Thioredoxin-2 Inhibits Mitochondria-Located ASK1-Mediated Apoptosis in a JNK-Independent Manner
Apoptosis signal-regulating kinase 1 (ASK1) mediates cytokines and oxidative stress (ROS)-induced apoptosis in a mitochondria-dependent pathway. However, the underlying mechanism has not been defined. In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of endothelial cells (ECs) where it binds to cytosolic (Trx1) and mitochondrial thioredoxin (Trx2), respectively. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for binding of Trx1 and Trx2, respectively. Mutation of ASK1 at C250 enhanced ASK1-induced JNK activation and apoptosis, whereas mutation of ASK1 at C30 specifically increased ASK1-induced apoptosis without effects on JNK activation. We further show that a JNK-specific inhibitor SP600125 completely blocks TNF induced JNK activation, Bid cleavage, and Bax mitochondrial translocation, but only partially inhibits cytochrome c release and EC death, suggesting that TNF induces both JNK-dependent and JNK-independent apoptotic pathways in EC. Mitochondria-specific expression of a constitutively active ASK1 strongly induces EC apoptosis without JNK activation, Bid cleavage, and Bax mitochondrial translocation. These data suggest that mitochondrial ASK1 mediates a JNK-independent apoptotic pathway induced by TNF. To determine the role of Trx2 in regulation of mitochondrial ASK1 activity, we show that overexpression of Trx2 inhibits ASK1-induced apoptosis without effects on ASK1-induced JNK activation. Moreover, specific knockdown of Trx2 in EC increases TNF/ASK1-induced cytochrome c release and cell death without increase in JNK activation, Bid cleavage, and Bax translocation. Our data suggest that ASK1 in cytoplasm and mitochondria mediate distinct apoptotic pathways induced by TNF, and Trx1 and Trx2 cooperatively inhibit ASK1 activities.
Thioredoxins (Trx) are cellular redox enzymes that have multiple functions in regulation of cell growth, apoptosis, and activation.1 Trx contains two redox-active cysteine residues in its catalytic center with a consensus amino acid sequence: cys-gly-pro-cys. Trx exists either in a reduced dithiol form or in an oxidized form. It participates in redox reactions by reversible oxidation of its active center dithiol to disulfide and catalyzes dithio-disulfide exchange reactions involving many thiol-dependent processes.1 Thus, Trx system is considered to constitute an endogenous antioxidant system in addition to the glutathione and superoxide dismutase systems. Two isoforms of Trx have been identified in mammalian cells: cytosolic and mitochondrial Trx (Trx1 and Trx2, respectively). Trx1 and Trx2 are encoded by distinct nuclear genes and Trx2 contains a mitochondrial targeting signal peptide.2–4 The mitochondrion is the major source of ROS generated during physiological respiration and pathological conditions during inflammation in response to cytokines.5 Therefore, the mitochondrial antioxidant systems including Trx2, Trx2 reductase, mitochondrial Trx peroxidase, and manganese superoxide dismutase (MnSOD) are critical in regulating mitochondrial ROS-induced cytotoxicity. Consistently, Trx2-deficient cells show an accumulation of intracellular ROS and mitochondria-dependent apoptosis.3 Conversely, overexpression of Trx2 confers resistance to ROS-induced cell death.2,4 Furthermore, genetic ablation of Trx2 in mice causes massively increased apoptosis in E10.5-day embryos, leading to embryonic lethality.6 Both Trx1- and Trx2-dependent systems have been reported to prevent oxidative stress-induced cytotoxicities. It has been shown that Trx1 prevents cell apoptosis by scavenging reactive oxygen species (ROS) to protect against oxidative stress. It also acts antiapoptotically by regulating the activities of transcription factors such as NF-κB and AP-1, and by directly binding and inhibiting the activity of the proapoptotic protein apoptosis signal-regulating kinase 1 (ASK1).1,7
