Thioredoxin Binding Protein Research Articles

Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges.

We pursued several strategies for expressing either full-length Sus scrofa diacylglycerol kinase (DGK) alpha or the catalytic domain (alphacat) in Escherichia coli. Alphacat could be extracted, refolded, and purified from inclusion bodies, but when subjected to analytical gel filtration chromatography, it elutes in the void volume, in what we conclude are microscopic aggregates unsuitable for x-ray crystallography. Adding glutathione S-transferase, thioredoxin, or maltose binding protein as N-terminal fusion tags did not improve alphacat's solubility. Coexpressing with bacterial chaperones increased the yield of alphacat in the supernatant after high-speed centrifugation, but the protein still elutes in the void upon analytical gel filtration chromatography. We believe our work will be of interest to those interested in the structure of eukaryotic DGKs, so that they know which expression strategies have already been tried, as well as to those interested in protein folding and those interested in chaperone/target-protein interactions.

Growth inhibition of pancreatic cancer cells by histone deacetylase inhibitor belinostat through suppression of multiple pathways including HIF, NFkB, and mTOR signaling in vitro and in vivo

Pancreatic ductal adenocarcinoma is a devastating disease with few therapeutic options. Histone deacetylase inhibitors are a novel therapeutic approach to cancer treatment; and two new pan-histone deacetylase inhibitors (HDACi), belinostat and panobinostat, are undergoing clinical trials for advanced hematologic malignancies, non-small cell lung cancers and advanced ovarian epithelial cancers. We found that belinostat and panobinostat potently inhibited, in a dose-dependent manner, the growth of six (AsPc1, BxPc3, Panc0327, Panc0403, Panc1005, MiaPaCa2) of 14 human pancreatic cancer cell lines. Belinostat increased the percentage of apoptotic pancreatic cancer cells and caused prominent G2 /M growth arrest of most pancreatic cancer cells. