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*
Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Taejon, Republic of Korea;
Laboratory of Cell Biology, Yonsei Medical Research Center, Yonsei University College of Medicine, Seoul, Republic of Korea; and
Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammalian TRX has many biological functions. A classic function is to
act as a hydrogen donor for ribonucleotide reductase, which is
essential for DNA synthesis (5). Human TRX is identical to
adult T cell leukemia-derived factor, which has been characterized as a
growth factor secreted by human T lymphotropic virus I-transformed
leukemic cell lines (6). TRX is secreted from cells using
a leaderless pathway (7, 8, 9) and stimulates the
proliferation of lymphoid cells, fibroblasts, and a variety of human
solid tumor cell lines (10, 11, 12, 13, 14). Furthermore, the stable
transfection of the human TRX gene increases cell proliferation
(15). In addition, TRX is an essential component of the
early pregnancy factor (16) and inhibits HIV expression in
macrophages (17). TRX has been found to modulate the
DNA-binding activity of transcription factors, including TFIIIC, BZLF1,
NF-
B, and glucocorticoid receptor, and to indirectly modulate AP-1
activity through Ref-1 (18, 19, 20, 21, 22, 23). In addition, TRX is also
known to act as a powerful antioxidant by reducing reactive oxygen
species (ROS), and protects against
H2O2-, TNF-
-induced
cytotoxicity, in which generation of ROS is thought to participate
(24, 25). Recently, TRX was found to be a physiological
inhibitor for apoptosis signal-regulating kinase 1 (ASK-1), which is a
pivotal component in cytokine- and stress-induced apoptosis
(26). While the present study was in progress, Nishiyama
et al. (27) reported that human TRX-binding
protein-2/vitamin D3 up-regulated protein 1
(VDUP1), originally reported as an up-regulated gene in HL-60 cells
treated with 1,25-dihydroxyvitamin D3
(28), is a negative regulator of TRX function. They also
demonstrated that TRX-binding protein-2/VDUP1 inhibits the reducing
activity of TRX via interacting catalytic active center of
TRX.
In the present study, we report the isolation and characterization of the mouse homologue for human VDUP1 (hVDUP1) (28). To date, little has been known about the cellular function of VDUP1. Mouse VDUP1 (mVDUP1) interacted with TRX to modulate its biological functions. The possible roles of mVDUP1 in oxidative stress were investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A yeast two-hybrid screen was performed according to the methods of Gyuris et al. (29). To construct the bait plasmid, a DNA fragment coding human TRX was amplified by RT-PCR from human keratinocyte cDNA library (30) and subcloned between the EcoRI and BamHI sites of pEG202 in the correct frame. After confirming that the bait plasmid itself could not activate transcription from the GAL1-GAL10 promoter in the reporter plasmid pSH18–34, we screened a mouse A20 B cell cDNA library constructed in pYESTrp (Invitrogen, Groningen, The Netherlands). Approximately three million colonies were screened. The isolation of positive clones and subsequent analyses were conducted, as described elsewhere (29).
cDNA cloning
The retrieved sequence for partial mVDUP1 in the yeast two-hybrid screen was subcloned into mammalian expression vector pFLAG-CMV, and named Flag-mVDUP1134–395(134–395). For the construction of full-length mVDUP1 cDNA, the 5' missing part of the sequence was deduced from EST database search, and amplified by RT-PCR from A20 B cell cDNA library using the following oligonucleotide primers: 5'-GCCAAGCTTATGGTGATGTTCAAGAAGAT-3' and 5'-TTCACTAGATCTCACTTCG-3'. The amplified product digested with HindIII and NspV was inserted into Flag-mVDUP1134–395(134–395) in the correct orientation and frame, which was confirmed by DNA sequencing, and named Flag-mVDUP1. Also, the full-length mVDUP1 was subcloned into pLXSN (Clontech, Palo Alto, CA). To obtain cDNA for human TRX, its open reading frame was amplified by RT-PCR from human keratinocyte cDNA library using the following primers: 5'-GCCGAATTCAAGATGGTGAAGCAGATC-3' and 5'-GGCGGATCCTTAGACTAATTCATTAATGGTG-3'. The amplified product digested with EcoRI and BamHI was subcloned into pBluescript KS (+). Again, the cDNA for TRX was subcloned into pEBG vector (31), which codes for GST-fused TRX or pFLAG-CMV (Eastman Kodak, Rochester, NY), respectively, thus named GST-TRX and Flag-TRX. TRX mutant lacking reducing activity was made by substituting two redox-active cysteine for serine (23) using the following primers: 5'-CCACGTGGTCTGGGCCTTCCAAAATGAT-3' and 5'-ATCATTTTGGAAGGCCCAGACCACGTGG-3', and named GST-TRX (CS). The mutagenesis was done using an ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s protocol.
Cell culture and transfection
NIH 3T3 and mouse lung fibroblast cells were maintained in DMEM supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin. Then the cells were placed into the quiescent state by reducing the serum concentration to 0.5% for 1 day before stimulation with TGF-ß1 or H2O2. The T cell hybridoma cells, KMls-8 (32), and 293 cells were maintained in RPMI supplemented with 10% FBS. For transient transfection assays, cells grown in 60-mm dishes were transfected with appropriate expression plasmids using the calcium phosphate method. For stable transfection (NIH 3T3), cells grown in 35-mm dishes were transfected with pLXSN-mVDUP1 as well as pLXSN using lipofectamine (Life Technologies, Grand Island, NY). Forty-eight hours after transfection, we began to select stable transfectants (0.5 mg/ml of G418).
