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Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri [S. J. W., A. B., C. M. B., S. M., A. L., D. R. S., P. C. G., D. G.], and Departments of Anesthesia [K. H.] and Biological Responses, Institute for Virus Research [J. Y.], Kyoto University Hospital, Kyoto University, Kyoto 606-8507, Japan
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ABSTRACT |
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INTRODUCTION |
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A long-standing hypothesis in radiation biology states that the IR-induced activation of cellular signaling pathways is initiated at the cytoplasmic membrane (4 , 11 , 12) . In recent years, several studies have demonstrated that genes encoding growth factors and cytokines involved in cytoplasmic membrane signaling are targets of IR (4 , 13 , 14) . Because c-Fos and c-Jun are nuclear proteins that interact to form the activated AP-1 transcription factor, they are not likely to bridge the physical barrier between the cytoplasm and the nucleus in the IR-induced signaling pathway (6 , 15) . Instead, upstream signaling factors probably exist to pass the cytoplasmic signal into the nucleus.
One potential candidate for such a role is TRX, a small, ubiquitous, multifunctional protein containing a redox-active disulfide/dithiol within the conserved active site -Cys-Gly-Pro-Cys- (16 , 17) . TRX, also known as adult T cell leukemia-derived factor, has both intracellular and extracellular functions and is a key regulator of cellular signaling in response to various cellular stresses (18, 19, 20) . Similar to AP-1, TRX is an inducible factor that shows cytoprotective activity against oxidative stress-induced apoptosis (21 , 22) and exhibits autocrine growth-promoting effects (23, 24, 25) . Agents such as phorbol 12-myristate 13-acetate and IR are known to generate intracellular free radicals such as ·OH and O2·- (26, 27, 28) that are believed to mediate many of their biological effects. In tissue culture cells treated with phorbol 12-myristate 13-acetate, TRX is imported into the nucleus and forms a physical interaction with the Ref-1 gene product, Ref-1 (29) . Ref-1 (also designated APE, HAP-1, and APEX) functions as both a nuclear DNA repair enzyme and as a reversible regulator of the DNA binding activity of several nuclear transcription factors, including AP-1, by altering the oxidation/reduction (redox) state of specific cysteine residues located in the basic DNA binding region of these transcription factors (15 , 30, 31, 32) . One possible role for the transient induction of AP-1 may be the initiation of protective or reparative cellular responses to the damaging effects of cellular stressing agents, including IR (6 , 10) . Therefore, the passage of redox signals through a series of sulfhydryl switches from TRX to Ref-1, finally resulting in the activation of the AP-1 transcription factor, might represent an important pathway for the transmission of radiation-induced signaling from the cytoplasm to the nucleus.
In vitro Ref-1 activity is induced by chemical reducing agents such as DTT; however, in the absence of such reducing agents, other cytoplasmic cellular signaling factors, such as TRX, TRX reductase, and NAD(P)H are hypothesized to activate the Ref-1 protein (15 , 31 , 33) . These findings implicate Ref-1 as a central target protein in posttranslational, redox-sensitive signaling cascades that use thiol-containing proteins to activate specific sets of redox-sensitive proteins that could serve to induce alterations in gene expression in response to oxidative stress. Because IR generates intracellular free radicals and irradiated cells have been shown to respond to this insult by increasing the production of NADPH via the pentose cycle (34) , it seems logical to investigate the relationship between IR and redox-sensitive signaling proteins in the cellular responses to IR.
The current study investigates the role of TRX and Ref-1 in the transduction of IR-induced signals from the cytoplasm to the nucleus, resulting in increased AP-1 DNA binding activity in HeLa and Jurkat cells. IR-induced subcellular relocalization of TRX from the cytoplasm to the nucleus in response to IR was demonstrated by Western blot analysis of nuclear extracts and confirmed by indirect immunofluorescence cell staining. A physical interaction between TRX and Ref-1 after exposure to IR was demonstrated using transient transfection two-hybrid assays. The addition of Ref-1 and TRX from irradiated cells to nonirradiated nuclear extracts was shown to activate the DNA binding activity of AP-1. Furthermore, alterations in the redox state of TRX and Ref-1 immunoprecipitated from irradiated cells were shown to be involved in the ability of these proteins to activate AP-1 DNA binding activity. Immunodepletion of Ref-1 from nuclear extracts showed inhibition of inducible AP-1 DNA binding activity in response to IR. Overall, these results suggest that the molecular cascade leading to AP-1 activation in response to IR involves the passage of redox signals through TRX from the cytoplasm to the nucleus, followed by interaction with Ref-1, possibly through a physical interaction between Ref-1 and TRX.
