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J Biol Chem, Vol. 274, Issue 31, 21645-21650, July 30, 1999


Identification of Thioredoxin-binding Protein-2/Vitamin D3 Up-regulated Protein 1 as a Negative Regulator of Thioredoxin Function and Expression*

Akira Nishiyama, Minoru MatsuiDagger , Satoshi Iwata, Kiichi Hirota§, Hiroshi Masutani, Hajime Nakamura, Yasushi Takagi, Hiroshi Sono, Yasuhiro Gon, and Junji Yodoi

From the Department of Biological Responses, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D3 up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1alpha ,25-dihydroxyvitamin D3 caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thioredoxin (TRX)1 is a 12-kDa ubiquitous protein that has disulfide reducing activity (1, 2). TRX has two redox-active cysteine residues in its consensus sequence (Trp-Cys-Gly-Pro-Cys) and serves as a general disulfide oxido-reductase. The two cysteine residues can be reversibly oxidized to form a disulfide bond and reduced by the action of TRX reductase and NADPH (3). TRX catalyzes the reduction of disulfide bonds in multiple substrate proteins.

The TRX system (TRX, TRX reductase, and NADPH) is widely conserved in almost all species from bacteria to higher eukaryotes and has a wide variety of biological functions. TRX was originally identified as a hydrogen donor of ribonucleotide reductase in Escherichia coli (4) and is an essential protein subunit of the phage T7 DNA polymerase complex (5). The simultaneous deletion of two TRX genes of Saccharomyces cerevisiae prolonged the cell cycle (6). The TRX homologue gene of Drosophila was identified as a gene in the deadhead locus, which is required for female meiosis and early embryonic development (7). In the human system, TRX has been cloned as adult T-cell leukemia-derived factor, produced by human T-cell leukemia virus-I-transformed T-cells, or as interleukin-1-like factor from Epstein-Barr virus transformed cells (8, 9). Human TRX has been reported to be involved in cell activation (10, 11) and cell growth promotion (12, 13). TRX expression seems essential for early development of the mouse embryo, because targeted disruption of the mouse TRX gene caused early embryonic lethality (14).

Accumulating evidence has indicated the importance of the regulation of reduction and oxidation (redox regulation) in various biological phenomena (15). The TRX system and the glutathione system constitute major thiol reducing systems (16). The reduced condition is preferable for DNA binding activity of various transcriptional factors such as AP-1 (17), NF-kappa B (18), polyomavirus enhancer-binding protein 2/AML1 (19), glucocorticoid receptor (20), and estrogen receptor (21). TRX plays an important role in the regulation of protein-nucleic acid interactions through the redox regulation of cysteine residue(s) (22, 23). The direct physical association between TRX and redox factor-1/AP-endonuclease has been demonstrated to play a key role in the AP-1 transcriptional activity (23, 24). Cellular redox status is also important in the regulation of apoptosis (25, 26). Recently, TRX has been isolated using a yeast two-hybrid system as a binding protein of a mitogen-activated protein kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) (27).

In order to investigate the molecular mechanism of TRX-dependent redox regulation in mammalian cells, we have identified TRX-binding proteins. A TRX-binding protein designated as thioredoxin-binding protein-2 (TBP-2) is identical to vitamin D3 up-regulated protein 1 (VDUP1). VDUP1 was originally reported as an up-regulated gene in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3 (28). Although several homologous sequences of VDUP1 from mammalian species have been reported, the function of VDUP1 remains unclear. In this paper, we report how TBP-2/VDUP1 interacts with TRX and modulates the function and the expression of TRX in vitro and in vivo.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids-- Standard methods were used for DNA and RNA manipulations (29). TRX mutant C32S/C35S, which lacks reducing activity, was made by substituting two redox-active cysteine residues for serine residues (24, 30). A cDNA of TRX or TRX C32S/C35S was fused in-frame to pGBT9 (CLONTECH) or pGEX4T-2 (Amersham Pharmacia Biotech). TRX cDNA fragment was excised by EcoRI from lambda gt10-TRX vector and ligated into pcDNA3 (Invitrogen) (9). pACT-cl.13 and pACT-cl.29 were isolated from positive colonies of yeast two-hybrid screening and contained partial coding sequences of TBP-2. The open reading frame of the TBP-2/VDUP1 was amplified by polymerase chain reaction using the following oligonucleotide primers: 5'-GGAATTCGATGGTGATGTTCAAGAAGATC-3' and 5'-CCGCTCGAGTCACTGACAATTGTTGTTGA-3'. To prepare protein expression vectors, the TBP-2/VDUP1 open reading frame was ligated in-frame to pGADGH (CLONTECH), pcDNA3.1/His B (Invitrogen), pGEX4T-3, pRSET B (Invitrogen), or pEGFP-C1 (CLONTECH). pcDNA3.1/His B-lacZ was purchased from Invitrogen.