Northern blot and RT-PCR analysis
KMls-8 cells were exposed to various treatments, as follows: PMA
(50 nM), ionomycin (1 µg/ml),
H2O2 (200 µM), 
-ray
(20 Gy), and UV (100 J/m2) for the indicated
times. NIH 3T3 cells were exposed to heat shock at 42°C for the
indicated times. Mouse lung fibroblast cells were incubated with 1
ng/ml TGF-ß1 for the indicated times. To prepare the tissues blot,
male BALB/c mice were sacrificed. Total RNAs were isolated using
Tri-Reagent (Molecular Research Center, Cincinnati, OH). Thirty
micrograms/lane of total RNA was fractionated by denaturing agarose gel
electrophoresis and transferred to nylon membranes
(GeneScreenPlus; NEN Life Science Products,
Boston, MA). The blots were hybridized overnight at 65°C in the
ExpressHyb solution (Clontech), and properly washed. For RT-PCR,
aliquots (3 µg) of total RNA were transcribed into cDNA at 37°C for
1 h in a total volume of 25 µl with 2.5 U of Moloney murine
leukemia virus reverse transcriptase. PCRs were then performed with
1/20 vol of the reverse-transcription reaction for amplification.
Amplifications were performed in a total volume of 30 µl
containing 0.5 U of Taq DNA polymerase and 10 pmol of
primers specific for mVDUP1 (5'-GCCGAATTCAAGATGGTGAAGCAGATC-3' and
5'-GGCGGATCCTTAGACTAATTCATTAATGGTG-3'), TRX
(5'-GTGGTGGACTTCTCTGCTAC-3' and 5'-GCTGGTAGCTGGTTACACTT-3'), IL-6
(5'- CTCAGCCCTGAGAAAGGAGA-3' and 5'-AAAGCTGCGCAGAATGAGAT-3'),
TGF-ß1 (5'-CGGGAGGCCAGCCGCGGGAC-3' and
5'-GGGTTGTGTTGGTTGTAGAGGGC-3'), ASK-1
(5'-CGTGGACTTCTGGATGGATT-3' and 5'-GACCTGGTTGCTCAGGTCTC-3'), or
ß-actin (5'-GTGGGGCGCCCCAGGCACCA-3' and
5'-CTCCTTAATGTCACGCACGATTTC-3'). Amplifications were performed with 25
cycles for ß-actin and 30 cycles for the others. The amplification
profile included denaturation at 95°C for 1 min, primer annealing at
55°C for 1 min, and extension at 72°C for 1 min. After PCR,
reaction mixtures were analyzed by agarose gel electrophoresis.
Immunocytochemical analysis
Forty-eight hours after transfection, HeLa cells were detached from dishes, and transferred onto sterile glass slides. Cells grown on glass slides were washed with PBS, dried, and fixed for immunostaining using a freshly prepared solution of cold methanol:acetone (1:1) for 20 min. After fixation, cells were dried and incubated in blocking buffer (1% BSA in PBS) for 20 min. Cells were incubated with either anti-Flag M2 mAb (Eastman Kodak) or normal mouse IgG at 1/100 dilution in blocking buffer for 1 h, and washed five times with PBS. Cells were incubated with goat FITC-conjugated anti-mouse IgG (Becton Dickinson, San Jose, CA) at 1/200 dilution in blocking buffer for 30 min, and were extensively washed again with PBS. Cells were mounted with glycerol for examination on a confocal laser-scanning microscope. The confocal microscope system was composed of a Leica TCS 4D connected to a Leica DAS upright microscope (Leica Lasertech, Heidelberg, Germany).
Precipitation of GST-fusion proteins and Western blot analysis
The 293 cells were transfected with various combinations of expression vectors, as indicated in the text. Twenty-four hours after transfection, cells were harvested in PBS, pelleted, and lysed in lysis buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.1% Nonidet P-40, plus protease inhibitors) for 1 h. After lysis, aliquots of cell lysates were incubated with glutathione-Sepharose (Pharmacia, Piscataway, NJ) for 2 h at 4°C. These beads were then washed five times with lysis buffer. The proteins were recovered by boiling in SDS-PAGE sample buffer. The eluted proteins were separated on SDS-PAGE, and either Coomassie stained or transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blot was subjected to Western analysis with anti-Flag Ab M2, or anti-hemagglutinin Ab 12CA5 (Boehringer Mannheim, Mannheim, Germany) using an enhanced chemoluminoscence (ECL) system (Amersham Pharmacia Biotech, Piscataway, NJ).
In vitro kinase activity assay
Aliquots (500 µg) of total cell lysates were
immunoprecipitated with Ab to JNK or ASK (PharMingen, San Diego, CA),
and incubated with GST-c-Jun 1–79(1–79) or myelin basic protein as a
direct substrate in the presence of
[-32P]ATP. After the reaction, the reaction
mixtures were separated in a 12% polyacrylamide gel electrophoresis
and autoradiographed.
TRX-reducing assay
The insulin disulfide reduction assay was essentially performed as described elsewhere (27), with a slight modification. Transiently transfected cells were lysed in lysis buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 300 mM NaCl, 10 mM EDTA, 0.1% Nonidet P-40, plus protease inhibitors). Cell extracts (20 µg) were preincubated at 37°C for 20 min with 2 µl of DTT activation buffer composed of 50 mM HEPES (pH 7.6), 1 mM EDTA, 1 mg/ml BSA, and 2 mM DTT in a total volume of 70 µl to reduce TRX. Then 40 µl of reaction mixture containing 200 µl of 1 M HEPES (pH 7.6), 40 µl of 0.2 M EDTA, 40 µl of NADPH (40 mg/ml), and 500 µl of insulin (10 mg/ml) was added. The reaction began with the addition of 10 µl of rat TRX reductase (100 A412 U/ml), and incubation continued for 20 min at 37°C. The reaction was stopped by the addition of 0.5 ml of 6 M guanidine-HCl and 1 mM DTNB (3-carboxy-4-nitrophenyl disulfide), and the absorbance at 412 nm was measured.