Apoptosis signal-regulating kinase 1 (ASK1) is one of several MAP3Ks that are activated in response to proinflammatory stimuli, ROS, and cellular stress leading to activation of MAP2K-JNK/p38 cascades.8 ASK1-induced apoptosis has been extensively studied. ASK1 is a 170-kDa protein that is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. Several cellular inhibitors including Trx1 have been shown to bind to and inhibit ASK1 apoptotic activity in resting cells.7 Trx1 directly associates with ASK1 in the N-terminal domain of ASK1 and inhibits its kinase activity. Deletion of the N-terminal 648 amino acids of ASK1 (ASK1-ΔN) causes constitutive ASK1 kinase activity, as it does in other MAP3Ks. This confirms that Trx1 inhibits ASK1 via the N-terminal inhibitory domain.7 Interestingly, only the reduced form of Trx1 binds to the N-terminal part of ASK1 and blocks activation of ASK1 by TNF.7,9 Neither the oxidized form (intramolecular disulfide between C32 and C35) nor the redox-inactive form (the double-mutation at catalytic sites C32 and C35) of Trx1 binds to ASK1. Apoptotic stimuli (TNF, ROS, or serum starvation) activate ASK1 in part by oxidizing Trx1 to release it from ASK1.7,9 We have recently shown that a single Cys residue in the catalytic site of Trx1 (C32 or C35) is critical for ASK1-binding. Furthermore, Trx1-C32S and Trx1-C35S constitutively bind to ASK1 even in the presence of hydrogen peroxide in vitro or of TNF in vivo, most likely because they cannot be oxidized to form a disulfide bond between the two catalytic cysteines leading to its release from ASK1.10 These data suggest that Trx1 is critical for the regulation of ASK1 activity.
Studies from ASK1 knockout mice have shown that ASK1 is a critical mediator in TNF, ROS, and stress-induced cell death.11 Moreover, overexpression of ASK1 induces a mitochondria-dependent apoptotic pathway.12 However, the underlying mechanism by which ASK1 mediates mitochondria-dependent apoptotic pathway is not fully understood. JNK, a downstream target of ASK1, has been shown to activate proapoptotic Bcl-2 family protein such as Bim/Bak/Bax, leading to release of proapoptotic factors such as cytochrome c and cell death.13,14 It is not clear whether or not the proapoptotic activity of ASK1 is solely dependent on downstream targets such as JNK. In the present study, we show that ASK1 localized in both cytoplasm and mitochondria of EC and forms a complex with Trx1 and Trx2 in resting state. Proapoptotic stimuli TNF and oxidative stress dissociate Trx1/Trx2 from ASK1, leading to enhanced mitochondria-dependent apoptosis characterized by cytochrome c release, caspase-3 activation, and nuclear fragmentation. Conversely, overexpression of Trx1 and Trx2 synergistically block TNF/ROS/ASK1-induced cell death. Furthermore, our data demonstrate that a specific interaction between ASK1 and Trx2 in mitochondria plays a pivotal role in regulation of cellular survival and apoptosis.
Materials and Methods
An expanded Materials and Methods section is provided in the online data supplement available at http://circres.ahajournals.org.
Confocal immunofluorescence microscopy, cell transfection and reporter gene assay, immunoprecipitation and immunoblotting, ASK1 and JNK kinase assays, GST-Trx pull-down assay, RNAi constructs and RNase protection assay, and quantitation of apoptosis have been described previously.10,15
ASK1 and Trx2 Form a Complex in Mitochondria That Is Dissociated in Response to TNF/ROS
To determine the underlying mechanism by which ASK1 mediated a mitochondria-dependent apoptosis, we examined subcellular localization of endogenous ASK1 by immunogold electron microscopy (EM). Human EC (HUVEC) was stained with anti-ASK1 (rabbit) and a mitochondria marker (mouse monoclonal) followed by goat anti-rabbit-5 nm gold particles and goat anti-mouse-15 nm gold particles. ASK1- and mitochondria-gold particles were visualized under EM. As shown in Figure 1A, a mitochondrial marker was detected only in mitochondria (m) but not in rough endoplasmic reticulum (rER) (left panel) or nucleus (N) (middle panel). ASK1 is stained inside mitochondria (Δ) as well cytoplasm (*). High-power views clearly show that ASK1 is colocalized with mitochondria marker (right panel). Similar results were obtained in human EC line EAhy926 (data not shown). TNF treatment (10 ng/mL for 15 minutes) does not alter the localization of ASK1 in mitochondria (data not shown).