Yeast Two-hybrid System-- The two-hybrid library screening was performed using the yeast MATCHMAKER two-hybrid system (CLONTECH) and human lymphocyte MATCHMAKER cDNA library (B-cell population of Epstein-Barr virus-transformed peripheral blood lymphocytes, CLONTECH) according to the manufacturer's instructions. pGBT9-TRX was used as the bait plasmid. Plasmids were introduced to yeast strains HF7c or SFY526 by a polyethylene glycol/lithium acetate method (31, 32). Colonies were grown on selective synthetic medium and were tested for histidine prototrophy or beta -galactosidase activity using 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside.

Cloning of TBP-2 cDNA-- The lambda ZAP II human placenta cDNA library (Stratagene) was screened with [alpha -32P]dCTP-labeled DNA probes derived from the XhoI fragment of pACT-cl.29. Positive plaques were phagemid-rescued by VCSM13 helper phage (Stratagene) according to the manufacturer's instruction. A 2.9-kilobase pair cDNA in pBluescript was sequenced on both strands.

Preparation of Recombinant Proteins-- In vitro translated proteins were prepared using a TNT-coupled rabbit reticulocyte translation system (Promega) and [35S]methionine (Amersham Pharmacia Biotech). Bacterially expressed His6-tagged recombinant protein was prepared under denaturing conditions according to the instructions provided in the QIAexpressionist booklet (Qiagen). E. coli strain BL21 (DE3) pLysS transformed with pRSET-TBP-2/VDUP1 was treated for 4 h with 1 mM isopropyl-beta -D-thiogalactoside. His6-tagged protein was purified by use of a Ni2+-nitrilotriacetic acid-agarose column. Glutathione S-transferase (GST) fusion proteins were prepared as follows: E. coli strain XL1 Blue MRF' transformed with each pGEX expression vector was treated for 4 h with 1 mM isopropyl-beta -D-thiogalactoside. Pelleted cells were lysed in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 150 mM NaCl, and protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 5 µg/ml aprotinin) by sonication. After centrifugation, supernatants were applied to glutathione Sepharose CL-6B columns (Amersham Pharmacia Biotech). GST fusion proteins were either eluted with the above buffer containing 10 mM reduced glutathione or immobilized on the beads and stored at -80 °C. Recombinant TRX was produced and provided by Ajinomoto Co. Inc. (Basic Research Laboratory, Kawasaki, Japan) (33).

Antibodies-- Anti-TBP-2/VDUP1 antibody was prepared by immunization of bacterially expressed His6-TBP-2/VDUP1 protein as described before (14). The immune serum was further purified by affinity columns coupled with the GST-TBP-2/VDUP1 protein and protein A Sepharose CL-6B (Amersham Pharmacia Biotech) (34). Anti-human TRX monoclonal antibodies (TRX-11 mAb and 21 mAb) were produced and provided by Fujirebio (Tokyo, Japan). These antibodies recognize different sites of TRX, respectively (35). Anti-Xpress antibody (Invitrogen) is a monoclonal antibody that recognizes the amino acid sequence Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys, which is contained in the N terminus of His6-tagged protein that was expressed with pcDNA3.1/His.

Cell Culture-- COS-7 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical) with heat-inactivated 10% fetal calf serum (Life Technologies, Inc.); HL-60 and Jurkat cells were cultured in RPMI 1640 medium (Life Technologies, Inc.) with heat-inactivated 10% fetal calf serum. COS-7 and HEK293 cells were transfected with Superfect transfection reagent (Qiagen) according to the manufacturer's instruction. After 24 h of transfection, cells were harvested and used for further studies. For the HL-60 differentiation, cells were seeded at 1 × 105 cells/ml and treated with 100 nM calcitriol (1alpha ,25-dihydroxyvitamin D3, Wako Pure Chemical Ind.) for 3 days.

