduced Trx-1 available in endothelial cells.31 Our data now identify actin as a new physiological binding partner for Trx-1 in endothelial cells. Recently, overexpressed Trx-1 has been shown to associate with actin in tumor cells.32 Interestingly, Trx-1 and actin do not form a trimeric complex with ASK-1 in endothelial cells. These findings suggest that at least 2 different pools of Trx-1 exist, one binding to actin and another one binding to ASK-1. Under conditions of oxidative stress, Trx-1 is released out of both complexes, leading to stress fiber formation and ASK-1 activation, which subsequently result in the induction of endothelial cell apoptosis (findings here and15). Actin has been demonstrated to be essential for effects induced by steady laminar flow, as cytoskeletal rearrangements are necessary for signal transduction.33 On the contrary, formation of rigid actin bundles (so-called stress fibers) is observed during apoptosis.27 Therefore, it is tempting to speculate that laminar flow increases Trx-1–actin interactions as one new mechanism to inhibit stress fiber formation, to allow cytoskeletal changes and thereby to protect against apoptosis induction. This hypothesis is also underscored by the finding that impaired migratory capacity correlates with robust stress fiber formation,34 which interferes with the dynamic reorganization of the actin cytoskeleton required for migrating cells.35,36 Furthermore, under conditions of oxidative stress, misassembled focal adhesion complexes and stress fibers are formed, leading to intense membrane blebbing, a hallmark of apoptosis.27 These data suggest that drastic actin bundle formation induced by oxidative stress marks cells for the apoptotic process. In this study, we found that treatment with H2O2 induces stress fibers, and once the fibers have been formed, the cell will undergo apoptosis. Because Trx-1 interacts predominantly with nonpolymerized actin, one could speculate that Trx-1 protects actin from polymerization by direct interaction. Thus, the Trx-1–actin interaction may protect endothelial cells from stress fiber–dependent apoptosis induction by preventing a misassembly of focal adhesions and, thus, induction of membrane blebbing. Of note, inhibition of stress fiber formation also resulted in reduced Trx-1 degradation. As Trx-1 is degraded by cathepsin D in lysosomes under conditions of oxidative stress,22 the interaction of Trx-1 with nonpolymerized actin might not allow its transport to these organelles. Therefore, the Trx-1–actin interaction seems to result in a mutual protection of the 2 proteins.
Another potential mechanism of how stress fiber formation could be inhibited is the S-nitros(yl)ation of actin. It has been observed in different cell types that S-nitros(yl)ation of actin reduces its polymerization and thereby leads to shorter actin filaments.37,38 Because Trx-1 can also be S-nitros(yl)ated in endothelial cells,24 a transnitros(yl)ation from Trx-1 to actin may occur, which in turn could prevent rigid bundle formation of actin. An involvement of actin in transnitros(yl)ation has already been investigated by Dalle-Donne et al, who reported that S-nitros(yl)ated actin acts as nitric oxide (NO) donor, showing a fast and potent vasodilating activity already at extremely low concentrations, suggesting that NO can indeed shuttle from S-NO to SH groups.37
Taking our data together, we present here for the first time evidence that actin, as a new binding partner for Trx-1 in endothelial cells, protects Trx-1 from degradation. Oxidative stress induces stress fiber formation, followed by Trx-1 degradation and apoptosis induction in endothelial cells. Stress fiber formation is required for Trx-1 degradation and apoptosis induction. The Trx-1–interaction protects Trx-1 from degradation and actin from enhanced bundle/stress fiber formation. Thus, maintaining the Trx-1–actin interaction is one prerequisite to protect endothelial cells from apoptosis.
Acknowledgments
We thank Diane Schmiegelt for expert technical assistance.
Sources of Funding
This study was supported by the
Disclosures
None.
Footnotes
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