Flow-cytometric analysis of apoptosis
NIH 3T3 cells stably transfected either with pLXSN-mVDUP1 or pLXSN were plated in 24-well plates (2 x 105/well), allowed to adhere overnight, and then given the indicated concentrations of H2O2. At the end of indicated times of incubation, attached cells were harvested by trypsinization. The cells then received propidium iodide (PI, 5 µg/ml), followed by flow cytometry analysis to simultaneously monitor PI uptake (FL-2 channel) and cell size (forward light scatter). The cells that displayed both a reduction in cell size and a high permeability to PI were understood to be dead, as defined previously (33).
DNA fragmentation assay
After treatment with 5 µg/ml of anisomycin for the indicated time, KMls-8 cells (5 x 106 cells) were lysed in 0.5 ml of 10 mM Tris-Cl (pH 8), 0.1 M EDTA, 20 µg/ml pancreatic RNase, and 0.5% SDS for 30 min at 37°C. Proteinase K (final concentration is 100 µg/ml) was then added to the cell lysate, and the lysate was further incubated at 50°C for 3 h. After cooling the cleared lysate to room temperature, the lysate was extracted once with an equal volume of phenol/chloroform (1:1), and the aqueous phase was precipitated with an equal volume of isopropanol in the presence of 0.5 M NaCl. The precipitate was spun down by centrifugation and the pellet was washed with 70% ethanol, dried, and solubilized in 100 µl of 10 mM Tris (pH 7.5), 1 mM EDTA, and 50 µg/ml RNase A. The genomic DNA solution was incubated at 37°C for 1 h, and 5-µl aliquot was separated on a 1% agarose gel, stained with ethidium bromide, and photographed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have suggested that TRX may function as a modulator of kinase activity such as ASK-1 in the redox-signaling process (26). To get an insight into the roles of TRX in signal transduction, we searched for additional TRX-interacting proteins using a yeast two-hybrid screen. By screening cDNA libraries derived from mouse A20 B cells, multiple cDNA clones were isolated (data not shown). Although they were classified into two groups on the basis of AluI and HaeIII digestion patterns, all of the cloned genes were identified to be a single gene only differing in size, as demonstrated by the DNA sequence analysis.
Comparative analysis of the DNA sequence of the cloned gene based on
the GenBank query revealed that it had the partial form of the mouse
homologue for a previously known gene as VDUP1 (28), whose
function is not known. To construct the complete mVDUP1 cDNA, the 5'
missing sequence was deduced from EST database, amplified by PCR, and
then connected to partial cDNA of mVDUP1. mVDUP1 mRNA is predicted
to encode a protein of 395 aa that is overall 94% identical to its
human counterpart (Fig. 1
A,
GenBank accession number AF173681). Database searches indicated that
mVDUP1 does not contain any significant functional motifs and
patterns.
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B). To determine whether the partial form of
mVDUP1134–395(134–395) lacking the N-terminal portion
interacts with TRX in mammalian cells, GST precipitation assay was
performed. GST-TRX successfully coprecipitated the partial form of
mVDUP1, demonstrating that the portion of mVDUP1 covering aa 134–395
involves in its binding to TRX (Fig. 1C). We also examined
the interaction between mVDUP1 and GST-TRX (CS), a
Cys32 and Cys35 mutant that
has lost reducing activity due to the mutation of cysteine to serine in
the redox-active center. As shown in Fig. 1D, mVDUP1 did not
interact with GST-TRX (CS) mutant, suggesting that their
interaction is made via the active site of TRX.
A recent report by Nishiyama et al. (27) demonstrated that
overexpression of hVDUP1 by transfecting cells with
VDUP1 cDNA or by treating cells with 1,25-dihydroxyvitamin
D3 inhibits the reducing activity of TRX.
Furthermore, they showed that recombinant hVDUP1 directly inhibits the
reducing activity of TRX. Consistent with these observations,
transfection of mVDUP1 cDNA into 293 cells reduced the endogenous
reducing activity of TRX or the activity of cotransfected TRX cDNA
(data not shown). In either case, the activity of TRX was decreased to
nearly 50% when mVDUP1 was overexpressed, and its activity
was more decreased when TRX reductase was omitted from the assay
system. These data all indicate that mVDUP1 acts as an inhibitor of
TRX-reducing activity via directly interacting catalytic active center
of TRX.
Tissue distribution and subcellular localization of mVDUP1
To determine the tissue distribution of mVDUP1, we
performed a Northern blot analysis using male BALB/c mouse tissues.
mVDUP1 transcript was detected in various tissues, including heart,
lung, thymus, spleen, kidney, testis, and skeletal muscle (Fig. 2A). mVDUP1 was expressed in
relatively low amounts in the brain and liver, while TRX was
significantly expressed in the liver. mVDUP1 is predicted to localize
in the cytoplasm by computational analysis (PSORT II, National
Institute of Basic Biology, Genomenet). To confirm the localization, we
transiently transfected the vector, pFlag-mVDUP1 into the HeLa cell and
probed it with Flag Ab. mVDUP1 is mainly found in the cytoplasm (Fig. 2B, right panel), which is consistent with the
location of TRX (21).