In Vitro Binding Assay and Immunoaffinity Purification-- In vitro translated proteins were diluted with Nonidet P-40 buffer containing 150 mM NaCl, 1.0% Nonidet P-40, and 50 mM Tris-HCl, pH 7.5. For immunoprecipitation, after preclearing by use of protein G-Sepharose (Zymed Laboratories Inc.), samples were incubated with antibody for 2 h and with protein G-Sepharose for an additional 20 min at 4 °C. For in vitro binding assay using GST fusion protein, samples were incubated with GST fusion protein. Then samples were centrifuged and washed five times with Nonidet P-40 buffer. The precipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.

The anti-human TRX monoclonal antibody (TRX-11 mAb) and mouse IgG1 (MOPC21, Sigma) were covalently coupled to Affi-Gel 10 beads (Bio-Rad), according to the manufacturer's instruction. Cells were washed and lysed with Nonidet P-40 buffer containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 5 µg/ml aprotinin). Then 10 mg of lysate was applied to each antibody column. Proteins were eluted with 100 mM glycine-HCl, pH 2.5. Eluted samples were neutralized with <FR><NU>1</NU><DE>10</DE></FR> volume of 1 M Tris-HCl, pH 8.0, and dialyzed with 50 mM ammonium acetate, pH 7.5. Samples were concentrated by evaporation and subjected to Western blotting analysis.

Reducing/Oxidizing Reagent Treatment-- GST-TRX was treated with reducing/oxidizing reagents according to previous report (27). Breifly, GST-TRX immobilized on the beads was treated with Nonidet P-40 buffer containing reducing/oxidizing reagents for 15 min at room temperature. Treated beads were centrifuged and washed five times with degassed Nonidet P-40 buffer by batch method. Then in vitro translated TBP-2/VDUP1 protein was diluted with Nonidet P-40 buffer and added to the treated beads. After incubation for 2 h, samples were centrifuged and washed five times with Nonidet P-40 buffer. The precipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis and visualized by autoradiography.

Western Blotting and Northern Blotting-- Cell extracts were prepared with lysis buffer (150 mM NaCl, 1.0% Nonidet P-40, 1.0% sodium deoxycolate, 0.1% SDS, 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 5 µg/ml aprotinin). SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described previously (36).

Total RNA from cultured cells was extracted using TRIzol reagent (Life Technologies, Inc.) according to the manufacturer's instruction. Electrophoresis and Northern blotting were performed as described previously (37).

Insulin Reducing Assay-- To estimate the reducing activity of TRX, insulin reducing assay was performed according to a previous report (36) with slight modifications. In our assay, yeast TRX reductase was used and was able to reduce recombinant human TRX. Yeast TRX reductase was provided by Oriental Yeast Co. Ltd. (Tokyo, Japan). The decrease in absorbance at 340 nm was recorded by use of a THERMOMAX micro plate reader (Molecular Devices) to detect maximal NADPH consumption rate (Vmax, millioptical density at 340 nm/min). As a control, samples were incubated with the reaction mixture without insulin. Each value was calculated according to a method previously reported (36).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Screening of Genes Encoding TRX-binding Protein by Yeast Two-hybrid System-- We used the yeast two-hybrid system to clone genes encoding TRX-binding protein using a cDNA library of B cell population of Epstein-Barr virus-transformed human peripheral blood lymphocyte. Among approximately 1.8 × 106 yeast transformants screened, nine colonies showed histidine prototrophy and beta -galactosidase activity. Isolated plasmids were classified by restriction enzyme excision or DNA sequencing into three groups that were designated as thioredoxin-binding proteins. Because double transformants with pGBT-TRX and each plasmid belonging to one group, TBP-2, showed strongly positive phenotypes of histidine prototrophy and beta -galactosidase activity, we chose TBP-2 for further study (Fig. 1). Data base searching of pACT-cl.29 revealed that TBP-2 has homology with TRT407-2 and vitamin D3 up-regulated protein 1 (VDUP1). TRT407-2 was reported as a gene screened by an RNA fingerprinting method in mink Mv1Lu cells (38), and VDUP1 was reported as an up-regulated gene in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3 (28). Another candidate (TBP-1) was identical to human p40phox, a cytosolic phagocyte oxidase component (39). We attempted to clone the full-length cDNA of TBP-2 for further study.


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  		Fig. 1.   Screening of TRX-binding protein by yeast two-hybrid system. pGBT9-TRX was co-transfected to yeast strain HF7c with the indicated plasmid. The growth of yeast transformants with pGBT9-TRX on selective synthetic medium without histidine (upper panel) or with histidine (lower panel) is shown. pACT-cl.13 and pACT-cl.29 each have an insert of 2.2- and 1.4-kilobase pair cDNA that belonged to TBP-2, respectively.

Isolation of TBP-2 cDNA-- To isolate the full-length cDNA of TBP-2, a human placenta cDNA library was screened, using probes derived from the insert DNA of pACT-cl.29. DNA sequencing analysis of isolated 2.9-kilobase pair cDNA revealed that TBP-2 was identical to VDUP1.

Interaction between TRX Protein and TBP-2/VDUP1 Protein-- To examine the interaction between TRX protein and TBP-2/VDUP1 protein, an in vitro binding assay was performed. 35S-Labeled proteins were prepared by in vitro translation. 35S-Labeled His6-TBP-2/VDUP1 protein was co-immunoprecipitated with TRX by anti-TRX monoclonal antibodies (Fig. 2A, lane 1) but not by control mouse IgG1 (Fig. 2A, lane 2). 35S-Labeled His6-beta -galactosidase protein was not co-immunoprecipitated with TRX (Fig. 2A, lane 3). In addition, 35S-labeled TRX was co-immunoprecipitated with in vitro translated His6-TBP-2/VDUP1 protein by anti-Xpress antibody but not by control mouse IgG1 (Fig. 2B).


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  		Fig. 2.   Interaction between TRX and TBP-2/VDUP1. A, 35S-labeled His6-TBP-2/VDUP1 protein was mixed with cold recombinant human TRX and immunoprecipitated using anti-TRX antibodies (mixture of TRX-11 mAb and 21 mAb) (lane 1) or control IgG1 (lane 2). 35S-Labeled His6-beta -galactosidase was used for immunoprecipitation with anti-TRX antibodies (lane 3). In vitro translation products, 35S-His6-TBP-2/VDUP1 (lane 4) and 35S-His6-beta -galactosidase (lane 5) are shown. 35S-His6-TBP-2/VDUP1 and 35S-His6-beta -galactosidase were prepared by use of an in vitro translation method. lacZ, beta -galactosidase. B, 35S-labeled human TRX was mixed with cold His6-TBP-2/VDUP1 and immunoprecipitated using anti-Xpress antibody (lane 1) or control IgG1 (lane 2). In vitro translation product of TRX is shown in lane 3. 35S-Labeled human TRX and His6-TBP-2/VDUP1 were prepared by use of an in vitro translation method. 4 µg of each antibody was used for each sample. Precipitated samples were subjected to SDS-polyacrylamide gel electrophoresis and visualized by means of autoradiography. C, detection of TRX-TBP-2/VDUP1 complex from Jurkat cell lysate. Jurkat cell lysate was prepared with Nonidet P-40 buffer, and TRX-TBP-2/VDUP1 complex was purified using each immunoaffinity column. Lane 1, anti-TRX antibody (TRX-11 mAb); lane 2, control IgG1. Eluted samples were concentrated and analyzed by Western blotting using the affinity purified anti-TBP-2/VDUP1 antibody. Jurkat cell lysate (50 µg/lane, lane 3) are shown as a positive control.

We then analyzed whether TRX interacts with TBP-2/VDUP1 in vivo. Cell lysates from human Jurkat cells were applied to an affinity column of either anti-TRX monoclonal antibody or control IgG1. Eluted proteins were subjected to Western blotting analysis with affinity purified anti-TBP-2/VDUP1 antibody. TBP-2/VDUP1 was detected in eluates from the anti-TRX antibody column (Fig. 2C, lane 1) but not from the control IgG1 column (lane 2). Therefore, these results demonstrated the interaction of TRX with TBP-2/VDUP1 in vitro and in vivo.