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MATERIALS AND METHODS |
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supplemented with 10% HI calf serum, Jurkat cells (human
leukemia cell line) were grown in RPMI 1640 supplemented with 5% HI
fetal bovine serum, and Cos-1 (African green monkey kidney) cells were
grown in DMEM supplemented with 10% HI FCS with penicillin and
streptomycin (100 units/ml). Cells were grown in a humidified 5%
CO2 atmosphere at 37°C. Prior to irradiation,
cells were serum starved for 48 h with 1% HI calf or fetal bovine
serum and irradiated with 10 Gy using unfiltered X-rays from a GE
Maxitron 250 kVp X-ray machine containing an enclosed incubator.
Control, nonirradiated cells were placed into a similar incubator.
Nuclear and Cytoplasm Subcellular Fractionation.
Cells at various time points after exposure to IR were washed twice
with PBS, scraped with 750 µl of PBS-2.5 mM EDTA, and
centrifuged at 14,000 rpm for 2 min at 4°C. The cell pellet was
suspended in 400 µl of buffer A. After incubating for 15 min on ice,
25 µl of 10% NP40 were added. The cellular suspension was vortexed
for 10 quick mixings and centrifuged at 14,000 rpm for 5 min at 4°C.
The supernatant (cytoplasmic fraction) was transferred to a new
microcentrifuge tube, and the supernatant and cell pellets were stored
at -80°C overnight. The nuclear pellet was thawed on ice for 15 min
and suspended in 60 µl of extraction buffer [10 mM HEPES
(pH 7.2), 422 mM NaCl, 0.5 mM
phenylmethylsulfonyl fluoride, 0.1 mM EGTA, and 5.3%
glycerol], incubated at 4°C for 30 min, and spun for 10 min at
14,000 rpm. The supernatant (nuclear fraction) was transferred to a new
microcentrifuge tube and stored at -80°C. Verification of the
subcellular fractions was determined by immunoblotting both the nuclear
and cytoplasmic extracts with Ref-1, a nuclear protein, and I-
B, a
cytoplasmic protein (data not shown). Protein concentrations were
determined using the Bradford method (per the manufacturer’s
specification; Bio-Rad Laboratories, Hercules, CA).
SDS-PAGE and Western Blot Analysis.
Equal amounts of protein (20 µg) from nuclear or cytoplasmic cellular
extracts were mixed with 5x Laemmli lysis buffer (3)
and boiled for 5 min. Samples were separated on denaturing
SDS-polyacrylamide gels and transferred to nitrocellulose filter paper
using a semidry apparatus (Owl Scientific Plastics, Inc., Portsmouth,
NH). The nitrocellulose filter was prepared and developed as described
previously (27)
, and Western blot analysis was performed
using an anti-TRX antibody (American Diagnostica, Inc., Greenwich, CT).
EMSA.
EMSAs were performed as described previously using a
32P-radiolabeled oligonucleotide corresponding to
the consensus AP-1 DNA binding site (27)
. Nuclear extracts
(10 µg) were incubated with poly(deoxyinosinic-deoxycytidylic acid)
for 10 min on ice, followed by the addition of radiolabeled
oligonucleotide (200,000 cpm of radiolabeled probe/reaction) and
incubation at 25°C for 20 min. No reducing agents were added to the
EMSAs or the buffers used to make nuclear extracts. Samples were run on
a 4.5% nondenaturing PAGE gel, dried, and exposed to a phosphorimager
screen using a STORM 840 Phosphorimager (Molecular Dynamics, Sunnyvale,
CA). For immunodepletion experiments, cells were lysed by the addition
of 1 ml of E1A lysis buffer [ELB: 50 mM HEPES (Sigma), pH
7.2, 250 mM NaCl (Fisher Scientific), 2 mM EDTA
(Sigma), and 0.1% NP40 (BDH Chemicals Ltd.)]. Cellular lysates were
precleared with 25 µl of protein A (Santa Cruz Biotechnology, Inc).
Immunodepletion of Ref-1 and TRX was performed immediately after the
extracts were prepared by adding 25 µl of protein A (Santa Cruz
Biotechnology, Inc.) and 5 µl of anti-Ref-1 antibody (polyclonal;
Santa Cruz Biotechnology, Inc.) or 5 µl of anti-TRX antibody
(polyclonal; American Diagnostic, Inc.). These reactions were shaken
slowly at 4°C for 2 h, and the IP was spun at 12,000 rpm for 1
min and washed in 750 ml of E1A buffer at 4°C three times. For the
immunoprecipitation experiments where immunoprecipitated TRX (Fig. 3B)
or Ref-1 (Fig. 5B)
was added to various
cellular extracts, the immunoprecipitated complex was added to 20 µg
of EMSA cellular extract for 45 min at 4°C with gentle shaking, and
EMSA was performed (27
, 35)
. Chemical modification of
immunoprecipitated Ref-1 and TRX was performed by adding either diamide
(80 µM; Sigma D-3648, 98% pure), NEM (50
µM; Sigma E-3876, 98% pure), or DTT (8
mM; Sigma D-9163, 99% pure) to
immunoprecipitation/protein A pellet for 30 min at 4°C. All chemicals
were filter sterilized and used without further purification. To remove
the free residual chemicals from the immunoprecipitation reaction, the
pellets were washed eight times in E1A buffer at 4°C. Briefly, for
each wash 750 ml of E1A buffer were added to the
immunoprecipitation/protein A pellet, followed by gentle shaking by
tapping the Eppendorf tube against the countertop for 30 s,
followed by spinning at 12,000 rpm for 1 min. The
immunoprecipitation/protein A pellet was added to 20 µg of EMSA
cellular extract for 45 min at 4°C with gentle shaking, and EMSAs
were performed (27
, 35) .
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Transient Cotransfections and Mammalian Two-Hybrid Assay.
A cDNA of TRX fused in-frame with the transcription factor VP-16
downstream of the CMV promoter (pCMV-TRX-VP-16) and a cDNA of Ref-1
fused in-frame with the DNA binding domain of Gal-4 (pCMV-Ref-1-Gal-4)
were used (29)
. Cos-1 cells were plated at
2 x 106 cells per 100-mm plate,
serum starved (0.5% FCS) for 8 h, and transfected via calcium
phosphate precipitation. In each transfection, 2 µg of pCMV-TRX-VP-16
and/or pCMV-Ref-1-Gal-4 were transfected with the reporter construct,
ptk-3x-Gal-4-LUC containing three copies of the Gal-4 binding site
upstream of the LUC gene in ptk-LUC (29)
. As an
internal control, 1 µg of the ß-gal expression plasmid
(pCMV-ß-gal) was used. Transfected cells were exposed to 10 Gy of IR
36 h after transfection and harvested after 10 h. LUC
activity was determined using a luminometer (Zylux Corp., Maryville,
TN). ß-gal activity was determined (Promega Corp., Madison, WI), and
the relative-fold induction of LUC activity was calculated by
normalizing to the ß-gal activity.
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RESULTS |
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, Lane 1 versus Lane 2),
reached a peak induction at 2 h (Lane 3), and returned
to nearly baseline at 6 h (Lane 6). Supershift
experiments with anti-Fos and anti-Jun antibodies and competition
experiments with a cold AP-1 oligonucleotide confirmed the specificity
of the AP-1 complex in this system (27)
. These experiments
were repeated in Jurkat cells with a similar induction of AP-1 binding
at 2–4 h after IR (Fig. 1B
, Lane 1
versus Lanes 2–6). Transient assays with
2x-AP-1-LUC were also done and confirmed a similar in induction in
AP-1-dependent gene expression (data not shown) as has been shown by
others (10)
. Thus, an increase in AP-1 DNA binding
activity is noted in both cell lines in response to IR.
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HeLa and Jurkat cells were irradiated, and the nuclear and cytoplasmic
cellular extracts were prepared by a subcellular fractionation.
Increases in nuclear TRX protein levels in response to IR were observed
in both HeLa and Jurkat cells (Fig. 2A)
. These increases were first seen at 1 h after
radiation (Fig. 2
A, Lane 2), reached a maximum at roughly
3 h (Lane 4), and returned to nearly baseline at 6 h (Lane 7). In addition, HeLa cells with or without exposure
to IR were examined by an indirect immunofluorescence method using
antibodies raised against TRX. TRX was located predominantly in the
cytoplasm of nonirradiated cells and appeared to accumulate in the
nucleus 1 h after exposure to IR (Fig. 2B)
. Nuclear TRX
was no longer seen after 6 h (data not shown). There appears to be
a close temporal correlation between the nuclear localization of TRX
and the induction of AP-1 DNA binding, as shown in Fig. 2A
.
These results indicate that IR may activate a cellular signaling
pathway(s) that results in the translocation of TRX into the nucleus.
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, increasing the amount of the cytoplasmic fraction from
irradiated cells appears to increase AP-1 DNA binding activity in a
dose-dependent manner up to 4 µg of extract. The increase in DNA
binding activity appears to be saturated above this amount. These
results suggest that a cytoplasmic factor from irradiated cells
increases AP-1 DNA binding activity in extracts from unirradiated
cells. Because TRX is predominantly a cytoplasmic protein that is
translocated to the nucleus upon exposure to IR (Fig. 2A)
,
TRX may participate in the induction of AP-1 DNA binding after IR.
To further investigate the role of TRX in the activation of AP-1 DNA
binding activity, TRX was immunoprecipitated from the cytoplasmic
extracts obtained from irradiated HeLa cells. The IP TRX/protein A
pellet was combined with 10 µg of nuclear extract from nonirradiated
HeLa cells, and EMSA was performed 40 min later. Control, IP protein A
(Fig. 3B
, Lane 1) and IP TRX (Lane 3),
both without any nuclear extract, are shown as controls. Nonirradiated
nuclear extract was also run as a control to establish the baseline
AP-1 DNA binding activity (Lane 2) and is similar to the
result shown in Fig. 3A
, Lane 1. When cytoplasmic
TRX IP from irradiated cells was added to nonirradiated nuclear cell
extract, a marked increase in AP-1 DNA binding activity was seen (Fig. 3
B, Lane 4). Western blot analysis of the IP TRX using
anti-Ref-1 antibody demonstrated an absence of Ref-1 protein in the IP
TRX (data not shown). This result combined with the results showing
translocation of TRX to the nucleus (Fig. 2)
is consistent with the
concept that cytoplasmic TRX activates AP-1 DNA binding by passing an
IR-induced signal from the cytoplasm to the nucleus.
Redox Alterations in the TRX Protein That Regulates AP-1 DNA
Binding Activity.
To determine whether the oxidation/reduction status of TRX protein
regulates AP-1 DNA binding activity, TRX was immunoprecipitated from
irradiated cells and split into five equal fractions that were
subsequently treated with various chemicals that alter protein thiol
oxidation/reduction (redox) status. After three washing steps with 750
ml of E1A buffer and repelleting, the IP TRX was treated with diamide
(a sulfhydryl oxidizing agent), NEM (a sulfhydryl alkylating agent), or
DTT (a sulfhydryl reducing agent) for 30 min on ice and washed eight
times to remove any residual chemical. The effects of adding TRX IP
from HeLa cells to the control nuclear extract from nonirradiated cells
that were not treated with chemicals are shown as a control (Fig. 4A
, Lane 1). The addition of diamide or NEM to IP
TRX decreased AP-1 DNA binding activity (Fig. 4
A, Lanes 2 and
3). In contrast to treatment with diamide and NEM, treatment of
the IP TRX with DTT increased AP-1 DNA binding compared with the
control sample (Fig. 4
A, Lane 1 versus Lane 4).
Finally, a fraction of IP TRX was treated with diamide, washed eight
times to remove any free chemical, and subsequently treated with DTT.
This treatment appeared to restore the ability of the IP TRX to induce
AP-1 DNA binding (Fig. 4
A, Lane 1 versus Lane 5). These
results suggest that alterations in the redox status of TRX play a
central role in the regulation AP-1 DNA binding after IR.
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, Lanes 3–4 and 5–6
versus Lanes 1–2). Cotransfection of
p3x-Gal-4/tk-LUC with both CMV-Ref-1/Gal-4 and CMV-TRX/VP-16 in
nonirradiated cells resulted in a 6-fold increase in LUC activity (Fig. 4
B, Lane 7). Ten h after exposure to 10 Gy of IR, an
additional increase in LUC activity to 11-fold above baseline was
observed (Fig. 4
B, Lane 8). These results suggest that a
physical interaction between TRX and Ref-1 occurs and that this
interaction is enhanced in response to IR.
Immunodepletion of Ref-1 Impairs Inducible, but not Basal, AP-1 DNA
Binding Activity.
Ref-1 has been shown to regulate AP-1 activity through a
redox-sensitive mechanism (27
, 31
, 32
, 35)
; therefore, the
role of Ref-1 in the regulation of IR-induced increases in AP-1 DNA
binding activity was examined. Irradiated cellular extracts were
depleted of Ref-1 by immunodepletion with anti-Ref-1 antibody to
determine the role of Ref-1 in the induction of AP-1 DNA binding
activity after IR. HeLa cell extracts from the experiments shown above
(Fig. 1)
were treated with the addition of protein A and anti-Ref-1
antibody, followed by gentle shaking at 4°C for 2 h and spinning
at 12,000 rpm for 1 min to pellet out the Ref-1 antibody complex.
Western blot analysis confirmed the removal of Ref-1 protein from the
extracts as well as the presence of Ref-1 in the IP complex (data not
shown). The continued presence of c-Fos/c-Jun proteins in the
Ref-1-depleted extracts as well as the lack of c-Fos/c-Jun protein in
the IP complex was established by Western analysis (data not shown).
Immunodepletion of Ref-1 from nonirradiated cells did not alter the
levels of AP-1 DNA binding activity detected by EMSA (Fig. 5A
, Lane 1 versus Lane 3). These results
suggest that the presence of Ref-1 in the extracts was not required for
AP-1 DNA binding activity in nonirradiated cells. Nuclear extracts from
cells harvested 1 h after exposure to IR and immunoprecipitated
with protein A only (Fig. 5A
, Lane 2) were used
as a positive control, and similar to Fig. 1
(Lanes 2–4), a
significant increase in AP-1 DNA binding activity was observed. In
contrast, no increase in AP-1 DNA binding activity was observed in
nuclear extracts from irradiated cells immunodepleted of Ref-1 and
isolated at 1 and 2 h after irradiation (Fig. 5A
,
Lanes 4 and 5). These results indicate that the
presence of Ref-1 is required for the IR-induced increase in AP-1 DNA
binding activity.
Restoration of IR-induced AP-1 DNA Binding Activity after Addition
of Ref-1 IP to Immunodepleted Extracts.
