Thioredoxin-2 Inhibits Mitochondria-Located
ASK1-Mediated Apoptosis in a JNK-Independent Manner
Rong Zhang, Rafia Al-Lamki, Lanfang Bai, Jeffrey W. Streb, Joseph M. Miano, John Bradley, Wang Min
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Abstract—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. Mitochondriaspecific 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. (Circ Res. 2004;94:1483-1491.)
Key Words: thioredoxin 䡲 ASK1 䡲 mitochondria 䡲 TNF-␣ 䡲 apoptosis
T
hioredoxins (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 peroxi-
dase, 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 E10.5day 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-␬B 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
Original received February 17, 2004; revision received April 19, 2004; accepted April 20, 2004.
From the Interdepartmental Program in Vascular Biology and Transplantation (R.Z., L.B., W.M.), Boyer Center for Molecular Medicine, Department
of Pathology, Yale University School of Medicine, New Haven, Conn; Department of Medicine (R.A.-L., J.B.), University of Cambridge, UK; and Center
for Cardiovascular Research (J.W.S., J.M.M.), University of Rochester, NY.
Correspondence to Dr Wang Min, Interdepartmental Program in Vascular Biology and Transplantation, Department of Pathology, Yale University
School of Medicine, BCMM 454, 295 Congress Ave, New Haven, CT 06510. E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000130525.37646.a7
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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-⌬N) 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
Results
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 (⌬) 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)
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Figure 1. ASK1 is localized inside mitochondria where it associates with Trx2. A, Colocalization of endogenous ASK1 and a mitochondrial marker in HUVECs. HUVECs were stained with anti-ASK1 (5 nm gold) and anti-mitochondria marker (15 nm gold). m indicates
mitochondria; rER, rough endoplasmic reticulum; N, nucleus; ⌬, mitochondrial ASK1; and *, cytoplasmic ASK1. Original magnifications:
Left and mid, ⫻52兩000; right, ⫻105兩000). B, TNF induces dissociation of Trx2 from ASK1. HUVECs were either left untreated or treated
with TNF (10 ng/mL for 15 minutes). ASK1, TrxR2, Trx1, and Trx2 protein in cytoplasmic and mitochondrial fractions were determined
by respective antibodies. Trx1-ASK1 and Trx2-ASK1 complexes were determined by immunoprecipitation with ASK1 followed by Western blot with anti-Trx1 and anti-Trx2 antibodies, respectively. C, Association of ASK1 with Trx2 in vitro. BAEC lysates expressing
HA-ASK1 were applied to GST-Trx1 or Trx2 protein beads. Bound ASK1 was determined by Western blot with HA. GST-fusion proteins
were detected by Western blot with anti-GST. D, Cys-90 at the catalytic site of Trx2 is critical for ASK1 binding. GST-Trx2 fusion proteins (WT, C90S, or C93S) were used for a pull-down assay in the absence (⫺) or presence (⫹) of 1 mmol/L H2O2. Bound ASK1 and
GST-fusion proteins were determined.
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).
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Figure 2. Effects of mutation at Trx-binding sites
on ASK1-mediated JNK activation and apoptosis.
A and B, Domain mapping of ASK1 for Trx1 (A)
and Trx2 (B) binding. Truncated expression constructs for ASK1-N (aa1–697), ASK1-N1 (aa1–501),
N2 (aa1–301), and N3 (aa1–73) were generated.
BAEC lysates expressing truncated ASK1-N were
used to determine Trx1-ASK1 and Trx2-ASK1
interaction by an in vitro pull-down assay using
GST-Trx1 (A) or GST-Trx2 (B), respectively.
Trx1-CS (double-mutations at C32 and C35) was
used as a negative control. Bound ASK1-N was
detected by Western blot with anti-Flag. C through
E, Mapping the critical Cys residue in ASK1 for
binding of Trx1 or Trx2. BAEC lysates expressing
ASK1-WT or ASK1-CS mutants were used for a
pull-down assay. Bound ASK1 was detected by
Western blot with anti-HA. f, ASK1-C250S and
ASK1-C30S have increased basal ASK1 activities
compared with ASK1-WT. BAECs were transfected with control vector (⫺), ASK1-WT, ASK1C30S, or ASK1-C250S. ASK1 protein was determined by Western blot with anti-HA. ASK1 activity
was determined by an in vitro kinase assay using
GST-MKK4 as a substrate. Relative ASK1 activities
are shown by taking ASK1-WT as 1.0. G, ASK1C250S (but not ASK1-C30S) increases JNK activation. BAECs were transfected with ASK1 constructs in the presence of a JNK-dependent
reporter gene (Materials and Methods). Relative
luciferase activities are presented from mean of
duplicate samples by taking vector control as 1.