Effect of Redox Status of TRX on TRX-TBP-2/VDUP1 Interaction-- Because TRX is a redox active protein, we next tested whether TRX-TBP-2/VDUP1 interaction is influenced by the redox status of TRX. GST-fused TRX was pretreated with reducing/oxidizing reagent, dithiothreitol, hydrogen peroxide, or diamide (a sulfhydryl-specific oxidant) and subjected to an in vitro binding assay. Whereas the TRX-TBP-2/VDUP1 interaction was unaffected by treatment with dithiothreitol, the interaction was markedly inhibited by treatment with hydrogen peroxide or diamide, suggesting that the reduced form of TRX is critically important for the interaction (Fig. 3).


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  		Fig. 3.   Effects of redox status of TRX on interaction with TBP-2/VDUP1. GST-TRX immobilized on the beads was treated with Nonidet P-40 buffer containing each concentration of reducing/oxidizing reagents for 15 min at room temperature and washed with degassed Nonidet P-40 buffer and subjected to a binding assay with 35S-labeled His6-TBP-2/VDUP1 protein as described under "Materials and Methods." DTT, dithiothreitol.

Analysis of Association between TRX and TBP-2/VDUP1 Using Mutant TRX-- To examine whether TRX-TBP-2/VDUP1 interaction requires intact redox active sites of TRX, we used mutant TRX C32S/C35S in which redox active cysteine residues are substituted to serine residues. In an in vitro binding assay, 35S-labeled His6-TBP-2/VDUP1 protein was co-precipitated with GST-TRX but not with GST-TRX C32S/C35S (Fig. 4A). Using a yeast two-hybrid system to test the interaction in vivo, S. cerevisiae strain HF7c was transformed with pGADGH-TBP-2/VDUP1 and either pGBT9-TRX or pGBT9-TRX C32S/C35S. Transformed colonies of pGBT9-TRX grew well on synthetic medium lacking histidine, whereas transformed colonies of pGBT9-TRX C32S/C35S failed to grow (Fig. 4B). These data showed that these redox active cysteine residues are required for the TRX-TBP-2/VDUP1 interaction.


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  		Fig. 4.   Interaction of TBP-2/VDUP1 with mutant TRX. A, GST fusion proteins immobilized on the beads were used for an in vitro binding assay with 35S-labeled His6-TBP-2/VDUP1 protein. The assay was performed as described under "Materials and Methods." Lane 1, GST; lane 2, GST-TRX; lane 3, GST-TRX C32S/C35S. B, yeast two-hybrid analysis using TRX mutant. pGBT9-TRX or pGBT-TRX C32S/C35S was co-transformed with pGADGH or pGADGH-TBP-2/VDUP1. The growth of yeast transformants on selective synthetic medium without histidine (upper panel) or with histidine (lower panel) is shown. Yeast strain HF7c was used in this experiment.

Effects of TBP-2/VDUP1 on TRX Reducing Activity-- TRX has a disulfide reducing activity and cleaves a disulfide bond in substrates such as insulin. We examined the effect of TBP-2/VDUP1 on TRX activity by the insulin reducing assay (36, 40). In our experiments, yeast TRX reductase was used and was able to reduce recombinant human TRX (data not shown). As shown in Fig. 5, a significant decrease of the reducing activity of TRX was observed in cellular extracts of COS-7 or HEK293 cells transiently transfected with a green fluorescent protein (GFP)-fused TBP-2/VDUP1 protein expression vector (Fig. 5A), in comparison with those of cells transfected with a GFP expression vector (Fig. 5A, lane 1). Similar results were obtained in experiments using His6-TBP-2/VDUP1 expression vector (data not shown).


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  		Fig. 5.   Effect of TBP-2/VDUP1 for the reducing activity of TRX. A, TRX activity in cells transiently transfected with TBP-2/VDUP1 expression vector. TRX activity of the cell extract (10 µg) of COS-7 (left panel) or HEK293 cells (right panel) transiently transfected with the indicated amount of GFP-TBP-2/VDUP1 expression vector was determined by use of an insulin reducing assay. Plasmids (total 10 µg/plate) were introduced to cells cultured in 10-cm dishes. Total amount of plasmid was adjusted to 10 µg with pEGFP-C1. Activities for samples are shown relative to Vmax of control (pEGFP-TBP-2/VDUP1 0 µg, lane 1), which is assigned as 100%. Data shown are representative of two-independent experiments. The results are the means ± S.D. of three samples. B, the effect of recombinant TBP-2/VDUP1 protein on TRX reducing activity. TRX activities of each sample were determined by use of an insulin reducing assay. Reduced TRX (0.5 µM) was incubated with the indicated concentration of GST (open bar) or GST-TBP-2/VDUP1 protein (closed bar) for 15 min at 25 °C. The reducing activity of TRX was measured in 0.1 M Tris-HCl (pH 8.0), 2 mM EDTA, 0.2 mM NADPH, 9.9 units/ml yeast TRX reductase, 140 µM insulin, 1 mM glutathione at 25 °C. Activities for samples are shown relative to Vmax of control (lane 1), which is assigned as 100%. Data shown are representative of two independent experiments. The results are the means ± S.D. of three samples.