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Because the TRX is one of the key components in redox regulation
and its gene expression is regulated by a variety of stress stimuli, we
tested the effects of oxidative stress, including
H2O2 on mVDUP1 gene
expression. The mVDUP1 mRNA expression was strongly induced
by 200 µM H2O2 treatment
in murine T cell hybridoma, KMls-8, at 12 h, but calcium
ionophore down-regulated the basal expression of mVDUP1, while TRX gene
expression was up-regulated by both treatments (Fig. 3A). There was a
marked increase of mVDUP1 mRNA at 24 h after TGF-ß1 treatment
(Fig. 3B), and exposing cells to -rays or UV increased
mVDUP1 gene expression moderately until 5 h after exposure (Fig. 3C). Meanwhile, heat shock elevated mVDUP1 gene expression
significantly at 1 h (Fig. 3D). Interestingly, when NIH
3T3 cells were overgrown (>90% confluent), mVDUP1 expression was
rapidly increased (Fig. 3E). Based on these data of gene
express regulation, mVDUP1 gene expression seems to be readily
regulated by a variety of stress, suggesting that mVDUP1 may be
actively involved in the stress responses.
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Binding mVDUP1 with TRX requires a TRX active site, indicating
that mVDUP1 might compete with other TRX-binding proteins for TRX
binding. The PAG has been initially identified by differential cloning
between untransformed and ras-transformed cells
(34). Its expression is higher in cells having a higher
level of proliferation. PAG is also known to be a thiol-specific
antioxidant that reduces hydrogen peroxide in the presence of TRX as an
immediate electron donor (35), thus renamed as
peroxiredoxin I. As shown in Fig. 4A, the interaction between
TRX and PAG was reduced up to 40% when mVDUP1 was overexpressed.
Another well-known TRX-interacting protein is ASK-1, which has been
identified as a mitogen-activated protein kinase kinase kinase and is
required for oxidative stress-induced apoptosis (36). TRX
is a physiological inhibitor of ASK-1, thus seeming to be essential for
survival (26). The binding between TRX and ASK-1 was
significantly reduced when mVDUP1 was overexpressed, suggesting that
mVDUP1 can compete with ASK-1 for binding to TRX (Fig. 4B).
To confirm the effects of endogenous VDUP on the binding of TRX and
ASK-1, HL-60 cells were treated with 1,25-dihydroxyvitamin
D3 to induce the VDUP1 gene expression. As
reported previously (27, 28), 1,25-dihydroxyvitamin
D3 treatment increased the hVDUP1 expression
(Fig. 5A) and decreased the
reducing activity of TRX (Fig. 5B). In this situation, ASK-1
activity was increased (Fig. 5C), further indicating that
excess binding of VDUP1 to TRX induces the release of ASK-1 from its
inhibitor, TRX, to become activated.
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The physiological function of PAG was suggested to protect against
apoptosis induced by serum deprivation, ceramide, and etoposide
(37). This antiapoptotic function might be due to the
peroxidase activity of PAG with the use of electrons from TRX. Thus, we
speculate that cells would be more vulnerable to oxidative stress if
mVDUP1 prevents the interaction between TRX and PAG. In addition,
mVDUP1 also interferes with the interaction between TRX and ASK-1,
probably rendering cells more sensitive to oxidative stress. To
investigate this functional property of mVDUP1 in oxidative stress, we
established a stable mVDUP1-transfected NIH 3T3 cell line. The
overexpression of mVDUP1 was confirmed by RT-PCR analysis (Fig. 6A). There was no difference
in cell morphology or spontaneous cell growth between the
vector-transfected cell line and mVDUP1-transfected cell line in the
presence of 10% serum. However, mVDUP1 transfectants grew more slowly
than vector controls in the presence of TGF-ß1 or in the absence of
serum (Fig. 6B), implying that overexpression of mVDUP1
makes cells more susceptible to growth inhibition in response to
stress. When murine KMls-8 T cells were treated with anisomycin, which
is known to induce JNK activation and apoptosis in T cells
(38), the correlation between DNA fragmentation and mVDUP1
expression was observed (Fig. 6C). In line with these
observations, more apoptotic cell death was observed in mVDUP1
transfectants than in vector controls when cells were treated with
H2O2 (Fig. 6D:
at 50 µM, control 19.6 ± 4.6%, mVDUP1 31.9 ± 6.5%; at
100 µM, control 39.8 ± 14.4%, mVDUP1 73.3 ± 19.4%,
respectively).
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A). It has been reported that
JNK is activated by ASK-1 (36), and its activity is
sensitive to H2O2 treatment
(40). In this regard, JNK activity was assayed after
H2O2 treatment. As shown in
Fig. 7B, mVDUP1 transfectants showed elevated JNK activity
compared with vector controls. Taken together, these results indicate
that VDUP1 expression rendered cells more vulnerable to oxidative
stress.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 (20, 23). Secreted TRX
has cytokine-like functions and functions as a potent costimulatory
molecule outside cells (11, 40, 41).
In an effort to find the regulatory factor of TRX, mVDUP1 was found to
interact with TRX through its active site of TRX. This interacting with
active site of TRX implies the biological significance of mVDUP1 in
regulating TRX function. This study has demonstrated two functional
aspects of mVDUP1 in this regard. First, mVDUP1 inhibits TRX functions
by blocking the active site, as shown in the case of hVDUP1
(27). Insulin-reducing assay demonstrated that mVDUP1
inhibits TRX-reducing activity (data not shown), which interacts with
the active site of TRX (Fig. 1). Second, mVDUP1 renders cells more
sensitive to oxidative stress probably by competing with cellular
factors, such as ASK-1 and PAG for binding to TRX. Saitoh et al.