To confirm that Ref-1 from irradiated cells must be present in the HeLa
cell extracts for IR-induced increases in AP-1 DNA binding activity to
occur, IP containing Ref-1 from irradiated or nonirradiated cells was
reintroduced to the Ref-1-immunodepleted extracts from irradiated cells
harvested 2 h after IR (Fig. 5B)
. When Ref-1 IP from
irradiated cells was added back to Ref-1 without extracts from
irradiated cells (Fig. 5
B, Lane 3), the IR-induced increase
in AP-1 DNA binding was restored (Fig. 5
B, compare
Lane 3 with Lane 2). When Ref-1 IP from
nonirradiated cells was added to Ref-1 without irradiated cell extracts
(Fig. 5
B, Lane 5), a small, but reproducible increase in
AP-1 DNA binding was observed (Fig. 5
B, compare Lane
5 with Lane 2). Interestingly, when Ref-1 IP from
irradiated cells was added to Ref-1 without extracts from nonirradiated
cells (Fig. 5
B, Lane 4), AP-1 DNA binding increased to
levels seen in cells exposed to IR (Fig. 5
B, compare
Lane 4 with Lane 1). Equal amounts of IP Ref-1
from control and irradiated cells were confirmed by Western analysis
(data not shown). This ruled out the possibility that the induction of
AP-1 DNA binding was attributable to increased levels of IP Ref-1
protein. In addition, Western blot analysis confirmed the continued
presence of c-Fos and c-Jun proteins in the Ref-1-depleted extracts as
well as the absence of c-Fos and c-Jun protein in the IP complex (data
not shown). Finally, we confirmed that recombinant bacterially
expressed His-tagged Ref-1 protein also activates AP-1 DNA binding
activity (data not shown), as shown by others (31
, 32)
.
These results indicate that the presence of Ref-1 from IR-treated cells
is required to activate inducible, but not basal, AP-1 DNA binding.
These experiments are consistent with previous studies investigating
Ref-1 activation of AP-1 in response to oxidative stress, including
12-O-tetradecanoylphorbol-13-acetate and
H2O2 (4
, 10
, 14)
.
Redox Alterations in the Ref-1 Protein from Irradiated Cells
Regulates AP-1 DNA Binding Activity.
In Fig. 4A
, it was demonstrated that TRX regulation of AP-1
DNA binding is altered by chemical oxidation and reduction. It was also
shown that TRX physically interacts with Ref-1. Thus, the role of redox
status in Ref-1 regulation of AP-1 DNA binding activity was examined.
Ref-1 from irradiated cells was immunoprecipitated and treated in the
presence of either diamide (a sulfhydryl oxidizing agent), NEM (a
sulfhydryl specific alkylating agent), or DTT (a sulfhydryl reducing
agent) and washed eight times to remove any free chemical. The IP Ref-1
was then added to nonirradiated Ref-1-immunodepleted cell extracts, and
AP-1 DNA binding activity was examined. Ref-1 IP from irradiated cells
without chemicals is shown as a control (Fig. 5C
, Lane
1). The addition of diamide or NEM to IP Ref-1 from irradiated
cells (Fig. 5
C, Lanes 2 and 3)
decreased AP-1 DNA binding activity. Ref-1 IP from irradiated cells and
treated with DTT significantly increased AP-1 DNA binding compared with
Ref-1 IP from irradiated cells that were not treated with chemicals
(Fig. 5
C, Lane 1 versus Lane 4). These results suggest that
alterations in redox status of the Ref-1 protein from irradiated cells
play a central role in the enhancement of AP-1 DNA binding activity
after exposure to IR.
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DISCUSSION |
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Key contributors in altering the intracellular redox potential are ROIs. ROIs can be formed by a variety of extracellular stressing agents including multiple exogenous genotoxic agents, such as inflammatory cytokines, chemical carcinogens, chemotherapeutic agents, and irradiation (9 , 26 , 28) . Alterations in steady-state levels of ROIs and the subsequent alteration of the intracellular redox potential are considered to be a primary mechanism regulating cellular signaling factors that link external stimuli with signal transduction in cellular response to stress (40) . It has been shown that IR results in the generation of multiple ROIs including superoxide (O2·-), hydrogen peroxide (H2O2), hydroxyl radical (·OH), and organic hydroperoxides (ROOH; Refs. 26 and 28 ). Because IR generates ROIs and activates redox-sensitive signaling factors, such as AP-1, the role of TRX and/or Ref-1 in bridging the physical barrier between the cytoplasm and the nucleus and regulating the induction of AP-1 in response to IR was examined.
