Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Deficiency of Thioredoxin Binding Protein-2 (TBP-2) Enhances TGF-β Signaling and Promotes Epithelial to Mesenchymal Transition

  • So Masaki,

    Affiliation Laboratory of Infection and Prevention, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan

  • Hiroshi Masutani,

    Affiliation Laboratory of Infection and Prevention, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan

  • Eiji Yoshihara,

    Affiliation Laboratory of Infection and Prevention, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan

  • Junji Yodoi

    yodoi@virus.kyoto-u.ac.jp

    Affiliations Laboratory of Infection and Prevention, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan, Department of Bioinspired Sciences, Center for Cell Signaling Research, Ewha Womans University, Seoul, South Korea

Deficiency of Thioredoxin Binding Protein-2 (TBP-2) Enhances TGF-β Signaling and Promotes Epithelial to Mesenchymal Transition

  • So Masaki, 
  • Hiroshi Masutani, 
  • Eiji Yoshihara, 
  • Junji Yodoi
PLOS
x

Abstract

Background

Transforming growth factor beta (TGF-β) has critical roles in regulating cell growth, differentiation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) of various cancer cells. TGF-β-induced EMT is an important step during carcinoma progression to invasion state. Thioredoxin binding protein-2 (TBP-2, also called Txnip or VDUP1) is downregulated in various types of human cancer, and its deficiency results in the earlier onset of cancer. However, it remains unclear how TBP-2 suppresses the invasion and metastasis of cancer.

Principal Findings

In this study, we demonstrated that TBP-2 deficiency increases the transcriptional activity in response to TGF-β and also enhances TGF-β-induced Smad2 phosphorylation levels. Knockdown of TBP-2 augmented the TGF-β-responsive expression of Snail and Slug, transcriptional factors related to TGF-β-mediated induction of EMT, and promoted TGF-β-induced spindle-like morphology consistent with the depletion of E-Cadherin in A549 cells.

Conclusions/Significance

Our results indicate that TBP-2 deficiency enhances TGF-β signaling and promotes TGF-β-induced EMT. The control of TGF-β-induced EMT is critical for the inhibition of the invasion and metastasis. Thus TBP-2, as a novel regulatory molecule of TGF-β signaling, is likely to be a prognostic indicator or a potential therapeutic target for preventing tumor progression.

Introduction

Transforming growth factor-β (TGF-β) has dual functions in cancer [1]. TGF-β acts as a tumor suppressor in the early stage of tumor development, and contradictorily, promotes the invasion and metastasis of tumor cells in the late stage. Recently, many studies have shown that TGF-β promotes cancer progression by inducing Epithelial-mesenchymal transition (EMT), which is a crucial process to acquire the ability to execute the invasion-metastasis steps of cancer [2], [3]. TGF-β induces the expression of several transcription factors driven to EMT [4], including Snail/SNAI1 [5] and Slug/SNAI2 [6], which act directly or indirectly as a repressor of E-Cadherin. The loss of E-Cadherin is a fundamental event in EMT [7], [8].

Thioredoxin binding protein-2 (TBP-2), also known as thiredoxin interacting protein (Txnip) [9] or Vitamin D3 upregulated protein 1 (VDUP1) [10], has been identified as a negative regulator of thioredoxin (TRX) [11] and is mainly localized in nucleus [12]. TBP-2 is a member of α-arrestin protein family, and contains two PPxY motifs, which are known to interact with WW domain-containing proteins including Nedd4 family of E3 ubiquitin ligases [13], [14]. TBP-2 has a variety of biological functions in cell proliferation [15], cell apoptosis [16], immune response [17], [18], [19], glucose and lipid metabolism [9], [20], [21], [22], [23], [24].

There is the growing evidence that TBP-2 plays as a suppressor of cancer. TBP-2 is downregulated in various human cancer cells [25], [26]. TBP-2 overexpression inhibits proliferation via cell cycle arrest [12], [27], [28], [29] and promotes apoptosis [30]. In human T cell lymphocyte virus type 1 (HTLV-I)- infected T cells, TBP-2 regulates cell growth and its expression is associated with responsiveness to IL-2-dependent growth [31], and plays a key role in glucocorticoid-induced cell death [32]. In vivo studies, TBP-2 overexpression suppressed tumor growth and metastasis of the transplanted tumor. Point mutation or knock out of TBP-2 gene in mice show the higher incidence of hepatocellular carcinoma [33], [34]. TBP-2 knock out mice also shows the earlier onset of N-butyl-N- (4-hydroxybutyl) nitrosamine (BBN)-induced bladder carcinoma [35].

thumbnail
Figure 1. Deficiency of TBP-2 enhances the transcriptional activity of TGF-β signaling.

(A) Effect of TBP-2 deficiency on the transcriptional activity of TGF-β was examined using 9×CAGA-MLP-Luc, TGF-β responsive luciferase reporter, in WT (Wild Type: TBP-2+/+) and TBP-2−/− MEFs with or without TGF-β (0.5 ng/ml). (B) The efficiency of TBP-2 knockdown by TBP-2 siRNA and negative control (N.C.) in A549 and MDA-MB-231 cells was determined by quantitative real-time PCR at 36 hours after transfection. (C) Effect of TBP-2 knockdown on the transcriptional activity of TGF-β was examined using 9×CAGA-MLP-Luc in A549 and MDA-MB-231 cells with or without TGF-β. N.C. means negative control. The error bars show mean ± SD. *P<0.05, **P<0.01, ***P<0.001, versus control (t-test).

https://doi.org/10.1371/journal.pone.0039900.g001

These results collectively support that TBP-2 deficiency contributes to the progression and metastasis of cancer, however, detail mechanisms of TBP-2 in this process has not been sufficiently elucidated. In the late stage of cancer cells, TBP-2 expression is downregulated and TGF-β elicits cancer malignancy driving EMT. This correlation provides the hypothesis that TBP-2 regulates TGF-β-associated cancer development in the late stage.

In the present study, we examined the role of TBP-2 in TGF-β signaling. TBP-2 deficiency increased TGF-β signaling by enhancing Smad2 phosphorylation levels, and upregulated TGF-β-induced expression of Snail or Slug, resulting in acceleration of TGF-β-driven EMT. These findings show a novel function of TBP-2, as a regulator of TGF-β signaling, and provide new insights to the mechanisms of TGF-β-induced EMT.

Results

TBP-2 Deficiency Enhances Transcriptional Activity of TGF-β Signaling

To investigate the function of TBP-2 in TGF-β signaling, we performed promoter assay using 9×CAGA-Luc (TGF-β-responsive promoter-reporter), which is the most frequently used reporter system for TGF-β/Smad signal transduction, in WT (Wild Type: TBP-2+/+) mouse embryonic fibroblasts (MEFs) and TBP-2−/− MEFs. The results showed that transcriptional activity in response to TGF-β is enhanced in TBP-2−/− MEFs compared with WT MEFs (Fig. 1A). The efficiency of TBP-2 knockdown in A549 and MDA-MB-231 cells was confirmed by real-time RT-PCR (Fig. 1B). All experiments with TBP-2 siRNA were done according to the same protocol. Knockdown of TBP-2 also resulted in enhancing TGF-β-induced transcriptional activity in A549, MDA-MB-231 (Fig. 1C) and 253J (data not shown) cell lines.

TBP-2 Deficiency Increases the mRNA Expression of TGF-β-targeted Genes

To further examine that TBP-2 regulates the expression of TGF-β-target genes, plasminogen activator inhibitor (PAI)-1 and Smad7, well known TGF-β-targeted genes, were quantified by real-time RT-PCR. TGF-β-mediated induction of PAI-1 and Smad7 is increased in TBP-2−/− MEFs (Fig. 2A), as well as A549 and MDA-MB-231 cells under the condition of TBP-2 knockdown (Fig. 2B).

thumbnail
Figure 2. Deficiency of TBP-2 upregulates mRNA of TGF-β targeted genes.

(A) TGF-β-induced mRNA expression of PAI-1 or Smad7, TGF-β targeted genes, in WT and TBP-2−/− MEFs was determined by quantitative real-time PCR. MEFs were cultured in the presence or absence of TGF-β (0.5 ng/ml) for 8 hours. (B) The effects of TBP-2 knockdown for TGF-β-induced mRNA expression of PAI-1 or Smad7 in A549 cells and MDA-MB-231 cells were determined by quantitative real-time PCR. A549 cells and MDA-MB-231 cells were cultured in the presence or absence of TGF-β (2.5 ng/ml for 6 hours and 1 ng/ml for 12 hours, respectively). N.C. means negative control. The error bars show mean ± SD. **P<0.01, N.S.: not significant.

https://doi.org/10.1371/journal.pone.0039900.g002

TBP-2 Deficiency Increases TGF-β-mediated Phosphorylation of Smad2

Next, we analyzed the level of TGF-β-mediated phosphorylation of Smad2 in WT and TBP-2−/− MEFs by the western blot analyses. The phospho-Smad2 protein level was declined at 20 hour-TGF-β stimulation in WT MEFs, but was continuously elevated in TBP-2−/− MEFs (Fig. 3A). Similarly, phospho-Smad2 levels were enhanced with TGF-β stimulation for 12, 24 and 36 hours in TBP-2 knockdown-A549 cells (Fig. 3B). In addition, total Smad2 protein levels went down for 4 hours, responding to TGF-β stimulation, but were unchanged between 4 to 20 hours in WT MEFs, whereas no significant differences from 0 to 20 hours with TGF-β stimulation in TBP-2−/− MEFs (Fig. 3A).

thumbnail
Figure 3. Deficiency of TBP-2 maintains the higher phosphorylation level of Smad2.

(A) WT and TBP-2−/− MEFs cells were stimulated with TGF-β (0.5 ng/ml) for the indicated times. p-Smad2, Smad2 and β-actin were analyzed by Western blot. (B) A549 cells under the condition of TBP-2 knockdown or not were stimulated with TGF-β (2.5 ng/ml) for the indicated times. p-Smad2, Smad2 and α-tubulin were analyzed by Western blot. N.C. means negative control.

https://doi.org/10.1371/journal.pone.0039900.g003

TBP-2 Deficiency Enhances the Induction of Snail and Slug by TGF-β

TGF-β induces the expression of transcriptional factors involved in EMT, including Snail and Slug. As the induction of Snail or Slug is a crucial step for EMT, the effect of TBP-2 knockdown on the induction of Snail and Slug by TGF-β was examined with real-time RT-PCR. The results showed that the TGF-β-responsive expression of Snail and Slug was enhanced with TGF-β stimulation for 6, 12 and 22 hours in A549 cells under the condition of TBP-2 knockdown (Fig. 4).

thumbnail
Figure 4. Knockdown of TBP-2 promotes Snail and Slug induction by TGF-β.

Induction of Snail (A) or Slug (B) transcription was examined in A549 cells under the condition of TBP-2 knockdown (black bars) or not (gray bars) cultured with TGF-β (2.5 ng/ml) for the indicated times. Snail or Slug mRNA were determined by quantitative real-time PCR. N.C. means negative control. The error bars show mean ± SD. *P<0.05, **P<0.01, ***P<0.001, versus control (t-test).

https://doi.org/10.1371/journal.pone.0039900.g004

TBP-2 Deficiency Promotes TGF-β-induced EMT

Then, we evaluated the effects of TBP-2 knockdown in TGF-β-induced EMT. Knockdown of TBP-2 promoted TGF-β-induced morphological changes in A549 (Fig. 5) and 253J cells (data not shown). In the presence of 2.5 ng/ml TGF-β for 24 or 36 hours, TGF-β-driven spindle-like morphology was significantly observed in TBP-2 knockdown-A549 cells. To quantify the morphological changes, we measured the length of the longest diagonal line of each cell. TBP-2 knockdown-cells with TGF-β stimulation significantly lengthened more than control cells (Fig. S1). Consistently, the depletion of E-Cadherin, an epithelial marker, was quickened, and similarly the induction of vimentin, a mesenchymal marker, was elevated in TBP-2 knockdown-A549 cells (Fig. 6). These results indicate that TBP-2 deficiency accelerates the TGF-β-driven EMT phenotype.

thumbnail
Figure 5. Knockdown of TBP-2 accelerates TGF-β-induced cell morphological changes.

A549 cells transfected with TBP-2-targeting siRNA (TBP-2) or negative control siRNA (N.C.) were cultured in the presence of TGF-β (2.5 ng/ml) for 0, 12, 24 and 36 hours. Photos were taken at the indicated times.

https://doi.org/10.1371/journal.pone.0039900.g005

thumbnail
Figure 6. Knockdown of TBP-2 accelerates TGF-β-driven E-Cadherin degradation.

A549 cells transfected with TBP-2-targeting siRNA (TBP-2) or negative control siRNA (N.C.) were treated with TGF-β (2.5 ng/ml) for 0, 12, 24 and 36 hours. E-Cadherin, Vimentin, TBP-2 and α-tubulin were analyzed by Western blot.

https://doi.org/10.1371/journal.pone.0039900.g006

Discussion

In this study, we demonstrated that deficiency of TBP-2 increases TGF-β-responsive transcriptional activity and upregulates Smad2 phosphorylation levels, resulting in the acceleration of TGF-β-induced EMT.

TBP-2 deficiency contributes to upregulate transcriptional activities for several stimuli or ligands. We or other groups reported that peroxisome proliferator activated receptor (PPAR) or insulin target genes are upregulated in TBP-2−/− mice, and that TBP-2 negatively regulates PPAR transcriptional activity in vitro [23]. TBP-2 deficiency might maintain the level of transcriptional activities with the imperfection of biological feedback.

TBP-2 deficiency also results in the enhancement of phosphorylation of signal transducers. Regarding the relationship between TBP-2 and cell signaling, it was reported that phosphorylation of ERK is enhanced in TBP-2-KO mice [36] bladders during BBN-induced bladder carcinogenesis [35]. Our previous study showed that TBP-2 is a negative regulator of TRX [11], and other group reported that overexpression of TRX elevates the ERK1/2 phosphorylation levels [37]. These reports suggest that TBP-2 deficiency facilitates TRX activity, resulting in enhancement of the phosphorylation levels of signal transducer, such as ERK1/2. However, TBP-2 deficiency did not change the protein levels of TRX in the presence or absence of TGF-β (data not shown), so that TRX might not be related to the regulation of TGF-β by TBP-2.

The re-expression of TBP-2 using expression vector in TBP-2−/− MEFs failed to rescue the knock out effects of TBP-2 on the CAGA promoter. We also performed the experiments on the gain-of-function of TBP-2 using expression vector in A549 and MDA-MB-231 cell lines. The results unexpectedly showed that the overexpression of TBP-2 did not lead to the opposite of the loss-of-function results (data not shown). These results might be caused by the difficulty in controlling the expression level of TBP-2 within the physiological range. Since TBP-2 is a multifunctional protein targeting several molecules, the superabundant expression of TBP-2 might cause unexpected effects, which should be dissected in our future study.

It has been also reported that TBP-2 deficiency promotes TNF-α-induced NF-κB activity [34], that TBP-2 inhibits mTOR activity by binding REDD1 protein [38], and that TBP-2 deficiency enhances the phosphorylation of Akt in response to insulin [16], [24]. The present study shows that TBP-2 deficiency enhances TGF-β-mediated Smad2 phosphorylation level. These findings suggest that TBP-2 act as a crucial feedback regulator for various biological responses. TBP-2 might be essential for protein phosphatases or protein degradation systems.

TBP-2 deficiency enhanced TGF-β signaling and upregulated Smad7 expression (Fig. 1 and 2). Smad7, one of inhibitory Smads, plays an essential role in the negative feedback regulation of TGF-β signaling [39], however, TBP-2 deficiency enhanced TGF-β-mediated Smad2 phosphorylation (Fig. 3) irrespective of increasing Smad7 expression. In the negative feedback of TGF-β signaling, Smad7 requires to bind to Smad ubiquitin regulatory factor 2 (Smurf2), HECT type E3 ligases containing WW domain [39], [40]. Smad7-Smurf2 complex binds to the activated TGF-β receptors, and induces their degradation [41], [42]. In addition, Smurf2 also decreases the protein levels of Smad2 in response to TGF-β stimulation. Our results showed that total Smad2 protein levels went down for 4 hours, responding to TGF-β stimulation in WT MEFs, but no significant differences in TBP-2−/− MEFs. TBP-2 contains two PPxY motifs, which are reported to interact with WW domain. TBP-2 interacts with Smurf2 in co-immnoprecipitation assay (data not shown), providing the hypothesis that TBP-2 is required for functions of Smurf2 in the negative feedback of TGF-β signaling. The significance of TBP-2-Smurf2 interaction has been entirely unclear and will be examined in detail.

In conclusion, we demonstrated that TBP-2 deficiency enhances Smad2 phosphorylation level, resulting in acceleration of TGF-β-driven EMT. Our findings show a novel mechanisms of cancer suppression associated with TBP-2 and provide new insights into TGF-β-mediated EMT. TBP-2 is likely to be a prognosis indicator by monitoring TBP-2 expression in tumor, and a potential therapeutic target in the inhibition of EMT.

Materials and Methods

Reagents and Antibodies

TGF-β1 was purchased from R&D systems. Stealth small interfering RNA (siRNA) for TBP-2 (UCAAUUCGAGCAGAGACAGACACCC) and a negative control were purchased from Invitrogen. The antibodies used were as follows: anti-phospho-Smad2 (Ser465/467) (138D4) and anti-Smad2 (L16D3) antibodies were purchased from Cell Signaling. Anti-Txnip antibody and Anti-Vimentin were from MBL. Anti-E-Cadherin antibody was from Transduction Laboratories. Anti-β-actin antibody was from Santa Cruz. Anti-α-tubulin antibody was from Sigma.

Cell Culture

Primary wild-type and TBP-2−/− mouse embryonic fibroblasts (MEFs) were generated as previously described [24]. Human lung adenocarcinoma cell line A549 was obtained from Health Science Research Bank. Human breast cancer cell line MDA-MB-231 was from DS Pharma Biomedical. MEFs, A549 and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin antibiotics, and 2 mM L-glutamine. The culture was maintained at 37°C with 5% CO2.

RNA Interference

All knockdown assay using siRNAs were performed with Lopofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. The cells were used after 36 hours from transfection.

Transient Transfection and Luciferase Reporter Assay

Cells were transiently transfected with pGL3 9×CAGA-MLP-Luc and pRL-TK (Promega) using TransIT-LT1 (Takara) according to the manufacturer’s instruction. pRL-TK was used as a control of the efficiency of transfection. At the same time of transfection, cells were under the condition of serum deprivation. After 20 hours of transfection, cells were stimulated with TGF-β for 20 hours. Luciferase activity was measured with the Dual-Luciferase reporter system (Promega).

RNA Isolation, RT-PCR and Real-time Quantitative PCR

Total RNAs were extracted using TRIzol (Invitrogen), and were reverse-transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) according to the manufacturer’s instruction. Real-time PCR was performed with Power STBR Green PCR Master Mix (Applied Biosystems), using β-actin as an internal control for normalization. Fluorescent detection and data analyses were performed using ABI 7500 Sequence Detection System (Applied Biosystems). Primers for PCR analyses were listed in Table S1.

Immunoblotting Analysis

For western blotting, the cells were lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche) and phosphatase inhibitor (Nacalai Tesque). The lysate were boiled with Laemmli Smaple Buffer (BIO-RAD) at 95°C for 3 minutes. The samples were subjected to SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary anti-mouse- or anti-rabbit-immunoglobulin G (GE Lifesciences). Finally, chemiluminescence was detected using Chemi-Lumi One L kit (Nacalai Tesque), and luminescence images were analyzed by LAS 3000 or LAS 4000 (GE Lifesciences).

Supporting Information

Figure S1.

The length of the longest diagonal line of TBP-2 siRNA-A549 and control siRNA-A549 cells in the presence or absence of TGF-β (2.5 ng/ml for 36 hours). The length of each cell was calculated from expanded photos (200 cells).

https://doi.org/10.1371/journal.pone.0039900.s001

(TIF)

Table S1.

Primer sequences for real-time PCR analyses.

https://doi.org/10.1371/journal.pone.0039900.s002

(TIFF)

Acknowledgments

We thank Ms. Suzuyo Furukawa for technical assistance, Dr. Yoshiyuki Matsuo for technical supports and helpful discussion, and Dr. Daizo Koinuma for valuable discussion. We also thank Dr. Koji Nishizawa and Dr. Hiroyuki Nishiyama for kindly providing 253J cells, and Dr. Kohei Miyazono for kindly providing pGL3 9×CAGA-MLP-Luc vector. We are deeply grateful to Dr. Koichi Ikuta and Dr. Kohei Miyazono for the critical review of the manuscript.

Author Contributions

Conceived and designed the experiments: SM. Performed the experiments: SM. Analyzed the data: SM HM JY. Contributed reagents/materials/analysis tools: EY. Wrote the paper: SM HM.