(26) reported that TRX is known as an inhibitor of ASK-1,
a mammalian mitogen-activated protein kinase kinase kinase that
delivers the apoptotic signal by activating JNK and p38 pathways. TRX
(CS) mutant or oxidized TRX failed to bind to ASK-1. Furthermore, they
demonstrated that H2O2
treatment (0.5–5 mM) decreases the TRX-ASK-1 interaction,
but increases ASK-1 activity and apoptosis, which is inhibited by
N-acetyl-L-cysteine
treatment (26). PAG, originally described as a
proliferating associated gene, is reported as an
antioxidant and antiapoptotic gene that requires TRX to carry out its
peroxidase function. mVDUP1 also competes with PAG for the binding site
of TRX, which can inhibit the antiapoptotic function of PAG. mVDUP1, as
an inhibitor of TRX-ASK-1 or TRX-PAG interaction, inhibits cell growth
due to inducing apoptosis in the presence of oxidative stress. However,
mVDUP1 has no peroxidase activity, and structural and functional
similarity with PAG or ASK-1 (data not shown). Biochemical
analysis is required for the regulation of ASK-1 and PAG enzymatic
activity by mVDUP1-TRX interaction. Moreover, overexpression of mVDUP1
made cells more sensitive to IL-6 expression and JNK activation
in response to oxidative stress (Fig. 6), further confirming the
prooxidant functions of mVDUP1.
mVDUP1 is a homologue for the hVDUP1 cDNA, which was originally
reported as an up-regulated gene by 1,25-dihydroxyvitamin
D3 (27). 1,25-dihydroxyvitamin
D3 induces myeloid cell differentiation
(42), and inhibits fibroblast proliferation
(43). In addition, mVDUP1 expression was decreased in rat
mammary tumors, and up-regulation of mVDUP1 by 1,25-dihydroxyvitamin
D3 treatment inhibited tumor cell growth, having
suggested the critical roles of mVDUP1 in mediating the inhibitory
effects of 1,25-dihydroxyvitamin D3 on tumor
cell growth (44). In addition, a dominant-negative mutant
TRX reverses the transformed phenotype of human breast cancer cells
(15). Taken together, mVDUP1, as an inhibitor of TRX, may
have an antitumor effect in certain types of tumors.
Another important aspect of mVDUP1 in response to stress is that mVDUP1
mRNA is readily inducible by various stress stimuli, including
H2O2, TGF-ß1, and heat
shock. Comparing the regulation of TRX gene expression, mVDUP1 is more
dramatically induced by 200 µM
H2O2 treatment (Fig. 3),
which induced cell death at this concentration (Fig. 5). Based on our
and Saitoh’s (26) observations, it seems that
H2O2 induces cell death or
stress response at least by inducing the dissociation of ASK-1 from TRX
and by increasing the gene expression of mVDUP1 more than that of
TRX.
Collectively, our observations and previous reports suggest that mVDUP1
induced by stress mediates stress responses by suppressing TRX
functions. It has been known that TGF-ß1 can stimulate as well as
inhibit cell proliferation, depending on cell types and culture
condition (45). The effects of TGF-ß1 on the
proliferation of murine lung fibroblast cells were also dependent on
serum concentration. At serum-reduced condition (<2% serum), TGF-ß1
increased cell proliferation. However, at higher serum concentration
(>5% serum), it did not increase it (data not shown). The effects of
TGF-ß1 on mVDUP1 expression were dependent on cell types. TGF-ß1
up-regulated VDUP-1 expression in murine lung fibroblasts (Fig. 3B) and in TGF-ß1-induced differentiated erythroid cells,
but had no effects in KMls-8 cells (data not shown). In addition,
up-regulation of mVDUP1 gene expression occurred at 24 h after
TGF-ß1 treatment in murine lung fibroblasts, while other stimuli such
as H2O2, UV, and heat shock
induced mVDUP1 gene expression within 12 h after treatment,
implying that TGF-ß1-induced mVDUP1 gene expression may display the
indirect regulatory responses, i.e., feedback regulation, of TGF-ß1
on cell function. Recently, it was reported that TGF-ß1-induced SnoN
oncoprotein participates in negative feedback regulation of TGF-ß1
signaling (46). In addition, 1,25-dihydroxyvitamin
D3, an inducer of VDUP1, can modulate TGF-ß1
signaling both positively and negatively, depending on molecular
interplay of smad proteins (47). It has been reported that
vitamin D3 modulates intracellular calcium flux
and protein kinase C activity (48, 49). In thyroid cells,
vitamin D3 decreases calcium uptake
(50), but it increases intracellular calcium level in
promyelocytic HL-60 cells to differentiate into monocytic cells
(48). Calcium ionophore suppressed the gene expression of
mVDUP1 in KMls-8 cells, indicating intracellular calcium level is
critical factor for mVDUP1 expression, as shown in calcium regulation
by vitamin D3. More detailed studies on the roles
of mVDUP-1 in TGF-ß1 and vitamin D3 signaling
will be required.
In summary, we identified mVDUP1 as an interacting factor with TRX. It locates in the cytosol and is strongly induced by stress responses. mVDUP1 inhibits TRX functions, including insulin-reducing activity and TRX interaction with other factors such as PAG and ASK-1. These characters of mVDUP1 render cells more vulnerable to oxidative stress. Its anti-TRX function and expression pattern imply that mVDUP1 is a key modulator for stress responses to modify the redox status in the cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Inpyo Choi, Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Republic of Korea.