Localization of endogenous ASK1 in EC mitochondria was further determined by indirect immunofluorescence microscopy. ASK1 showed a punctate mitochondrial staining and was colocalized with Mitotracker (online Figure S1, in the online data supplement at http://circres.ahajournals.org). Similar data were obtained in bovine endothelial cells (BAECs) and human EC cell line EAhy.926 (data not shown). To determine whether ASK1 and Trx2 forms a complex in mitochondria, cytosolic and mitochondrial fractions from HUVECs were isolated. Mitochondrial protein Trx2 reductase and Trx2 are detected only in the mitochondrial fraction, whereas Trx1 was excluded from mitochondria and detected only in the cytoplasm. However, ASK1 is detected in both mitochondrial and cytosolic fractions (Figure 1B). We next monitored formation of ASK1-Trx2 complex and its regulation by apoptotic stimuli such as TNF and hydrogen peroxide. HUVECs were left untreated or treated with TNF as described previously.10 Subsequently, the association of ASK1 with Trx2 in mitochondria was determined by immunoprecipitation. As previously described, Trx1 binds to cytosolic ASK1 and is dissociated from ASK1 on TNF treatment (Figure 1B). Association of Trx2 with ASK1 in the mitochondrial fraction was readily detected in untreated EC cells (Figure 1B, −TNF). TNF treatment did not significantly alter the partition of ASK1 and Trx2 proteins between mitochondrial and cytosolic fractions. However, TNF significantly reduced the mitochondrial ASK1-Trx2 complex (Figure 1B, +TNF), suggesting that TNF induced a dissociation of Trx2 from ASK1. Similar results were obtained in BAECs with TNF (10 ng/mL) or H2O2 (1 mmol/L) (data not shown). These data suggest Trx2, like Trx1, binds to ASK1 in resting cells and is dissociated from ASK1 in response to TNF/ROS.
To further characterize interaction between Trx2 and ASK1, we performed an in vitro GST pull-down assay using GST-Trx2. BAEC lysates containing ASK1 protein was used for an in vitro pull-down assay. ASK1 binds to GST-Trx1 and GST-Trx2, but not GST alone (Figure 1C). Trx2 contains the two Cys located in the catalytic sites (Cys90 and Cys93) corresponding to Cys32 and Cys35. To determine their roles in ASK1 binding, we mutated the Cys residues to generate Trx2-C90S and Trx2-C93S. We examined association of ASK1 with Trx2 proteins in the presence or absence of 1 mmol/L H2O2. ASK1 bound to Trx2-WT and Trx2-C93S (but not to Trx-C90S) in the absence of H2O2 (Figure 1D), suggesting that Cys90 in Trx2 is critical for ASK1-binding. Addition of 1 mmol/L H2O2 disrupted the association of Trx2-WT with ASK1. In contrast, Trx2-C93S retained its association with ASK1 in the presence of H2O2 (Figure 1D). These data indicate that Trx2-C93S, like Trx1-C32S or C35S, forms a stable complex with ASK1, which is resistant to dissociation by ROS.