References

  1. 1. Pardali K, Moustakas A (2007) Actions of TGF-β as tumor suppressor and pro-metastatic factor in human cancer. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1775: 21–62.K. PardaliA. Moustakas2007Actions of TGF-β as tumor suppressor and pro-metastatic factor in human cancer.Biochimica et Biophysica Acta (BBA) - Reviews on Cancer17752162
  2. 2. Zavadil J, Böttinger EP (2005) TGF-β and epithelial-to-mesenchymal transitions. Oncogene 24: 5764–5774.J. ZavadilEP Böttinger2005TGF-β and epithelial-to-mesenchymal transitions.Oncogene2457645774
  3. 3. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial–mesenchymal transitions. Nature Reviews Molecular Cell Biology 7: 131–142.JP ThieryJP Sleeman2006Complex networks orchestrate epithelial–mesenchymal transitions.Nature Reviews Molecular Cell Biology7131142
  4. 4. Xu J, Lamouille S, Derynck R (2009) TGF-β-induced epithelial to mesenchymal transition. Cell Research 19: 156–172.J. XuS. LamouilleR. Derynck2009TGF-β-induced epithelial to mesenchymal transition.Cell Research19156172
  5. 5. Vincent T, Neve EPA, Johnson JR, Kukalev A, Rojo F, et al. (2009) A SNAIL1–SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial–mesenchymal transition. Nature Cell Biology 11: 943–950.T. VincentEPA NeveJR JohnsonA. KukalevF. Rojo2009A SNAIL1–SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial–mesenchymal transition.Nature Cell Biology11943950
  6. 6. Karen M. Hajra DY-SC, Fearon ER (2002) The SLUG Zinc-Finger Protein Represses E-Cadherin in Breast Cancer. Cancer Research 62: 1613–1618.DY-SC Karen M. HajraER Fearon2002The SLUG Zinc-Finger Protein Represses E-Cadherin in Breast Cancer.Cancer Research6216131618
  7. 7. Nieto MA (2002) The snail superfamily of zinc-finger transcription factors. Nature reviews Molecular cell biology 3: 155–166.MA Nieto2002The snail superfamily of zinc-finger transcription factors.Nature reviews Molecular cell biology3155166
  8. 8. Peinado H, Olmeda D, Cano A (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nature Reviews Cancer 7: 415–428.H. PeinadoD. OlmedaA. Cano2007Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?Nature Reviews Cancer7415428
  9. 9. Bodnar JS, Chatterjee A, Castellani LW, Ross DA, Ohmen J, et al. (2002) Positional cloning of the combined hyperlipidemia gene Hyplip1. Nature Genetics 30: 110–116.JS BodnarA. ChatterjeeLW CastellaniDA RossJ. Ohmen2002Positional cloning of the combined hyperlipidemia gene Hyplip1.Nature Genetics30110116
  10. 10. Chen KS, DeLuca HF (1994) Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3. biochimica et Biophysica Acta 1219: 26–32.KS ChenHF DeLuca1994Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3.biochimica et Biophysica Acta12192632
  11. 11. Nishiyama A, Matsui M, Iwata S, Hirota K, Masutani H, et al. (1999) Identification of Thioredoxin-binding Protein-2/Vitamin D3 Up-regulated Protein 1 as a Negative Regulator of Thioredoxin Function and Expression. The Journal of Biological Chemistory 274: 21645–21650.A. NishiyamaM. MatsuiS. IwataK. HirotaH. Masutani1999Identification of Thioredoxin-binding Protein-2/Vitamin D3 Up-regulated Protein 1 as a Negative Regulator of Thioredoxin Function and Expression.The Journal of Biological Chemistory2742164521650
  12. 12. Nishinaka Y, Masutani H, Oka Si, Matsuo Y, Yamaguchi Y, et al. (2004) Importin α1 (Rch1) Mediates Nuclear Translocation of Thioredoxin-binding Protein-2/Vitamin D3-up-regulated Protein 1. Journal of Biological Chemistry 279: 37559–37565.Y. NishinakaH. MasutaniSi OkaY. MatsuoY. Yamaguchi2004Importin α1 (Rch1) Mediates Nuclear Translocation of Thioredoxin-binding Protein-2/Vitamin D3-up-regulated Protein 1.Journal of Biological Chemistry2793755937565
  13. 13. Alvarez CE (2008) On the origins of arrestin and rhodopsin. BMC evolutionary biology 8: 222.CE Alvarez2008On the origins of arrestin and rhodopsin.BMC evolutionary biology8222
  14. 14. Patwari P, Chutkow WA, Cummings K, Verstraeten VLRM, Lammerding J, et al. (2009) Thioredoxin-independent Regulation of Metabolism by the -Arrestin Proteins. Journal of Biological Chemistry 284: 24996–25003.P. PatwariWA ChutkowK. CummingsVLRM VerstraetenJ. Lammerding2009Thioredoxin-independent Regulation of Metabolism by the -Arrestin Proteins.Journal of Biological Chemistry2842499625003
  15. 15. Kuljaca S, Liu T, Dwarte T, Kavallaris M, Haber M, et al. (2009) The cyclin-dependent kinase inhibitor, p21WAF1, promotes angiogenesis by repressing gene transcription of thioredoxin-binding protein 2 in cancer cells. Carcinogenesis 30: 1865–1871.S. KuljacaT. LiuT. DwarteM. KavallarisM. Haber2009The cyclin-dependent kinase inhibitor, p21WAF1, promotes angiogenesis by repressing gene transcription of thioredoxin-binding protein 2 in cancer cells.Carcinogenesis3018651871