The current work confirms that IR induces AP-1 DNA binding activity. In
addition, subcellular fractionation demonstrated that in response to
IR, TRX is transported into the nucleus, and this result was confirmed
via indirect immunofluorescence cell staining. Furthermore, TRX IP from
irradiated cells added to nonirradiated nuclear extracts
resulted in activation of AP-1 DNA binding activity. It was also shown
that the ability of TRX to stimulate AP-1 DNA binding activity was
abolished by chemical oxidation and increased by chemical reduction.
The results of these experiments strongly suggest that TRX becomes
reduced and is transported from the cytoplasm to the nucleus in
response to IR, resulting in the induction of AP-1 DNA binding
activity. For many years, it has been known that exposure to IR and
other oxidants results in an immediate increase in NADPH pools caused
by stimulation of glucose metabolism through the pentose cycle
(34)
. Because NADPH pools coupled with TRX reductase
provide the reducing equivalents for TRX, these results are consistent
with the hypothesis that IR-induced stimulation of NADPH production
could be linked to radiation-induced signal transduction through redox
changes in TRX (Fig. 6)
.
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Although the activation of transcription factors, such as AP-1,
ultimately occurs within the nucleus, a long-standing hypothesis in
radiation biology states that the initiation of cellular signaling
pathways resulting from irradiation exposure occurs at the cytoplasmic
membrane (4
, 9
, 14)
. This raises several interesting
questions regarding the cytoplasmic factors and pathways activated by
IR as well as the specific factors that pass the signal from the
cytoplasm to the nucleus. One mechanism of transcriptional regulation
involves the movement of specific signaling factors from one
subcellular location to another. Two examples of this are the
extracellular signal-regulated kinases, also known as mitogen-activated
protein kinases, and nuclear factor-B, both of which are rapidly
transported to the nucleus in response to exposure to IR (3
, 41)
. The results presented in this report identify TRX as
another signaling factor that is transported to the nucleus in response
to IR, resulting in the activation of nuclear transcription factors.
One of the intracellular functions of TRX is to initiate protein-nucleic acid interactions of nuclear transcription factors (29) . TRX has two redox-active cysteine residues, -Cys-Gly-Pro-Cys-, in an active center and participates in redox reactions through the reversible oxidation/reduction of these thiol residues (16 , 42 , 43) . In this regard, TRX serves as a source of reducing equivalents to alter protein function and subsequently regulate nuclear transcription factor activity. This suggests that alterations in intracellular oxidation/reduction reactions may induce alterations in the redox state of TRX initiating a signaling cascade (19 , 44) in response to a variety of environmental stresses.
Once TRX has reached the nucleus, at least one mechanism regulating AP-1 DNA binding activity involves the nuclear redox-sensitive signaling protein Ref-1 (3 , 35) . This signal transduction appears to occur through a critical cysteine residue (motif), located in the basic DNA binding domain of c-Fos/c-Jun, that is flanked by the basic amino acids lysine and arginine (KCR). The redox status of this critical cysteine plays a central role in the regulation of c-Fos/c-Jun (AP-1) DNA binding activity (3 , 15 , 31 , 32 , 45) . Interestingly, genetic analysis of Ref-1 using site-directed mutagenesis has identified a cysteine located at position 65 in the redox domain that is critical for the redox-sensitive activation of c-Fos/c-Jun DNA binding (46) . Therefore, these critical cysteines, located in the functional domains of TRX and Ref-1, appear to be targets for the passage of redox-sensitive cellular signals to transcription factors in response to stress.
In this model, the redox status of these critical cysteine residue(s) is strongly influenced by its local protein microenvironment (47) . Subtle changes in cellular redox potentially alter the ionization state of the cysteine sulfur molecule (-CH2-SH to -CH2-S-), resulting in profound changes in protein activity (23) . The critical cysteine(s) would act as a redox-sensitive "sulfhydryl switch" that reversibly modulates protein activity (31 , 32) . The redox potential then could be passed along specific signal transduction cascades using proteins with redox-sensitive sites (1 , 15) .