ASK1 Mutant Defective in Trx2-Binding Enhanced Apoptotic Activity Without Compromising JNK Activation
We have previously proposed that Trx1 binds to the N-terminal domain of ASK1 via a formation of an intermolecular disulfide bride.10 To define a Cys residue in ASK1 that might participate in this interaction, we generated a series of N-terminal truncation and site-specific mutant constructs of ASK1. By in vitro GST pull-down assay, we first defined the aa73–301 and aa1–73 in ASK1 are critical for association of Trx1 and Trx2, respectively (Figures 2A and 2B). We mutated the Cys residues within this region to Ser (C22, C30, C67, C120, C185, C200, 206, C225/226, and C250) and found that C30 is critical for Trx2-binidng, whereas C250 for Trx1-binding (Figures 2C and 2D). ASK1-C30S lost the ability to bind Trx2, and Trx1-defective mutant (ASK1-C250S) retains ability for Trx2-binding, and vice versa (Figure 2E). These data further support our model that Trx and ASK1 form a complex via an intermolecular disulfide bridge (Cys32 or Cys35 in Trx1 with Cys250 in ASK1; Cys90 in Trx2 with Cys30 in ASK1), and TNF/ROS likely disrupt these intermolecular disulfide bonds leading to Trx oxidation and dissociation from ASK1.
We reasoned that ASK1 mutants defective in Trx-binding (ASK1-C30S for Trx2 and ASK1-C250S for Trx1) should have increased activities in apoptosis and JNK activation. To test this hypothesis, HA-tagged ASK1 constructs were transfected into BAECs. Mutations either at C30 or at C250 did not significantly alter localization of ASK1 by subcellular fractionation analyses; online Figure S2C). Similar results were obtained from subcellular localization analysis of ASK1 proteins by confocal microscopy (not shown). To determine ASK1-induced JNK activation, BAECs were transfected with ASK1-WT, C30S, or C250S in the presence of a JNK-reporter gene. ASK1 protein expression and basal ASK1 activity were determined by Western blot and an in vitro kinase assay using GST-MKK4 as a substrate. ASK1-C250S and ASK1-C30S show higher basal activities than ASK1-T (Figure 2F), suggesting that Trx1 and Trx2 are endogenous inhibitor of ASK1. JNK activation was determined by JNK-dependent reporter gene assay. ASK1-C250S (but not ASK1-C30S) expression elicited a dramatic increase of JNK activation (Figure 2B). EC survival and apoptosis were determined by cell number and nuclear fragmentation (DAPI staining). In contrast to JNK activation, both ASK1-C30S and C250S had increased apoptotic activities compared with ASK1-WT (Figure 2C). These data suggest that Trx1 and Trx2 are critical regulators of ASK1.
TNF Induces Both JNK-Dependent and Independent Apoptotic Pathways
The data that ASK1-C30S increases cell death without JNK activation suggest that ASK1 in mitochondria may mediate a JNK-independent apoptotic pathway. We first determined if TNF induces a JNK-independent pathway in ECs. ECs were treated with TNF (10 ng/mL) plus cycloheximide (CHX, 10 μg/mL) in the presence or absence of JNK-specific inhibitor SP600125 (20 μmol/L). Previously, it has been shown that JNK-dependent events in TNF-induced apoptosis include cleavage of Bid and mitochondrial translocation of Bax. We examined JNK activation, Bid cleavage, and Bax translocation in ECs. JNK activation was determined by an in vitro kinase assay. Bid cleavage was determined by Western blot with anti-Bid, which recognizes both intact and truncated Bid. Bax translocation was determined by indirect immunofluorescence microscope with anti-Bax which specifically recognizes mitochondrial conformation of Bax. Results show that TNF(+CHX) in ECs strongly induces JNK activation, Bid cleavage, and Bax translocation (Figures 3A and 3B). Bid cleavage may include both caspase-8-cleaved tBid and JNK-dependent jBid.14 SP600125 diminishes TNF-induced JNK activity, Bid cleavage, and Bax translocation (Figure 3A and 3B). However, SP600125 only partially inhibits TNF-induced cytochrome c release and apoptosis (Figures 3C and 3D). As a control, caspase-3 inhibitor z-VAD (30 μmol/L) significantly blocks TNF(+CHX)-induced apoptosis in ECs (Figure 3D). These data suggest that TNF induces both JNK-dependent and JNK-independent apoptotic pathways. Cytochrome c release and capsase-3 activation appear to be common downstream events.16 The JNK-dependent (but not JNK-independent) apoptotic pathway involves in Bid cleavage in cytoplasm and translocation of Bax into mitochondria.