Similar results were obtained from 2 additional
experiments. H, ASK1-C30S defective in Trx2binding increases apoptotic activity. BAECs were
transfected with ASK1 constructs. Forty eight
hours after transfection, nucleus fragmentation was
visualized by DAPI staining. Apoptosis rate is
shown. Data are presented as mean of duplicates
from two independent experiments. *P⬍0.05.
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 (ASK1C250S) 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 JNKreporter 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 JNKdependent reporter gene assay. ASK1-C250S (but not ASK1C30S) 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 (Fig-
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Figure 3. TNF induces JNK-dependent and independent apoptotic pathways. HUVECs were pretreated with JNK inhibitor SP600125
(20 ␮mol/L) for 30 minutes followed by treatment with TNF (10 ng/mL) plus cycloheximide (CHX, 10 ␮g/mL) for 6 hours. A, SP600125
diminishes TNF-induced JNK activation and Bid cleavage. Total EC lysates were used to determine JNK activity by an in vitro kinase
assay using GST-c-Jun as a substrate (top) and Bid cleavage by Western blot with anti-Bid (bottom). B, SP600125 blocks TNF-induced
Bax translocation into mitochondria. Bax localization in treated ECs was determined by immunofluorescence microscope using antiBax, which specifically recognizes mitochondrial form of Bax. C, SP600125 only partially inhibits TNF-induced cytochrome c release.
Cytoplasm fractions from treated ECs were isolated as described and cytochrome c was determined by Western blot with anticytochrome c. D, SP600125 only partially inhibits TNF-induced EC apoptosis. HUVECs were treated with TNF⫹CHX in the presence of
JNK-specific inhibitor SP600125 (20 ␮mol/L) or caspase-3 inhibitor z-VAD (30 ␮mol/L) as indicated for 6 hours. EC apoptosis was
determined by DAPI staining for nuclei fragmentation. Apoptosis rate is shown. Data are presented as mean of duplicates from two
independent experiments. *P⬍0.05.
ure 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
␮g/mL) in the presence or absence of JNK-specific inhibitor
SP600125 (20 ␮mol/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 TNFinduced cytochrome c release and apoptosis (Figures 3C and
3D). As a control, caspase-3 inhibitor z-VAD (30 ␮mol/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-⌬N) in mitochondria by fusing the
Trx2 mitochondria targeting sequence to ASK1-⌬N
(mtASK1-⌬N) (Figure 4A). The mitochondrial targeting
sequence of Trx2 (aa1– 60) is critical for its localization in
mitochondria, and Trx2 with deletion of this sequence
(⌬Trx2) is expressed in the cytoplasm (online Figure S3A).
ASK1-⌬N lacking the N-terminal binding domain showed
exclusively cytoplasmic localization (online Figure S3B).
However, mtASK1-⌬N is specifically detected in mitochondria and colocalized with Trx2 (online Figure S3C). We then
examined ASK1-⌬N and mtASK1-⌬N–induced JNK activation, Bax translocation, and apoptosis. Although
mtASK1-⌬N shows as similar ASK1 basal activity to
ASK1-⌬N as measured by an in vitro kinase assay,
mtASK1-⌬N or mtASK1-⌬N-K709R (a kinase inactive mu-
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Figure 4. Mitochondria-located ASK1 specifically mediates a JNK-independent apoptotic pathway. A, Diagram for expression constructs of Trx2, ⌬Trx2, ASK1-⌬N, mtASK1-⌬N, and mtASK1-⌬N-K709R. Mt indicates mitochondria targeting signal sequence of Trx2.
Band C, JNK-dependent reporter gene was cotransfected with ASK1-⌬N, mtASK1-⌬N, and mtASK1-⌬N-K709R into BAECs. Expression and the basal ASK1 activity was determined by Western blot with anti-ASK1 and by an in vitro kinase assay using GST-MKK4 as
a substrate (B). JNK activities (relative luciferase activities) are presented from mean of duplicate samples by taking vector control as 1
(C). Similar results were obtained from 2 additional experiments. D, Bax translocation. BAECs were transfected with ASK1 constructs
as indicated. Bax translocation was determined by indirect immunofluorescence microscope using a double staining with anti-ASK1
(followed by anti-mouse IgG-FITC) and anti-Bax (followed by anti-rabbit-TRITC). Percentage of Bax staining in ASK1-positive cells is
presented. Data are presented as mean of duplicates from two independent experiments. E, EC apoptosis. BAECs were transfected
with ASK1 constructs in the presence of JNK inhibitor SP600125 (20 ␮mol/L) or caspase-3 inhibitor (z-VAD, 30 ␮mol/L). Twenty four
hours after transfection, apoptotic cells (with nuclear condensation) were determined after DAPI staining. Apoptosis rate is shown. Data
are presented as mean of duplicates from two independent experiments.
tant) did not induce JNK activation (Figures 4B and 4C).
ASK1-⌬N, but not mtASK1-⌬N or mtASK1-⌬N-K709R,
significantly induced Bax translocation into mitochondria
(Figure 4D). However, ASK1-⌬N or mtASK1-⌬N induced
comparable EC apoptosis (Figure 4E). ASK1-⌬N–induced
apoptosis is strongly inhibited by either caspase-3 inhibitor
z-VAD or JNK-specific inhibitor SP600125. However,
mtASK1-⌬N–induced apoptosis is significantly blocked by
z-VAD, but not by SP600125 (Figure 4E). mtASK1-⌬N 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 mitochondrialocated ASK1 specifically induces a JNK-independent apoptotic pathway.
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.
Trx2 Specifically Inhibits ASK1-Induced EC
Apoptosis With no Effects on ASK1-Induced
JNK Activation
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. TNFinduced JNK activation was significantly elevated in AdShTrx1, but not in Ad-ShTrx2 cells (Figure 6A). We then
examined TNF-induced apoptosis in Trx1 or Trx2knockdown cells. ECs expressing Trx1 or Trx2 shRNA were
treated with TNF (10 ng/mL) in the presence of CHX (10
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
Knockdown of Trx1 or Trx2 Sensitizes Cells to
ASK1-Induced Apoptosis
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Figure 5. Trx2 specifically inhibits ASK1-induced EC apoptosis with no effects on ASK1-induced JNK activation. BAECs
were transfected with VC alone (Ctrl) or ASK1 in the presence
or absence of Trx1, Trx2, or both. Forty eight hours after
transfection, JNK activity was determined by an in vitro
kinase assay (A). Relative JNK activity/ASK1 protein is presented from mean of duplicate samples from two independent experiments by taking ASK1 as 1. Expression of ASK1,
Trx1, and Trx2 was determined by Western blot with antiASK1, anti-Flag, and anti-V5, respectively. Cytosolic fractions
were isolated and ASK1-induced cytochrome c release was
detected by Western blot with anti-cytochrome c (B). C,
BAECs were transfected with VC, Trx1, Trx2, or both in the
absence or presence of ASK1. Nuclear fragmentation in cells
expressing transgenes (ASK1 and/or Trx) was determined and
total 50 cells from each group were examined. Apoptotic rate
is shown. Data are presented as means of duplicates from
three independent experiments.
␮g/mL) for 2 to 6 hours when EC apoptosis peaks. TNFinduced 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 Trx1and Trx2-knockdown cells (Figure 6d), consistent with that
Trx2 Inhibits ASK1
1489
Figure 6. Trx2 knockdown sensitizes cells to ASK1-mediated
responses. A, Knockdown of Trx1 (but not of Trx2) sensitizes
cells to TNF-induced JNK activation. EC infected with Ad-Shag,
Ad-ShTrx1, or Ad-ShTrx2 were treated with TNF (10 ng/mL for
15 minutes), and JNK activity was determined by an in vitro
kinase assay using GST-c-Jun as a substrate. Relative JNK
activities are presented from mean of duplicate samples by taking vector control as 1. Similar results were obtained from 2
additional experiments. B through E, Effects of Trx1 and Trx2
knockdown on TNF(⫹CHX)–induced Bid cleavage, Bax translocation, cytochrome c release, and cell death. EC infected with
Ad-Shag, Ad-ShTrx1, or Ad-ShTrx2 were. Forty eight hours after
infection, cells were treated with TNF (10 ng/mL) plus CHX (10
␮g/mL) for 2 to 6 hour. B, EC lysates at 2- and 6-hour time
points were used to determine Bid cleavage by Western blot
with anti-Bid. C, Bax translocation at 6-hour time point was
determined as in Figure 4. Percentage of Bax staining cells are
presented. Data are presented as mean of duplicates from two
independent experiments. D, Cytosolic fractions from EC at
6-hour time point of treatment were isolated, and cytochrome c
was detected by Western blot with anti-cytochrome c. Relative
levels of cytochrome c are shown (setting TNF⫹CHX-treated
Shag vector as 1.0). E, Apoptotic cells (with nuclear condensation) at 6-hour time point were determined by DAPI staining.
Apoptosis rate is shown. Data are presented as mean of duplicates from two independent experiments. *P⬍0.05.
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 Trx1knockdown cells compared with the control cells. These data
support that Trx2 specifically regulates the JNK-independent
intrinsic apoptotic pathway induced by TNF.
Discussion
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
1490
Circulation Research
June 11, 2004
Figure 7. Model for regulation of ASK1mediated apoptosis by Trx1 and Trx2.
Trx1 in cytoplasm, whereas Trx2 in mitochondria, forms a complex with ASK1 in
resting cells to retain ASK1 in an inactive
state. Proapoptotic stimuli (such as TNF
and ROS) dissociate Trx1 and Trx2 from
ASK1 leading to ASK1 activation. ASK1
in cytoplasm mediates a JNK-dependent
apoptotic pathway associated with JNK
activation, Bid cleavage, and Bax translocation. ASK1 in mitochondria mediates
a JNK-independent apoptotic pathway.
The two pathways appear to converge at
cytochrome c release and caspase-3
activation. Thus, Trx1 and Trx2 cooperatively inhibit ASK1-mediated apoptosis.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
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 JNKdependent 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 JNKindependent 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-TRAF2ASK1 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 possibil-
Zhang et al
ity is that ASK1 may target Bcl-2/Bax family proteins, which
have been implicated in regulation of mitochondrial voltagedependent 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.
Acknowledgments
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
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.
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Thioredoxin-2 Inhibits Mitochondria-Located ASK1-Mediated Apoptosis in a
JNK-Independent Manner
Rong Zhang, Rafia Al-Lamki, Lanfang Bai, Jeffrey W. Streb, Joseph M. Miano, John Bradley
and Wang Min
Circ Res. 2004;94:1483-1491; originally published online April 29, 2004;
doi: 10.1161/01.RES.0000130525.37646.a7
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2004 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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World Wide Web at:
http://circres.ahajournals.org/content/94/11/1483
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2004/06/02/94.11.1483.DC1
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Supplemental Materials (Zhang et al., Thioredoxin-2 inhibits mitochondria-located ASK1-mediated
apoptosis in a JNK-independent manner).
MATERIALS AND METHODS
Constructs Mammalian expression plasmids for V5-epitope tagged (at the C-terminus) Trx2 and
cytosolic form of Trx2 lacking the mitochondria targeting sequence (aa1-60) (∆Trx2) were generously
provided by Dr. Dean P. Jones (Emory University) 1. Trx1 and ASK1 constructs were described
previously
2,3
. A mitochondrial form of ASK1-∆N (mtASK1-∆N) was generated by inserting Trx2
mitochondrial targeting sequence (aa1-60) to the N-terminus of ASK1-∆N. The mutant ASK1 (CS
mutants), Trx2-C90S and Trx2-C93S were constructed by site-directed mutagenesis using
QuickchangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer’s protocol, and
mutations at the desired sites were confirmed by DNA sequencing. pShag vector were from Dr. Gregory
J. Hannon (Cold Spring Harbor Laboratory, NY) 4. The adenoviral Gateway vector (pAd/PL-DEST) and
ViraPower Adenoviral Expression System were purchased from Invitrogen (Invitrogen Corp., San
Diego, California, USA).
Antibodies. A rabbit polyclonal antibody against Trx1 or Trx2 was generated by immunizing rabbits
with GST-Trx1 or GST-Trx2 protein through Cocalico Biologicals Inc. (Reamstown, Pennsylvania,
USA). We obtained anti-ASK1 (H300), anti-Bax (N20) and anti-caspase 8 from Santa Cruz
Biotechnology. Anti-cytochrome c and anti-β-tubulin were purchased from BD Pharmingen. Anti-Bid
and anti-phospho-p38 were purchased from Cell Signaling. Anti-V5 was from Invitrogen, anti-HA was
from Roche and anti-Flag was from Sigma.
Zhang et al., Trx2 inhibits ASK1
1
Cells, cytokines and inhibitors. Human umbilical vein endothelial cells (HUVECs) and bovine aortic
ECs (BAECs) and were purchased from Clonetics Corp. (San Diego, California, USA). Human
umbilical vein EC (HUVEC) were cultured in modified M199 culture medium, containing 20% v/v
heat-inactivated bovine fetal calf serum (FCS), 100µg/ml heparin sodium salt, 30 µg/ml endothelial cells
growth supplement, 2 mM L-glutamine, 60 units/ml penicillin, and 0.5 µg/ml streptomycin at 370C, in
5% CO2 on gelatin-coated tissue culture plastic as described previously 5. Cells were used at passages 24. Human recombinant TNF was from R&D Systems Inc. (Minneapolis, MN) and used at 10 ng/ml.
Caspase-3 inhibitor (z-VAD), JNK-specific inhibitor SP600125 and p38 inhibitor SB203580 were
purchased from Calbiochem.
Immunogold electron microscopy. The cells were dislodged gently using a rubber policeman, fixed in
4% formaldehyde in 0.1M PIPES (Sigma, UK) buffer pH 7.5 for 30 minutes at 40C, washed three times
for 2 minutes each in 0.1M PIPES containing 2mM CaCl2 and centrifuged at 210 x g. The cells pellets
were then collected and processed for freeze substitution and low temperature embedding for
immunocytochemistry as described 6. Briefly, HUVEC were cryoprotected in 30% propylene glycol for
1 hour at 40C, and were frozen in melting propane cooled in liquid nitrogen, substituted against
methanol containing 0.1% uranyl acetate at -900C for 24 hours, at -70oC for 24 hours, and at -50oC for
24 hours. Cells were then impregnated with Lowicryl HM 20 resin over a period of 3 days and the resin
was polymerized by ultraviolet irradiation at a temperature of -50oC. Ultrathin sections (40-50 nm) were
cut on a Leica Ultracut-S (Leica, Vienna, Austria) ultramicrotome and mounted on Formvar-coated
nickel grids. The grids were incubated, section down, for 30 min at room temperature in blocking buffer
containing 10% FCS (fetal calf serum) in 0.1M Tris/HCI buffer pH 7.5 (TBS) to suppress non-specific
Zhang et al., Trx2 inhibits ASK1
2
antibody binding. Excess blocking buffer was removed and sections were incubated overnight at room
temperature, with rabbit polyclonal anti-human ASK1 (H-300; sc-7931; Santa Cruz Biotechnology) or
anti-human Trx2 antibody diluted at 1:5 in blocking buffer at a final concentration of 40 µg/ml. After
rinsing extensively with TBS, the grids were incubated for 1 hour at room temperature with goat antirabbit-5 nm colloidal gold particles (British Biocell, Cardiff, UK) diluted 1:100 in the blocking buffer.
For co-localization of ASK1 and mitochondria, grids were incubated with anti-ASK1 (rabbit polyclonal)
and anti-mitochondria (mouse monoclonal anti-p65 in mitochondria, MAB1273, Chemicon with 1:5
dilution) followed by goat anti-rabbit-5nm colloidal gold particles and goat anti-mouse-15 nm colloidal
gold particles. After thorough rinsing in TBS and in double distilled water, the grids were contraststained with uranyl-acetate and lead citrate for 3 seconds each. Grids were then viewed in a Philip CM
100 electron microscope at an accelerating voltage of 80 kV. Controls included omission of the primary
antibody or use of non-immune serum as negative controls.
Confocal immunofluorescence microscopy. Fixation, permeabilization, and staining of cultured
HUVEC and BACE were performed as described previously
7,8
. For mitochondria visualization, cells
were stained with 20 nM tetramethylrhodamine methyl ester (TMRM) (Molecular Probes, Eugene, OR)
for 30 min at 37˚C and washed twice with phosphate buffered saline (PBS). For indirect fluorescence
microscopy, the fluorescein isothiocyanate(FITC)-conjugated anti-IgG and tetramethyl-rhodamine
isothiocyanate (TRITC)-conjugated anti-IgG were purchase from Molecular Probes. Confocal
immunofluorescence microscopy was performed using an Olympus confocal microscope.
Cell transfection and reporter gene assay. Transfection of HUVEC was performed by the DEAEdextran method as described previously 9 and transfection of BAECs was performed by Lipofectamine
Zhang et al., Trx2 inhibits ASK1
3
2000 according to manufacturer’s protocol (Invitrogen Corp., San Diego, California, USA). For reporter
gene assay, cells were cultured at 90% confluence in 6-well plates and were transfected with total of 2.5
µg plasmid constructs including reporter gene (1 µg), renilla gene (0.5 µg) and various transgenes (1 µg)
as indicated. Cells were harvested at 36-48 h post-transfection and cell lysates (10 µl) were measured for
luciferase activity followed by renilla activity twice in duplicates with Promega reagents (Promega
Corp., Madison, Wisconsin, USA) using a Berthold luminometer (EG&G Wallac, Gaithersburg,
Maryland, USA). All data were normalized as relative luciferase light unit/renilla unit.
Preparation of subcellular fractions. Subcellular fractions from HUVEC and BAEC were prepared as
described 1. To obtain the cytosolic fraction, cells were washed with PBS and resuspended in 50 µl of
250 mM sucrose and 70 mM Tris (pH 7.0) with protease inhibitors mixture (Roche Diagnostics Corp.,
Indianapolis, Indiana, USA). 10 µl of 4 mg/ml digitonin was added followed by incubation at room
temperature for 2 min. 2 µl of Cells were stained with trypan blue followed by direct observation with
light phase contrast microscopy to obtain lysis of 90–95% of cells. This approach allows preparation of
cytosol that is essentially free of mitochondrial contamination 1. After centrifugation at 600 x g for 2
min at room temperature, the supernatant was collected as the cytosolic fraction. To prepare the
mitochondrial fraction, cells were washed once with PBS, resuspended in ice-cold hypotonic buffer (10
mM NaCl, 1.5 mM CaCl2, 10 mM Tris-Cl, pH 7.5, with protease inhibitors mixture, and kept on ice for
10 min. MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM EDTA, 5 mM Tris, pH 7.6) was then
added, and cells were homogenized using a Dounce homogenizer with 30 strokes. Disruption of plasma
membrane was monitored by trypan blue staining. After removing the nuclear fraction by two
successive centrifugations at 3000 x g for 10 min, the supernatant was centrifuged at 12,000 x g for 10
min. The pellet was collected as the mitochondrial portion and resuspended in lysis buffer (50 mM
Zhang et al., Trx2 inhibits ASK1
4
HEPES, pH 7.0, 500 mM NaCl, and 1% Nonidet P-40, supplemented with a mixture of protease
inhibitors. After measuring the protein concentrations (Bio-Rad reagents), the recombinant protein
contents in both fractions were analyzed by Western blot analysis for cytochrome c (BD PharMingen,
San Diego, California, USA), Trx1, Trx2 and ASK1 as indicated.
Immunoprecipitation and immunoblotting. HUVECs or BAECs after various treatments were
washed twice with cold PBS and lysed in 1.5 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM
NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM
sodium pyrophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM PMSF, 1 mM EDTA) for 20 min
on ice. Protein concentrations were determined with a Bio-Rad kit. For immunoprecipitation to analyze
protein interaction in vivo, 400 µg of cell lysate supernatant were precleared by incubating with 5 µg
normal rabbit serum and protein A/G agarose (Santa Cruz Biotechnology Inc., Santa Cruz, California,
USA) on rotator at 4˚C overnight. The lysates were then incubated with 5 µg of the first protein-specific
antiserum (e.g. anti-Trx1 or anti-Trx2 antibody) for 2 h with 50 µl of protein A/G agarose. Immune
complexes were collected after each immunoprecipitation by centrifugation at 14,000 x g for 10 min
followed by 4 washes with lysis buffer. The immune complexes were subjected to SDS-PAGE followed
by immunoblot (Immobilon P, Millipore, Milford, MA) with the second protein (e.g., ASK1)-specific
antibody (Santa Cruz Biotechnology Inc.). The chemiluminescence was detected using an ECL kit
according to the instructions of the manufacturer (Amersham Life Science, Arlington Heights, Illinois,
USA). For detection of Flag-tagged proteins (Trx1 and ASK1-N, anti-Flag M2 antibody (SigmaAldrich, St Louis, Missouri, USA) was used for Western blot. For detection of HA-tagged proteins
(ASK1) and V5-tagged proteins (Trx2), anti-HA (Roche Diagnostics Corp) and anti-V5 antibodies
(Invitrogen) was used for Western blot, respectively.
Zhang et al., Trx2 inhibits ASK1
5
ASK1 and JNK kinase assays. ASK1 and JNK assays were performed as described previously 2,3 using
GST-MKK4 and GST-c-Jun (1-80) fusion protein as a substrate, respectively. Briefly, total 400 µg cell
lysates were immunoprecipitated with 5 µg of antibody against ASK1 or JNK1 (Santa Cruz). The
immunoprecipitates were mixed with 10 µg GST-MKK4 or GST-c-Jun (1-80) suspended in the kinase
buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 25 mM β-glycerophosphate, 100 µM sodium
orthovanadate, 2 mM DTT, 20 µM ATP) containing 1 µl (10 µCi) of [γ-32P] ATP. The kinase assay was
performed at 25˚C for 30 min. The reaction was terminated by the addition of Laemmli sample buffer
and the products were resolved by SDS-PAGE (12%) followed by protein transferring to a membrane
(Immobilon P). The phosphorylated GST-MKK4 or GST-c-Jun (1-80) was visualized by
autoradiography. The membrane was further used for Western blot with anti-ASK1 or anti-JNK1.
GST-Trx pull-down assay. GST fusion protein preparation and GST pull-down assay were performed
as described previously
2,3
. Briefly, GST-Trx fusion proteins expressed in Escherichia coli XL-1 blue
were affinity purified on glutathione-Sepharose beads. 400 µg of cell lysates expressing ASK1 were
incubated overnight at 4˚C with 10 µg of GST-Trx bound to glutathione-Sepharose in the lysis buffer
containing either 1 mM DTT or 1 mM H 2O2. The beads were washed 4 times with the lysis buffer before
the addition of boiling Laemmli sample buffer. Bound ASK1 proteins were resolved on SDS-PAGE and
detected by Western blot with anti-ASK1 (for full-length ASK1) or anti-Flag antibody (for Flag-tagged
ASK1-N).
Adenoviral expression of RNAi for ShTrx. A short hairpin RNA (shRNA) targeted to human Trx1
(+53 to +80 bp from ATG) or Trx2 (+22 to + 49 bp from ATG) was selected based on the program
Zhang et al., Trx2 inhibits ASK1
6
(http://katahdin.cshl.org:9331/RNAi/) and double-strand oligonucleotides encoding the shRNA were
synthesized and cloned into the pShag vector. The sequence of the oligonucleotides used to create pShTrx1 were: Trx1-A: CAG TCT TGC TCT CGA TCT GCT TCA CCA TGA AGC TTG ATG GTG
AGG CGG ATC GGG AGC AAG ATT GCT TTT TTT T; Trx1-B: GAT CAA AAA AAA GCA ATC
TTG CTC CCG ATC CGC CTC ACC ATC AAG CTT CAT GGT GAA GCA GAT CGA GAG CAA
GAC TGC G; Trx2-A: CTT CCT GGA GAT GAC AGA GGC CAG GAA CGA AGC TTG GTT CTT
GGC CTC TGT CGT CTC CGG GGA GCC CTT TTT T; Trx2-B: GAT CAA AAA AGG GCT CCC
CGG AGA CGA CAG AGG CCA AGA ACC AAG CTT CGT TCC TGG CCT CTG TCA TCT CCA
GGA AGC G. The oligonucleotides were synthesized by Integrated DNA Technology Inc. (Skotie,
Illinois, USA) and were annealed/cloned into pShag vector 4. The clones were confirmed by DNA
sequencing. Expression of short hairpin RNA (ShRNA) for Trx is under control of the U6 promoter. The
U6-sh-Trx DNA fragment was subsequently cloned into pAd-PL-DEST by Gateway LR Clonase
Enzyme Mix (Invitrogen). The cloning and viral preparation were performed according to the
manufacturer’s protocol .
RNase protection assay. Total RNA from cultured cells was extracted with a Total RNA Isolation Kit
(Ambion Inc., Austin, Texas, USA). RNase protection assays (RPA) were performed using the
HybSpeed RPA kit (Ambion Inc.), per manufacturer’s directions. Each U6-hairpin cassette was cloned
into Not I-EcoR V cut pBluescript II (Promega), linearized with Nde I, and radiolabeled using the
Maxiscript T7 polymerase in vitro transcription kit (Ambion) per manufacturer’s instructions to generate
antisense riboprobes for use in RPA. Briefly, 10 µg of RNA per sample were hybridized with each
probe, digested with an RNase A/T mixture, and separated on 8% acrylamide/ 8M Urea gel followed by
autoradiography as described previously 3,10.
Zhang et al., Trx2 inhibits ASK1
7
Caspase 9 activity assay: Caspase 9 activity was measured with a Caspase 9 peptide substrate acetylASP-Glu-Val-Asp-7 amido-4-methylcoumarin (Ac-DEVD-AMC) (Sigma) as described previously for
caspase 3 3. Briefly, BAEC were harvested in a lysis buffer (25 mM Hepes, pH 7.4, 5 mM CHAPS, 5
mM DTT) and incubated on ice for 15-20 min followed by a centrifugation at 14,000 x g for 10-15 min
at 4˚C. For each reaction, 5 µl (200 µg) of cell lysate was incubated with 200 µl of 16 µM Caspase 9
peptide substrate acetyl-Val-Glu-His-Asp-7 amido-4-methylcoumarin (Ac-VEHD-AMC) in the assay
buffer (25 mM Hepes, pH 7.4, 5 mM EDTA, 0.1% CHAPS, 5 mM DTT). The reaction was incubated in
the dark for 1-1.5 h and fluorescence was measured in a fluorescence plate reader. The measured
fluorescence was used as an arbitrary unit.
Quantitation of apoptosis. Cell killing assayed was performed as described previously with a
modification
. For apoptosis assays, cell nuclei were stained with DAPI (2.5 µg/ml), and apoptotic
3,10
cell (nucleus fragmentation) were counted under UV microscope 3,10.
Statistical analysis. Data are presented as means (±SD). For Western blot, kinase activity, apoptosis and
reporter gene assays, experiments were performed at least twice with duplicates. Analysis of
densitometry was performed using NIH Image 1.60. Results were then normalized for comparison
among different experimental groups by arbitrarily setting the value of control cells to 1.0. Statistical
analyses were performed with StatView 4.0 package (ABACUS Concepts). Differences were analyzed
by unpaired 2-tailed Student t test. Values of p<0.05 were taken as significant.