The results presented in this report are consistent with the model that
IR results in environmental stress and the formation of ROIs that
initiates a cellular response pathway at the level of the cytoplasmic
membrane. IR has also been shown to stimulate NADPH production through
the pentose cycle and as such represents a possible mechanism linking
IR-induced changes in metabolism to activation of transcription factors
via TRX and Ref-1 (Fig. 6
; Ref. 28
). In this model, IR
induction of NADPH levels may activate a pathway involving TRX
reductase that is upstream of TRX (Fig. 6)
. Because reduced, but not
oxidized, TRX activates AP-1 DNA binding activity (Fig. 4A
,
Lanes 2 and 3 versus Lane
4), it would seem logical to suggest that an upstream factor such
as TRX reductase reduces TRX prior to nuclear translocation. Reduced
TRX interacts with Ref-1 (Fig. 4B)
and passes a redox signal
to Ref-1, which must also be in a reduced state to activate AP-1 DNA
binding (Fig. 5C)
. Finally, Ref-1 appears to pass a redox
signal to the AP-1 complex to induce DNA binding activity (Fig. 6)
. The
results of these experiments suggest a stress-activated,
redox-sensitive signaling pathway involving proteins containing
critical cysteine residues that are required for functional activity
(Fig. 6)
. The potential linkage between stress-activated metabolic
pathways for increasing NADPH synthesis and TRX/Ref-1 also provides a
plausible explanation for why IR and other oxidants can activate
transcription factors (i.e., AP-1) that require reduction to
become activated in vitro.
The discovery that TRX is a redox-sensitive cytoplasmic signaling factor activating AP-1 in response to IR identifies TRX as a potential target for radio-modifying agents. Furthermore, the subsequent determination of signaling factors upstream of TRX may also identify cellular targets for altering the inherent radiosensitivity of tumor cells. Finally, TRX nuclear translocation and interaction with Ref-1 firmly establishes a link between cytoplasmic events after radiation, resulting in the activation of nuclear transcription factors that potentially govern cellular responses to IR. Taken together with previous investigations (21) , these results support the concept that a common central pathway(s) mediating cellular responses to IR or other types of environmental or metabolic oxidative stress may involve redoxsensitive signaling pathways leading to AP-1 activation via Ref-1 and redox-sensitive cytoplasmic signaling factors such as TRX and TRX reductase.
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FOOTNOTES |
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1 Supported by NIH Grants 1 K08 CA72602-01 and PO1
CA75556 (to D. G.), Grant R01HL51469 (to D. R. S.), Grants RO1
CA49018 and PO1 CA75556 (to A. L.), and Grant CA69593 (to P. G.).
Grants ACS-IRG-58-010-43 and ACS RPG-00-292-01-TBE (to D. G.) were
from the American Cancer Society. 
2 These two authors contributed equally to this
work.
3 Present address: B180 Medical Laboratories, Free
Radical and Radiation Biology Program, University of Iowa, Iowa City,
IA 52242.
4 To whom requests for reprints should be
addressed, at Mallinckrodt Institute of Radiology, Washington
University School of Medicine, 4511 Forest Park Boulevard, Suite 411,
St. Louis, MO 63108. Phone: (314) 362-9781; Fax: (314) 362-9790;
E-mail: davidg{at}radonc.wustl.edu
5 The abbreviations used are: AP-1, activator
protein-1; IR, ionizing radiation; TRX, thioredoxin; Ref-1, redox
factor-1; HI, heat inactivated; EMSA, electrophoretic mobility shift
assay; IP, immunoprecipitate; NEM, N-ethylmaleimide;
CMV, cytomegalovirus; ß-gal, ß-galactosidase; LUC, luciferase; tk,
thymidine kinase; VP-16, etoposide; ROI, reactive oxygen
intermediate.
Received 5/12/00. Accepted 10/ 3/00.
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D. Daily, A. Vlamis-Gardikas, D. Offen, L. Mittelman, E. Melamed, A. Holmgren, and A. Barzilai Glutaredoxin Protects Cerebellar Granule Neurons from Dopamine-induced Apoptosis by Dual Activation of the Ras-Phosphoinositide 3-Kinase and Jun N-terminal Kinase Pathways J. Biol. Chem., June 8, 2001; 276(24): 21618 - 21626. [Abstract] [Full Text] [PDF]
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