Mitochondria-Located ASK1 Specifically Mediates a JNK-Independent Apoptotic Pathway
We hypothesize that cytosolic ASK1 specifically mediates JNK-dependent pathways, whereas mitochondrial ASK1 specifically mediates JNK-independent pathways. To test our hypothesis, we specifically expressed a constitutively active form of ASK1 (ASK1-ΔN) in mitochondria by fusing the Trx2 mitochondria targeting sequence to ASK1-ΔN (mtASK1-ΔN) (Figure 4A). The mitochondrial targeting sequence of Trx2 (aa1–60) is critical for its localization in mitochondria, and Trx2 with deletion of this sequence (ΔTrx2) is expressed in the cytoplasm (online Figure S3A). ASK1-ΔN lacking the N-terminal binding domain showed exclusively cytoplasmic localization (online Figure S3B). However, mtASK1-ΔN is specifically detected in mitochondria and colocalized with Trx2 (online Figure S3C). We then examined ASK1-ΔN and mtASK1-ΔN-induced JNK activation, Bax translocation, and apoptosis. Although mtASK1-ΔN shows as similar ASK1 basal activity to ASK1-ΔN as measured by an in vitro kinase assay, mtASK1-ΔN or mtASK1-ΔN-K709R (a kinase inactive mutant) did not induce JNK activation (Figures 4B and 4C). ASK1-ΔN, but not mtASK1-ΔN or mtASK1-ΔN-K709R, significantly induced Bax translocation into mitochondria (Figure 4D). However, ASK1-ΔN or mtASK1-ΔN induced comparable EC apoptosis (Figure 4E). ASK1-ΔN-induced apoptosis is strongly inhibited by either caspase-3 inhibitor z-VAD or JNK-specific inhibitor SP600125. However, mtASK1-ΔN-induced apoptosis is significantly blocked by z-VAD, but not by SP600125 (Figure 4E). mtASK1-ΔN did not induce p38 activation as determined by Western blot with anti-phopsho-p38, and mASK1-induced apoptosis was not inhibited by p38-specific inhibitor SB203580 (data not shown). These data strongly support that mitochondria-located ASK1 specifically induces a JNK-independent apoptotic pathway.
Trx2 Specifically Inhibits ASK1-Induced EC Apoptosis With no Effects on ASK1-Induced JNK Activation
To determine the effects of Trx2 on ASK1, Trx2 with a V5 epitope-tag at the C-terminus was transfected into BAECs, which have high transfection efficiency (>80%) compared with HUVECs (≈10%). The transfected Trx2, like the endogenous Trx2, showed a strong punctate mitochondrial staining, whereas Flag-tagged Trx1 displayed a smear of nonmitochondrial staining (online Figure S4A). Subcellular fractionation also showed that Trx2 is detected in the mitochondrial fraction whereas Trx1 in cytosolic fractions (online Figure S4B). We then determined the effects of Trx2 expression on ASK1-induced JNK activation as determined by an in vitro kinase assay and EC apoptosis. As previously demonstrated,15 overexpression of ASK1 induced JNK activation, cytochrome c release, and EC apoptosis (Figures 5A through 5C). Expression of Trx1 significantly inhibited these effects. Although no effect on ASK1-induced JNK activation was detected, expression of Trx2 significantly decreased ASK1-induced cytochrome c release and EC apoptosis (Figures 5B and 5C). In contrast, ASK-binding defective mutant Trx2-C90S diminished ability to inhibit ASK1-induced JNK activation and EC apoptosis (Figures 5B and 5C), suggesting that the inhibitory effects of Trx2 on ASK1 is dependent on the interaction between the two molecules.