3 Abbreviations used in this paper: TRX, thioredoxin; ASK-1, apoptosis signal-regulating kinase 1; VDUP1, vitamin D3 up-regulated protein 1; hVDUP1, human VDUP1; JNK, c-Jun N-terminal kinase; mVDUP1, mouse VDUP1; PAG, proliferation-associated gene; PI, propidium iodide; ROS, reactive oxygen species.
Received for publication August 16, 1999. Accepted for publication April 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 20:3821., a receptor-type protein tyrosine phosphatase. Gene 186:77.[Medline]
,25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells. Biochem. Biophys. Res. Commun. 102:937.[Medline]
,25-dihydroxyvitamin D3. J. Cell. Physiol. 172:284.[Medline]
. Am. J. Physiol. 276:993.
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 W.-J. Lee Bacterial-modulated host immunity and stem cell activation for gut homeostasis Genes & Dev., October 1, 2009; 23(19): 2260 - 2265. [Abstract] [Full Text] [PDF]
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P. Patwari, W. A. Chutkow, K. Cummings, V. L. R. M. Verstraeten, J. Lammerding, E. R. Schreiter, and R. T. Lee Thioredoxin-independent Regulation of Metabolism by the {alpha}-Arrestin Proteins J. Biol. Chem., September 11, 2009; 284(37): 24996 - 25003. [Abstract] [Full Text] [PDF]
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H. Cha-Molstad, G. Saxena, J. Chen, and A. Shalev Glucose-stimulated Expression of Txnip Is Mediated by Carbohydrate Response Element-binding Protein, p300, and Histone H4 Acetylation in Pancreatic Beta Cells J. Biol. Chem., June 19, 2009; 284(25): 16898 - 16905. [Abstract] [Full Text] [PDF]
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 J. Chen, H. Cha-Molstad, A. Szabo, and A. Shalev Diabetes induces and calcium channel blockers prevent cardiac expression of proapoptotic thioredoxin-interacting protein Am J Physiol Endocrinol Metab, May 1, 2009; 296(5): E1133 - E1139. [Abstract] [Full Text] [PDF]
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 T. Kato, Y. Shimono, M. Hasegawa, M. Jijiwa, A. Enomoto, N. Asai, Y. Murakumo, and M. Takahashi Characterization of the HDAC1 Complex That Regulates the Sensitivity of Cancer Cells to Oxidative Stress Cancer Res., April 15, 2009; 69(8): 3597 - 3604. [Abstract] [Full Text] [PDF]
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 A. Advani, R. E. Gilbert, K. Thai, R. M. Gow, R. G. Langham, A. J. Cox, K. A. Connelly, Y. Zhang, A. M. Herzenberg, P. K. Christensen, et al. Expression, Localization, and Function of the Thioredoxin System in Diabetic Nephropathy J. Am. Soc. Nephrol., April 1, 2009; 20(4): 730 - 741. [Abstract] [Full Text] [PDF]
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 S.-T. Pang, W.-C. Hsieh, C.-K. Chuang, C.-H. Chao, W.-H. Weng, and H.-H. Juang Thioredoxin-interacting protein: an oxidative stress-related gene is upregulated by glucose in human prostate carcinoma cells J. Mol. Endocrinol., March 1, 2009; 42(3): 205 - 214. [Abstract] [Full Text] [PDF]
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 Z. Lappalainen, J. Lappalainen, N. K. J. Oksala, D. E. Laaksonen, S. Khanna, C. K. Sen, and M. Atalay Diabetes impairs exercise training-associated thioredoxin response and glutathione status in rat brain J Appl Physiol, February 1, 2009; 106(2): 461 - 467. [Abstract] [Full Text] [PDF]
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 J. Chen, S. T. Hui, F. M. Couto, I. N. Mungrue, D. B. Davis, A. D. Attie, A. J. Lusis, R. A. Davis, and A. Shalev Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes FASEB J, October 1, 2008; 22(10): 3581 - 3594. [Abstract] [Full Text] [PDF]
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 T. Ebrahimian, M. R. Sairam, E. L. Schiffrin, and R. M. Touyz Cardiac hypertrophy is associated with altered thioredoxin and ASK-1 signaling in a mouse model of menopause Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1481 - H1488. [Abstract] [Full Text] [PDF]
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 C.-L. Chen, C.-F. Lin, W.-T. Chang, W.-C. Huang, C.-F. Teng, and Y.-S. Lin Ceramide induces p38 MAPK and JNK activation through a mechanism involving a thioredoxin-interacting protein-mediated pathway Blood, April 15, 2008; 111(8): 4365 - 4374. [Abstract] [Full Text] [PDF]
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 J. Chen, G. Saxena, I. N. Mungrue, A. J. Lusis, and A. Shalev Thioredoxin-Interacting Protein: A Critical Link Between Glucose Toxicity and {beta}-Cell Apoptosis Diabetes, April 1, 2008; 57(4): 938 - 944. [Abstract] [Full Text] [PDF]
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W. A. Chutkow, P. Patwari, J. Yoshioka, and R. T. Lee Thioredoxin-interacting Protein (Txnip) Is a Critical Regulator of Hepatic Glucose Production J. Biol. Chem., January 25, 2008; 283(4): 2397 - 2406. [Abstract] [Full Text] [PDF]
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 R. A. Davis Searching for Causality of Knocking Out Txnip: Is Txnip Missing in Action? Circ. Res., December 7, 2007; 101(12): 1216 - 1218. [Full Text] [PDF]