Legends to supplemental figures
Zhang et al., Trx2 inhibits ASK1
8
Fig.S1. Co-localization of endogenous ASK1 in mitochondria of HUVEC. HUVEC were stained
with 20 nM tetramethylrhodamine methyl ester (TMRM) for 30 min at 37˚C and washed twice with
phosphate buffered saline (PBS) followed by indirect immunofluorescence microscopy. Staining was
performed with anti-ASK1 antibody (rabbit polyclonal) followed by fluorescein isothiocyanate(FITC)conjugated anti-rabbit IgG (left). Mitotracker is shown in red (middle). The merging picture is shown on
the right.
Fig.S2. Subcellular localization of ASK mutants. ASK1-WT, C250S or C30S were transfected into
BAEC and subcellular fractionations were prepared. ASK1 protein in cytosolic (cyt) and mitochondrial
(mit) fractions was determined by Western blot with anti-HA. As a control, endogenous Trx2 protein
was determined by Western blot with anti-Trx2.
Fig.S3. Localization of Trx2, Trx2, ASK1- N and mtASK1- N. a. Localization of the wild-type
Trx2 and a truncated Trx2 lacking the mitochondria targeting sequence (∆Trx2) in EC. V5-tagged Trx2
and ∆Trx2 were transfected into BAEC. Trx2 and ∆Trx2 protein localization was determined by
immunofluorescence microscope with anti-V5. b-c. Localization of ASK1-∆N and mtASK1-∆N. ASK1∆N or mtASK1-∆Nwas co-transfected with V5-tagged Trx2 into BAEC. Co-localization of ASK1-∆N
(b) and mtASK1-∆N (c) with Trx2 was determined by immunofluorescence microscope with anti-ASK1
(followed by anti-rabbit IgG conjugated with FITC) and anti-V5 (followed by anti-mouse IgG
conjugated with TRITC). d. Distribution of Trx2, ∆Trx2, ASK1-∆N and mtASK1-∆N. Cytosolic and
mitochondrial fractions were prepared and protein expression was determined by Western blot with antiV5 (Trx2 and ∆Trx2) or anti-ASK1 (ASK1-∆N and mtASK1-∆N).
Zhang et al., Trx2 inhibits ASK1
9
FigS4. Localization and distribution of Trx1 and Trx2. a. Localization of Trx1 and Trx2. BAEC
were transfected with Flag-Trx1 and Trx2-V5. Localization of Trx1 and Trx2 was visualized by indirect
confocal microscopy using FITC-conjugated anti-Flag and anti-V5 respectively. b. Trx2 is detected in
mitochondrial whereas Trx1 in cytosolic fractions. Cytosolic (cyt) and mitochondrial (mit) fractions
were prepared as described. Trx1 and Trx2 were determined by Western blot with anti-Flag and anti-V5,
respectively.
FigS5. Characterization of Trx1 and Trx2-knockdown by shRNA. Expression of Trx1 ShRNA
specifically knockdown Trx1 protein (a) whereas Trx2 ShRNA knockdown Trx2 protein (b). HUVEC
were infected with adenovirus (multiple of infection = 100) harboring Shag, shTrx1 or shTrx2. Total
RNA was isolated and expression of Trx shRNA was evaluated by RNase protection assay (RPA), using
in vitro transcribed RNA spanning the Trx1 shRNA or Trx2 shRNA sequences as a probe (~ 400bp).
The protected shRNA corresponding to Trx1 or Trx2 (~29 nt) were detected by PAGE, indicating that
the transcribed shRNA was processed to siRNA (top panels). RNA from Shag was used as a control.
Total cell lysates from EC were used to determine Trx1 (a) and Trx2 (b) expression by Western blot
with anti-Trx1 and anti-Trx2, respectively. β-tubulin expression was used as a control. The relative
levels of Trx are shown below each lane. Relative levels of Trx expression are shown (setting Shag
vector as 1.0).
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Zhang et al., Trx2 inhibits ASK1
11
Fig.S2
VC
WT
cyt mit
cyt
mit
C250S
C30S
cyt mit
cyt
mit
ASK1
(anti-HA)
Trx2
(anti-Trx2)
Fig.S3
a-c: see separate Fig.S3a,b,c
cyt
mit
ASK1
Trx2
d.
∆Trx2 Trx2
∆N mtASK1-∆N
∆Trx2
(IB: V5)
Trx2
(IB: V5)
ASK1-∆N
mtASK1-∆N
Fig.S4
a. see separate Fig.S4a
b.
VC Trx1 Trx2
cyt
Trx1
(IB: Flag)
mit
Trx2
(IB: V5)
Fig.S5
a.
Shag
b.
ShTrx1
Shag ShTrx2
ShTrx2 (29 nt)
(RPA: Trx2)
ShTrx1 (29 nt)
(RPA: Trx1)
1.0
0.2
Trx1
(IB: Trx1)
β-tubulin
(IB: tubulin)
Trx2
(IB: Trx2)
1.0
0.25
β-tubulin
(IB: tubulin)