Knockdown of Trx1 or Trx2 Sensitizes Cells to ASK1-Induced Apoptosis
To further characterize the roles of Trx1 and Trx2 in TNF/ASK1-induced apoptosis, we used an RNA interference (RNAi) approach (see Materials and Methods). HUVECs were infected with adenovirus expressing shRNA to human Trx1 and Trx2. The expression of endogenous Trx1 and Trx2 was specifically downregulated (75% to 80%) using appropriate shRNAs (online Figures S5A and S5B). We next examined the effects of Trx knockdown on TNF-induced JNK activation measured by an in vitro kinase assay. TNF-induced JNK activation was significantly elevated in Ad-ShTrx1, but not in Ad-ShTrx2 cells (Figure 6A). We then examined TNF-induced apoptosis in Trx1 or Trx2-knockdown cells. ECs expressing Trx1 or Trx2 shRNA were treated with TNF (10 ng/mL) in the presence of CHX (10 μg/mL) for 2 to 6 hours when EC apoptosis peaks. TNF-induced Bid cleavage, Bax translocation, cytochrome c release, and cell death were determined as described. Knockdown of Trx1 (but not of Trx2) significantly sensitizes cells to TNF(+CHX)-induced Bid cleavage and Bax translocation (Figures 6B and 6C). However, TNF(+CHX)-induced cytochrome c release was significantly increased in both Trx1- and Trx2-knockdown cells (Figure 6d), consistent with that cytochrome c release is a common downstream regulated by Trx1 and Trx2. Thus, TNF(+CHX)-induced apoptosis (Figure 6E) were greatly increased in both Trx2- and Trx1-knockdown cells compared with the control cells. These data support that Trx2 specifically regulates the JNK-independent intrinsic apoptotic pathway induced by TNF.
Trx1 and Trx2 Regulate Distinct Apoptotic Pathways Induced by TNF and ASK1
Although Trx1 and Trx2 are major components of the cellular antioxidant system, distinct biological functions of Trx1 and Trx2 have been demonstrated by studies using genetically deficient mice and cells. Deletion of either Trx1 or Trx2 causes embryonic lethality,6,17 suggesting that they are not functionally redundant. In the present study, we demonstrate that Trx1 and Trx2 cooperatively regulate TNF/ASK1 apoptotic activities (Figure 7). First, Trx1 and Trx2 bind to ASK1 at different sites. Cys-250 and Cys-30 in the N-terminal domain of ASK1 are critical for association with Trx1 and Trx2, respectively. Second, Trx1 and Trx2 regulate distinct signaling events induced by TNF/ASK1 in distinct cellular compartments. ASK1 is localized in cytoplasm where it binds to Trx1 as well as in mitochondria where it associates with Trx2. We have previously demonstrated that association of Trx1 with ASK1 regulates ASK1-induced JNK activation and apoptosis in EC.10 Consistently, mutation of ASK1 that prevents Trx1-binding (ASK1-C250S), knockdown of Trx1, or expression of cytosolic form of ASK1 leads to a JNK-dependent apoptotic pathway (JNK activation, Bid cleavage, and Bax mitochondrial translocation). In contrast, cells expressing a mutant ASK1 with deficiency for Trx2-binding (ASK1-C30S), knockdown of Trx2 by RNA interference, or specific expression of ASK1 in mitochondria significantly increase apoptosis in a JNK-independent manner. Our data suggest that cytochrome c release and caspase-3 activation are common downstream events in JNK-dependent and JNK-independent pathways induced by TNF/ASK1. Taken together, ASK1-Trx2 complex in mitochondria represents a distinct pathway regulating vascular EC survival and death in response to proinflammatory cytokines and oxidative stress.