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 F. Turturro, G. Von Burton, and E. Friday Hyperglycemia-Induced Thioredoxin-Interacting Protein Expression Differs in Breast Cancer-Derived Cells and Regulates Paclitaxel IC50 Clin. Cancer Res., June 15, 2007; 13(12): 3724 - 3730. [Abstract] [Full Text] [PDF]
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 J. Nishimura, Y. Dewa, M. Muguruma, Y. Kuroiwa, H. Yasuno, T. Shima, M. Jin, M. Takahashi, T. Umemura, and K. Mitsumori Effect of Fenofibrate on Oxidative DNA Damage and on Gene Expression Related to Cell Proliferation and Apoptosis in Rats Toxicol. Sci., May 1, 2007; 97(1): 44 - 54. [Abstract] [Full Text] [PDF]
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M. Kobayashi-Miura, K. Shioji, Y. Hoshino, H. Masutani, H. Nakamura, and J. Yodoi Oxygen sensing and redox signaling: the role of thioredoxin in embryonic development and cardiac diseases Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2040 - H2050. [Abstract] [Full Text] [PDF]
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W. J. E. Tissing, M. L. den Boer, J. P. P. Meijerink, R. X. Menezes, S. Swagemakers, P. J. van der Spek, S. E. Sallan, S. A. Armstrong, and R. Pieters Genomewide identification of prednisolone-responsive genes in acute lymphoblastic leukemia cells Blood, May 1, 2007; 109(9): 3929 - 3935. [Abstract] [Full Text] [PDF]
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C. Berndt, C. H. Lillig, and A. Holmgren Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1227 - H1236. [Abstract] [Full Text] [PDF]
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 P. C. Schulze, H. Liu, E. Choe, J. Yoshioka, A. Shalev, K. D. Bloch, and R. T. Lee Nitric Oxide-Dependent Suppression of Thioredoxin-Interacting Protein Expression Enhances Thioredoxin Activity Arterioscler Thromb Vasc Biol, December 1, 2006; 26(12): 2666 - 2672. [Abstract] [Full Text] [PDF]
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P. Patwari, L. J. Higgins, W. A. Chutkow, J. Yoshioka, and R. T. Lee The Interaction of Thioredoxin with Txnip: EVIDENCE FOR FORMATION OF A MIXED DISULFIDE BY DISULFIDE EXCHANGE J. Biol. Chem., August 4, 2006; 281(31): 21884 - 21891. [Abstract] [Full Text] [PDF]
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 G.-H. Liu, J. Qu, and X. Shen Thioredoxin-mediated Negative Autoregulation of Peroxisome Proliferator-activated Receptor {alpha} Transcriptional Activity Mol. Biol. Cell, April 1, 2006; 17(4): 1822 - 1833. [Abstract] [Full Text] [PDF]
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 C. E. Filby, S. B. Hooper, F. Sozo, V. A. Zahra, S. J. Flecknoe, and M. J. Wallace VDUP1: a potential mediator of expansion-induced lung growth and epithelial cell differentiation in the ovine fetus Am J Physiol Lung Cell Mol Physiol, February 1, 2006; 290(2): L250 - L258. [Abstract] [Full Text] [PDF]
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G. Xiang, T. Seki, M. D. Schuster, P. Witkowski, A. J. Boyle, F. See, T. P. Martens, A. Kocher, H. Sondermeijer, H. Krum, et al. Catalytic Degradation of Vitamin D Up-regulated Protein 1 mRNA Enhances Cardiomyocyte Survival and Prevents Left Ventricular Remodeling after Myocardial Ischemia J. Biol. Chem., November 25, 2005; 280(47): 39394 - 39402. [Abstract] [Full Text] [PDF]
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 F. Vignols, C. Brehelin, Y. Surdin-Kerjan, D. Thomas, and Y. Meyer A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteins in vivo PNAS, November 15, 2005; 102(46): 16729 - 16734. [Abstract] [Full Text] [PDF]
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J.-H. Jeon, K.-N. Lee, C. Y. Hwang, K.-S. Kwon, K.-H. You, and I. Choi Tumor Suppressor VDUP1 Increases p27kip1 Stability by Inhibiting JAB1 Cancer Res., June 1, 2005; 65(11): 4485 - 4489. [Abstract] [Full Text] [PDF]
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 A. H. Minn, C. Hafele, and A. Shalev Thioredoxin-Interacting Protein Is Stimulated by Glucose through a Carbohydrate Response Element and Induces {beta}-Cell Apoptosis Endocrinology, May 1, 2005; 146(5): 2397 - 2405. [Abstract] [Full Text] [PDF]
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 S. S. Sheth, L. W. Castellani, S. Chari, C. Wagg, C. K. Thipphavong, J. S. Bodnar, P. Tontonoz, A. D. Attie, G. D. Lopaschuk, and A. J. Lusis Thioredoxin-interacting protein deficiency disrupts the fasting-feeding metabolic transition J. Lipid Res., January 1, 2005; 46(1): 123 - 134. [Abstract] [Full Text] [PDF]
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B. J. Deroo, S. C. Hewitt, S. D. Peddada, and K. S. Korach Estradiol Regulates the Thioredoxin Antioxidant System in the Mouse Uterus Endocrinology, December 1, 2004; 145(12): 5485 - 5492. [Abstract] [Full Text] [PDF]
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Y. Nishinaka, H. Masutani, S.-i. Oka, Y. Matsuo, Y. Yamaguchi, K. Nishio, Y. Ishii, and J. Yodoi Importin {alpha}1 (Rch1) Mediates Nuclear Translocation of Thioredoxin-binding Protein-2/Vitamin D3-up-regulated Protein 1 J. Biol. Chem., September 3, 2004; 279(36): 37559 - 37565. [Abstract] [Full Text] [PDF]