  16. 16. Chen J, Hui ST, Couto FM, Mungrue IN, Davis DB, et al. (2008) Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes. The FASEB Journal 22: 3581–3594.J. ChenST HuiFM CoutoIN MungrueDB Davis2008Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes.The FASEB Journal2235813594
  17. 17. Lee KN, Kang H-S, Jeon J-H, Kim E-M, Yoon S-R, et al. (2005) VDUP1 Is Required for the Development of Natural Killer Cells. Immunity 22: 195–208.KN LeeH-S KangJ-H JeonE-M KimS-R Yoon2005VDUP1 Is Required for the Development of Natural Killer Cells.Immunity22195208
  18. 18. Son A, Nakamura H, Okuyama H, Oka Si, Yoshihara E, et al. (2008) Dendritic cells derived from TBP-2-deficient mice are defective in inducing T cell responses. European Journal of Immunology 38: 1358–1367.A. SonH. NakamuraH. OkuyamaSi OkaE. Yoshihara2008Dendritic cells derived from TBP-2-deficient mice are defective in inducing T cell responses.European Journal of Immunology3813581367
  19. 19. Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J (2009) Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nature Immunology 11: 136–140.R. ZhouA. TardivelB. ThorensI. ChoiJ. Tschopp2009Thioredoxin-interacting protein links oxidative stress to inflammasome activation.Nature Immunology11136140
  20. 20. Hui TY, Sheth SS, Diffley JM, Potter DW, Lusis AJ, et al. (2004) Mice Lacking Thioredoxin-interacting Protein Provide Evidence Linking Cellular Redox State to Appropriate Response to Nutritional Signals. Journal of Biological Chemistry 279: 24387–24393.TY HuiSS ShethJM DiffleyDW PotterAJ Lusis2004Mice Lacking Thioredoxin-interacting Protein Provide Evidence Linking Cellular Redox State to Appropriate Response to Nutritional Signals.Journal of Biological Chemistry2792438724393
  21. 21. Parikh H, Carlsson E, Chutkow WA, Johansson LE, Storgaard H, et al. (2007) TXNIP Regulates Peripheral Glucose Metabolism in Humans. PLoS Medicine 4: 0868–0879.H. ParikhE. CarlssonWA ChutkowLE JohanssonH. Storgaard2007TXNIP Regulates Peripheral Glucose Metabolism in Humans.PLoS Medicine408680879
  22. 22. Hui ST, Andres AM, Miller AK, Spann NJ, Potter DW, et al. (2008) Txnip balances metabolic and growth signaling via PTEN disulfide reduction. Proceedings of the National Academy of Sciences of the United States of America 105: 3921–3926.ST HuiAM AndresAK MillerNJ SpannDW Potter2008Txnip balances metabolic and growth signaling via PTEN disulfide reduction.Proceedings of the National Academy of Sciences of the United States of America10539213926
  23. 23. Oka Si, Yoshihara E, Bizen-Abe A, Liu W, Watanabe M, et al. (2008) Thioredoxin Binding Protein-2/Thioredoxin-Interacting Protein Is a Critical Regulator of Insulin Secretion and Peroxisome Proliferator-Activated Receptor Function. Endocrinology 150: 1225–1234.Si OkaE. YoshiharaA. Bizen-AbeW. LiuM. Watanabe2008Thioredoxin Binding Protein-2/Thioredoxin-Interacting Protein Is a Critical Regulator of Insulin Secretion and Peroxisome Proliferator-Activated Receptor Function.Endocrinology15012251234
  24. 24. Yoshihara E, Fujimoto S, Inagaki N, Okawa K, Masaki S, et al. (2010) Disruption of TBP-2 ameliorates insulin sensitivity and secretion without affecting obesity. Nature Communications 1: 127.E. YoshiharaS. FujimotoN. InagakiK. OkawaS. Masaki2010Disruption of TBP-2 ameliorates insulin sensitivity and secretion without affecting obesity.Nature Communications1127
  25. 25. Butler LM, Zhou X, Xu WS, Scher HI, Rifkind RA, et al. (2002) The histone deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin. Proceedings of the National Academy of Sciences 99: 11700–11705.LM ButlerX. ZhouWS XuHI ScherRA Rifkind2002The histone deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin.Proceedings of the National Academy of Sciences991170011705
  26. 26. Goldberg SF, Miele ME, Hatta N, Takata M, Paquette-Straub C, et al. (2003) Melanoma Metastasis Suppression by Chromosome 6: Evidence for a Pathway Regulated by CRSP3 and TXNIP. Cancer Research 63: 432–440.SF GoldbergME MieleN. HattaM. TakataC. Paquette-Straub2003Melanoma Metastasis Suppression by Chromosome 6: Evidence for a Pathway Regulated by CRSP3 and TXNIP.Cancer Research63432440
  27. 27. Han SH, Jeon JH, Ju HR, Jung U, Kim KY, et al. (2003) VDUP1 upregulated by TGF-β1 and 1,25-dihydorxyvitamin D3 inhibits tumor cell growth by blocking cell-cycle progression. Oncogene 22: 4035–4046.SH HanJH JeonHR JuU. JungKY Kim2003VDUP1 upregulated by TGF-β1 and 1,25-dihydorxyvitamin D3 inhibits tumor cell growth by blocking cell-cycle progression.Oncogene2240354046
  28. 28. Nishinaka Y, Nishiyama A, Masutani H, Oka Si, Ahsan KM, et al. (2004) Loss of Thioredoxin-Binding Protein-2/Vitamin D3 Up-Regulated Protein 1 in Human T-Cell Leukemia Virus Type I-Dependent T-Cell Transformation: Implications for Adult T-Cell Leukemia Leukemogenesis. Cancer Research 64: 1287–1292.Y. NishinakaA. NishiyamaH. MasutaniSi OkaKM Ahsan2004Loss of Thioredoxin-Binding Protein-2/Vitamin D3 Up-Regulated Protein 1 in Human T-Cell Leukemia Virus Type I-Dependent T-Cell Transformation: Implications for Adult T-Cell Leukemia Leukemogenesis.Cancer Research6412871292