To confirm the inhibitory effect of TBP-2/VDUP1 on the reducing activity of TRX, we tested recombinant GST-TBP-2/VDUP1 protein in the insulin reducing assay. The reducing activity of TRX was repressed to less than 50% by the addition of 1 µM GST-TBP-2/VDUP1 protein, indicating that TBP-2/VDUP1 protein inhibits the disulfide reducing activity of TRX in vitro (Fig. 5B).

Effects of TBP-2/VDUP1 on TRX Expression-- We examined the effect of TBP-2/VDUP1 on TRX expression. As shown in Fig. 6, Western blotting analysis demonstrated that expression of TRX protein was significantly down-regulated in COS-7 cells transiently transfected with GFP-TBP-2/VDUP1 expression vector (Fig. 6). Similar results were obtained in experiments using His6-TBP-2/VDUP1 expression vector (data not shown). Thus, TBP-2/VDUP1 protein down-regulated TRX protein expression as well.


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  		Fig. 6.   Expression of TRX protein in TBP-2/VDUP1 overexpressed cells. Each lane contains 10 µg of cell lysate that was isolated from cells transfected with the indicated amount of GFP-TBP-2/VDUP1 expression vector. Plasmids (total 2 µg/well) were introduced to cells cultured in a 6-well plate. The total amount of plasmid was adjusted to 2 µg with pEGFP-C1. Western blotting was performed using a monoclonal anti-TRX antibody (TRX-11 mAb).

TRX and TBP-2/VDUP1 expression in HL-60 cells differentiated with 1alpha ,25-dihydroxyvitamin D3-- Because TBP-2/VDUP1 was originally reported as an up-regulated gene in 1alpha ,25-dihydroxyvitamin D3 treatment in HL-60 cells (28), we analyzed TBP-2/VDUP1 and TRX expression in the differentiation of 1alpha ,25-dihydroxyvitamin D3-induced HL-60 cells. There was a gradual increase of TBP-2/VDUP1 mRNA after treatment with 1alpha ,25-dihydroxyvitamin D3 (Fig. 7A). After 72 h, the TBP-2/VDUP1 mRNA was enhanced 9-fold over that before the treatment. In contrast, 72 h after the treatment, the TRX mRNA level markedly declined to less than 20% compared with that before the treatment. This inverted expression pattern was also observed in protein expression. The expression of TBP-2/VDUP1 protein was enhanced after treatment of 1alpha ,25-dihydroxyvitamin D3 (Fig. 7B). In contrast, 48 h after the treatment, the amount of TRX protein was reduced to half of that before treatment (Fig. 7B). We then analyzed the reducing activity of TRX in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3. Insulin reducing activity of the cell lysates decreased to 70% of the control value within 24 h and to 60% by 72 h (Fig. 8). Thus, TRX expression and its reducing activity were down-regulated in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3, whereas TBP-2/VDUP1 expression was up-regulated.


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  		Fig. 7.   Expression of TBP-2/VDUP1 and TRX in 1alpha ,25-dihydroxyvitamin D3-stimulated HL-60 cells. A, Northern blotting analysis of TBP-2/VDUP1 and TRX expression. Each lane contains 20 µg of total RNA that was isolated from cells treated with 1alpha ,25-dihydroxyvitamin D3. The blot was hybridized with 32P-labeled probes for TBP-2/VDUP1, TRX or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B, Western blotting analysis of TBP-2/VDUP1 and TRX expression. Each lane contains 20 µg of cell lysate from cells treated with 1alpha ,25-dihydroxyvitamin D3. Western blotting was performed using an anti-TBP-2/VDUP1 antibody or a monoclonal anti-TRX antibody (TRX-11 mAb).