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P. C. Schulze, J. Yoshioka, T. Takahashi, Z. He, G. L. King, and R. T. Lee Hyperglycemia Promotes Oxidative Stress through Inhibition of Thioredoxin Function by Thioredoxin-interacting Protein J. Biol. Chem., July 16, 2004; 279(29): 30369 - 30374. [Abstract] [Full Text] [PDF]
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T. Y. Hui, S. S. Sheth, J. M. Diffley, D. W. Potter, A. J. Lusis, A. D. Attie, and R. A. Davis Mice Lacking Thioredoxin-interacting Protein Provide Evidence Linking Cellular Redox State to Appropriate Response to Nutritional Signals J. Biol. Chem., June 4, 2004; 279(23): 24387 - 24393. [Abstract] [Full Text] [PDF]
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 J. Yoshioka, P. C. Schulze, M. Cupesi, J. D. Sylvan, C. MacGillivray, J. Gannon, H. Huang, and R. T. Lee Thioredoxin-Interacting Protein Controls Cardiac Hypertrophy Through Regulation of Thioredoxin Activity Circulation, June 1, 2004; 109(21): 2581 - 2586. [Abstract] [Full Text] [PDF]
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 D. G Simmons and T. G Kennedy Rat endometrial Vdup1 expression: changes related to sensitization for the decidual cell reaction and hormonal control Reproduction, April 1, 2004; 127(4): 475 - 482. [Abstract] [Full Text] [PDF]
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 S. J. Erkeland, M. Valkhof, C. Heijmans-Antonissen, A. van Hoven-Beijen, R. Delwel, M. H. A. Hermans, and I. P. Touw Large-Scale Identification of Disease Genes Involved in Acute Myeloid Leukemia J. Virol., February 15, 2004; 78(4): 1971 - 1980. [Abstract] [Full Text] [PDF]
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E. R. Tulman, C. L. Afonso, Z. Lu, L. Zsak, G. F. Kutish, and D. L. Rock The Genome of Canarypox Virus J. Virol., January 1, 2004; 78(1): 353 - 366. [Abstract] [Full Text] [PDF]
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H. Yamawaki, J. Haendeler, and B. C. Berk Thioredoxin: A Key Regulator of Cardiovascular Homeostasis Circ. Res., November 28, 2003; 93(11): 1029 - 1033. [Abstract] [Full Text] [PDF]
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 S. T. Winokur, Y.-W. Chen, P. S. Masny, J. H. Martin, J. T. Ehmsen, S. J. Tapscott, S. M. van der Maarel, Y. Hayashi, and K. M. Flanigan Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation Hum. Mol. Genet., November 15, 2003; 12(22): 2895 - 2907. [Abstract] [Full Text] [PDF]
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 B. McMorran, L. Town, E. Costelloe, J. Palmer, J. Engel, D. Hume, and B. Wainwright Effector ExoU from the Type III Secretion System Is an Important Modulator of Gene Expression in Lung Epithelial Cells in Response to Pseudomonas aeruginosa Infection Infect. Immun., October 1, 2003; 71(10): 6035 - 6044. [Abstract] [Full Text] [PDF]
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 E. J Stevenson, P. G Giresi, A. Koncarevic, and S. C Kandarian Global analysis of gene expression patterns during disuse atrophy in rat skeletal muscle J. Physiol., August 15, 2003; 551(1): 33 - 48. [Abstract] [Full Text] [PDF]
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 Y. Takahashi, Y. Ishii, A. Murata, T. Nagata, and S. Asai Localization of Thioredoxin-interacting Protein (TXNIP) mRNA in Epithelium of Human Gastrointestinal Tract J. Histochem. Cytochem., July 1, 2003; 51(7): 973 - 976. [Abstract] [Full Text] [PDF]
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 E. S. Roberts, M. A. Zandonatti, D. D. Watry, L. J. Madden, S. J. Henriksen, M. A. Taffe, and H. S. Fox Induction of Pathogenic Sets of Genes in Macrophages and Neurons in NeuroAIDS Am. J. Pathol., June 1, 2003; 162(6): 2041 - 2057. [Abstract] [Full Text] [PDF]
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S. F. Goldberg, M. E. Miele, N. Hatta, M. Takata, C. Paquette-Straub, L. P. Freedman, and D. R. Welch Melanoma Metastasis Suppression by Chromosome 6: Evidence for a Pathway Regulated by CRSP3 and TXNIP Cancer Res., January 15, 2003; 63(2): 432 - 440. [Abstract] [Full Text] [PDF]
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S.-T. Pang, K. Dillner, X. Wu, A. Pousette, G. Norstedt, and A. Flores-Morales Gene Expression Profiling of Androgen Deficiency Predicts a Pathway of Prostate Apoptosis that Involves Genes Related to Oxidative Stress Endocrinology, December 1, 2002; 143(12): 4897 - 4906. [Abstract] [Full Text] [PDF]
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P. C. Schulze, G. W. De Keulenaer, J. Yoshioka, K. A. Kassik, and R. T. Lee Vitamin D3-Upregulated Protein-1 (VDUP-1) Regulates Redox-Dependent Vascular Smooth Muscle Cell Proliferation Through Interaction With Thioredoxin Circ. Res., October 18, 2002; 91(8): 689 - 695. [Abstract] [Full Text] [PDF]
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Y. Wang, G. W. De Keulenaer, and R. T. Lee Vitamin D3-up-regulated Protein-1 Is a Stress-responsive Gene That Regulates Cardiomyocyte Viability through Interaction with Thioredoxin J. Biol. Chem., July 12, 2002; 277(29): 26496 - 26500. [Abstract] [Full Text] [PDF]
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