  29. 29. Jeon JH, Lee KN, Hwang CY, Kwon KS, You KH, et al. (2005) Tumor Suppressor VDUP1 Increases p27kip1 Stability by Inhibiting JAB1. Cancer Research 65: 4485–4489.JH JeonKN LeeCY HwangKS KwonKH You2005Tumor Suppressor VDUP1 Increases p27kip1 Stability by Inhibiting JAB1.Cancer Research6544854489
  30. 30. Kim SY, Shu HW, Chung JW, Yoon SR, Choi I (2007) Diverse Functions of VDUP1 in Cell Proliferation, Differentiation, and Diseases. Cellular & Molecular Immunology 4: 345–351.SY KimHW ShuJW ChungSR YoonI. Choi2007Diverse Functions of VDUP1 in Cell Proliferation, Differentiation, and Diseases.Cellular & Molecular Immunology4345351
  31. 31. Ahsan MK, Masutani H, Yamaguchi Y, Kim YC, Nosaka K, et al. (2005) Loss of interleukin-2-dependency in HTLV-I-infected T cells on gene silencing of thioredoxin-binding protein-2. Oncogene 25: 2181–2191.MK AhsanH. MasutaniY. YamaguchiYC KimK. Nosaka2005Loss of interleukin-2-dependency in HTLV-I-infected T cells on gene silencing of thioredoxin-binding protein-2.Oncogene2521812191
  32. 32. Chen Z, Lopez-Ramos DA, Yoshihara E, Maeda Y, Masutani H, et al. (2010) Thioredoxin-binding protein-2 (TBP-2/VDUP1/TXNIP) regulates T-cell sensitivity to glucocorticoid during HTLV-I-induced transformation. Leukemia 25: 440–448.Z. ChenDA Lopez-RamosE. YoshiharaY. MaedaH. Masutani2010Thioredoxin-binding protein-2 (TBP-2/VDUP1/TXNIP) regulates T-cell sensitivity to glucocorticoid during HTLV-I-induced transformation.Leukemia25440448
  33. 33. Sheth SS, Bodnar JS, Ghazalpour A, Thipphavong CK, Tsutsumi S, et al. (2006) Hepatocellular carcinoma in Txnip-deficient mice. Oncogene 25: 3528–3536.SS ShethJS BodnarA. GhazalpourCK ThipphavongS. Tsutsumi2006Hepatocellular carcinoma in Txnip-deficient mice.Oncogene2535283536
  34. 34. Kwon HJ, Won YS, Suh HW, Jeon JH, Shao Y, et al. (2010) Vitamin D3 Upregulated Protein 1 Suppresses TNF-α-Induced NF- B Activation in Hepatocarcinogenesis. The Journal of Immunology 185: 3980–3989.HJ KwonYS WonHW SuhJH JeonY. Shao2010Vitamin D3 Upregulated Protein 1 Suppresses TNF-α-Induced NF- B Activation in Hepatocarcinogenesis.The Journal of Immunology18539803989
  35. 35. Nishizawa K, Nishiyama H, Matsui Y, Kobayashi T, Saito R, et al. (2011) Thioredoxin-interacting protein suppresses bladder carcinogenesis. Carcinogenesis 32: 1459–1466.K. NishizawaH. NishiyamaY. MatsuiT. KobayashiR. Saito2011Thioredoxin-interacting protein suppresses bladder carcinogenesis.Carcinogenesis3214591466
  36. 36. Kwon HJ, Won YS, Yoon YD, Yoon WK, Nam KH, et al. (2011) Vitamin D3 up-regulated protein 1 deficiency accelerates liver regeneration after partial hepatectomy in mice. Journal of Hepatology 54: 1168–1176.HJ KwonYS WonYD YoonWK YoonKH Nam2011Vitamin D3 up-regulated protein 1 deficiency accelerates liver regeneration after partial hepatectomy in mice.Journal of Hepatology5411681176
  37. 37. Arai RJ, Ogata FT, Batista WL, Masutani H, Yodoi J, et al. (2008) Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases. Toxicology and applied pharmacology 233: 227–237.RJ AraiFT OgataWL BatistaH. MasutaniJ. Yodoi2008Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases.Toxicology and applied pharmacology233227237
  38. 38. Jin HO, Seo SK, Kim YS, Woo SH, Lee KH, et al. (2011) TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1. Oncogene 30: 3792–3801.HO JinSK SeoYS KimSH WooKH Lee2011TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.Oncogene3037923801
  39. 39. Ogunjimi AA, Briant DJ, Pece-Barbara N, Le Roy C, Di Guglielmo GM, et al. (2005) Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain. Molecular cell 19: 297–308.AA OgunjimiDJ BriantN. Pece-BarbaraC. Le RoyGM Di Guglielmo2005Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain.Molecular cell19297308
  40. 40. Inoue Y, Imamura T (2008) Regulation of TGF-β family signaling by E3 ubiquitin ligases. Cancer Science 99: 2107–2112.Y. InoueT. Imamura2008Regulation of TGF-β family signaling by E3 ubiquitin ligases.Cancer Science9921072112
  41. 41. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, et al. (2000) Smad7 Binds to Smurf2 to Form an E3 Ubiquitin Ligase that Targets the TGF-β Receptor for Degradation. Molecular Cell 6: 1365–1375.P. KavsakRK RasmussenCG CausingS. BonniH. Zhu2000Smad7 Binds to Smurf2 to Form an E3 Ubiquitin Ligase that Targets the TGF-β Receptor for Degradation.Molecular Cell613651375
  42. 42. Shi W, Sun C, He B, Xiong W, Shi X, et al. (2004) GADD34-PP1c recruited by Smad7 dephosphorylates TGF type I receptor. The Journal of Cell Biology 164: 291–300.W. ShiC. SunB. HeW. XiongX. Shi2004GADD34-PP1c recruited by Smad7 dephosphorylates TGF type I receptor.The Journal of Cell Biology164291300