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  		Fig. 8.   The reducing activity of TRX in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3. Cell extracts (10 µg) of HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3 were collected at indicated hours. The TRX activities were determined by use of the insulin reducing assay. Activities for samples are shown relative to Vmax of control (0 h), which is assigned as 100%. Data shown are representative of two-independent experiments. The results are the means ± S.D. of three samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In our search for interacting molecules with TRX, we isolated VDUP1 as a TBP-2 using a yeast two-hybrid system. We characterized TBP-2/VDUP1 as a TRX-binding protein. The interaction was dependent on the redox status of TRX. Moreover, the inhibitory effect of TBP-2/VDUP1 on the reducing activity of TRX and the expression was observed in TBP-2/VDUP1-overexpressed cells as well as HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3.

VDUP1 was originally reported as an up-regulated gene in HL-60 cells stimulated by 1alpha ,25-dihydroxyvitamin D3 (28). The function of VDUP1 is still unclear, although several homologous sequences from mammalian species have been reported. The rat VDUP1 homologue was isolated as a down-regulated gene by N-methyl-N-nitrosourea in rat mammary tumor (41). There are two transcripts homologous to VDUP1, TRT407-2 and TRT407-9, whose expressions were induced by cycloheximide and repressed by transforming growth factor-beta in mink Mv1Lu cells (38).

The interaction of TRX with TBP-2/VDUP1 was demonstrated both in vitro and in vivo. TRX treated with oxidizing reagents was incapable of binding with TBP-2/VDUP1. It should be noted that the effect of oxidizing reagents was detectable at a low concentration (10 µM), in which aggregation of TRX was avoided (42). Thus, only the reduced form of TRX appears to interact with TBP-2/VDUP1. There is a possibility that the presence of unreacted reagents has effects on the interaction between TRX and TBP-2/VDUP1. However, it seems unlikely because the GST-TRX beads used in the experiments were intensively washed after treatment with reducing/oxidizing reagents. In addition, the interaction was hardly inhibited by direct addition of the low concentration (10 µM) of reducing/oxidizing reagents to the TRX and TBP-2/VDUP1 reaction mixture.2 Therefore, TRX-TBP-2/VDUP1 interaction appears highly dependent on the redox status of TRX. In addition, we used substituted TRX to analyze the involvement of the active site of TRX in the interaction with TBP-2/VDUP1. TRX C32S/C35S was not able to interact with TBP-2/VDUP1 either in the in vitro binding assay or the yeast two-hybrid system. These results strongly suggest that the TRX active site is important for the TRX-TBP-2/VDUP1 interaction. Analysis of the crystal structure of TRX has indicated that the active-site conformation of TRX C32S/C35S is very similar to that of oxidized TRX (43). Thus, the incapability of the TRX C32S/C35S and oxidized TRX to interact with TBP-2/VDUP1 does not seem to be caused by a large scale conformational change. In addition, TBP-2/VDUP1 inhibited TRX activity in insulin reducing assay. If TBP-2/VDUP1 is a substrate for TRX, inhibitory effect was not observed in insulin reducing assay. Our result suggests that TBP-2/VDUP1 is not a substrate for TRX. Therefore, interaction of TRX with TBP-2/VDUP1 may be a direct protein-protein interaction manner. Physical interactions of TRX with other proteins have been reported. TRX has been isolated as an ASK1-binding protein using a yeast two-hybrid system (27). TRX directly binds to ASK1 and inhibits the apoptosis signal conducted by ASK1. These results suggest that the direct protein-protein interaction of TRX with its binding protein may be a basic mechanism of the redox regulation of cellular processes.

In the TBP-2/VDUP1-overexpressed cells, decrease of the reducing activity of TRX was observed. This result raised the possibility that TBP-2/VDUP1 affects enzymatic action of TRX or TRX expression. Therefore, we examined the effect of TBP-2/VDUP1 on insulin reducing activity of TRX and TRX protein expression. The insulin reducing assay using recombinant GST-TBP-2/VDUP1 protein clearly demonstrated the inhibitory effect of TBP-2/VDUP1 on the reducing activity of TRX. Structural interference caused by GST seems unlikely, because experiments using cell lysates transfected with His6-TBP-2/VDUP1 expression vector also showed the decreased TRX activity. Although recombinant TBP-2/VDUP1 protein demonstrated an inhibitory effect, the effect was not complete, probably because the TBP-2/VDUP1 protein concentration was insufficient. Additionally, suppression of TRX protein expression was observed in cells transiently transfected with TBP-2/VDUP1 expression vector. In addition to its inhibitory effect, TBP-2/VDUP1 may be involved in TRX protein expression, and the interaction of TBP-2/VDUP1 with TRX might be required for the suppression mechanism of TRX expression. Based on these findings, we hypothesize that TBP-2/VDUP1 inhibits the redox-regulatory action of TRX.