ASK1 Mediates Multiple Apoptotic Pathways
ASK1 can be activated by various proapoptotic stimuli including death receptors, DNA damaging agents, oxidants, and cellular stresses such as growth factor deprivation and endoplasmic reticulum (ER) stresses caused by protein aggregation,18 suggesting that ASK1 may mediate multiple apoptotic pathways. Indeed, it has been shown that ASK1 induced cytochrome c release and activation of caspase-9 and caspase-3, but not of caspase-8.12 In response to ER-stress such as protein aggregation, TRAF2 and ASK1 are recruited by an ER-transmembrane sensor IRE1 to form IRE1-TRAF2-ASK1 complex leading to JNK activation and apoptosis.19 In this study, we show that ASK1 is localized in both cytoplasm and mitochondria of vascular EC where it induces distinct apoptotic signaling events. Because we did not observe significant translocation of ASK1 from cytoplasm to mitochondria during apoptosis, we propose that ASK1 compartmentalization is critical for responses to various proapoptotic stimuli. Thus, cytoplasm-located ASK1 may mediate death receptor (Fas), Daxx, or ER stress-induced extrinsic apoptotic pathway. In contrast, ASK1 located in mitochondria may mediate cell death via an intrinsic apoptotic pathway in response to TNF, ROS, or DNA damaging agents.
Function of ASK1-Trx2 Complex in Mitochondria
Our data from immunogold EM clearly showed that ASK1 is localized in mitochondria in resting cells. This is different from JNK protein that translocate into mitochondria during apoptosis.20 ASK1 is colocalized with Trx2 and form a complex with Trx2 in mitochondria, suggesting that ASK1 likely resides inside mitochondria matrix where Trx2 is located. The mechanism by which ASK1 in mitochondria mediates TNF-induced apoptosis is not understood. Trx is one of the major systems combating against oxidative stress. It is conceivable that Trx1 in cytoplasm and Trx2 in mitochondria inhibit ASK1 activity by either scavenging ROS or by a direct interaction with ASK1. A direct association of Trx1 or Trx2 with ASK1 appears to be critical for regulation of ASK1 activity. It has been shown that TNF induces mitochondrial ROS production in HUVECs primarily occurs at the ubisemiquinine site.21 It is likely that ROS generated in the mitochondria in response to TNF induce dissociation of Trx2 from ASK1 leading to its activation. Interestingly, we have previously shown that TNF can be delivered into mitochondria of ECs where TNF receptors are present.22
ASK1 appears to directly target on regulators participated in cytochrome c release. It has been shown that Trx2 forms complexes with cytochrome c.3 Thus, ASK1 may directly regulate the formation and dissociation of Trx2-cytochrome c complex. In support of this model, Trx2-knockdown cells show sensitized cytochrome c release in response to TNF. The nature of ASK1-Trx2-cytochrome c complex and its regulation needs to be further investigated. Another possibility is that ASK1 may target Bcl-2/Bax family proteins, which have been implicated in regulation of mitochondrial voltage-dependent anion channel and release of cytochrome c.23,24 Alternatively, ASK1 may target other mitochondrial components such as the mitochondrial permeability transition pore (PTP). This is supported by the data that overexpression of Trx2 showed increased mitochondrial membrane potential and sensitivity toward rotenone, an inhibitor of complex I of the respiratory chain.2
In summary, our data demonstrate that Trx1 and Trx2 differentially regulate ASK1-mediated apoptotic events (Figure 7). It is conceivable that Trx1 and Trx2 cooperatively execute their antiapoptotic function against ASK1-mediated cell death. Our study should provide novel therapeutic approaches to treat apoptosis-associated cardiovascular diseases.
This work was supported by a grant from NIH 1R01HL65978-01 and AHA 0151259T to W.M., Medical Research Council (UK) to J.B. and R.A.-L., and NIH HL62572 to J.M.M. We thank Dr Jordan S. Pober (Yale University) for discussion.
Original received February 17, 2004; revision received April 19, 2004; accepted April 20, 2004.
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