We observed the decrease of TRX expression and the reducing activity in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3 whose TBP-2/VDUP1 expression was enhanced as described in a previous report (28). An intriguing possibility is that up-regulation of TBP-2/VDUP1 expression is involved in the decrease of TRX expression and reducing activity. The decrease of TRX mRNA also was observed in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3. 1alpha ,25-Dihydroxyvitamin D3 treatment or action of TBP-2/VDUP1 may be involved in the decrease of TRX mRNA. This inverted pattern of TRX and TBP-2/VDUP1 expression also was observed in cell cycle-synchronized cells.2 The suppression mechanism of TRX expression in HL-60 cells treated with 1alpha ,25-dihydroxyvitamin D3 and the involvement of TBP-2/VDUP1 should be further analyzed.

1alpha ,25-Dihydroxyvitamin D3 is an essential biologically active molecule and is important for regulation of calcium homeostasis and secretion of hormone (44, 45). 1alpha ,25-Dihydroxyvitamin D3 also is a potent inducer of myeloid leukemic cell differentiation (46, 47) and can inhibit the growth of cancer cells from several different tissues (48, 49). TRX has been reported to have cell-growth promoting effects (12, 13), although TRX is involved in growth inhibitory mechanism in some system (50). Human TRX was discovered as adult T-cell leukemia-derived factor from human T-cell lymphotropic virus-I transformed T-cells (9). Elevated levels of TRX expression has been observed in some human tumors (51, 52). In addition, TRX transfected cells exhibited severalfold increased colony formation in soft agarose, whereas a redox-inactive mutant TRX C32S/C35S acts in a dominant negative manner to inhibit proliferation (53). Accordingly, the decrease of TRX function due to the TRX-TBP2/VDUP1 interaction may be one of the mechanisms through which 1alpha ,25-dihydroxyvitamin D3 exerts its growth inhibitory effect (54).

In conclusion, we identified TBP-2/VDUP1 as a new TRX-binding protein. Stable interaction of TRX with TBP-2/VDUP1 was detected in vitro and in vivo. TBP-2/VDUP1 had an inhibitory effect on TRX-dependent reducing activity. Suppression of TRX protein expression also was observed in cells transfected with TBP-2/VDUP1 expression vector. Our results suggest the possibility that TBP-2/VDUP1 acts as an endogenous negative regulator of TRX, although the precise mechanism of this regulation is not clear yet. TRX-TBP-2/VDUP1 interaction may play an important role in the redox regulation of various cellular processes such as growth and differentiation of the cells sensitive to a variety of inducers, including 1alpha ,25-dihydroxyvitamin D3 responses.

    ACKNOWLEDGEMENTS

We thank S. Toyama for helpful technical advice, W. Brown for review of the manuscript, H. Takei and H. Yamanaka for communicating their results before publication, Y. Yamaguchi for technical assistance, and Y. Kanekiyo for secretarial help.

    FOOTNOTES

* This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a Grant-in-Aid of Research for the Future from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Lab. of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.

§ Present address: Dept. of Anesthesia, Kyoto University Hospital, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.

To whom correspondence should be addressed: Dept. of Biological Responses, Inst. for Virus Research, Kyoto University, 53, Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: 81-75-751-4024; Fax: 81-75-761-5766; E-mail: yodoi@virus.kyoto-u.ac.jp.

2 A. Nishiyama and J. Yodoi, unpublished data.

    ABBREVIATIONS

The abbreviations used are: TRX, thioredoxin; ASK1, apoptosis signal-regulating kinase 1; TBP, thioredoxin-binding protein; VDUP1, vitamin D3 up-regulated protein 1; GST, glutathione S-transferase; GFP, green fluorescent protein; mAb, monoclonal antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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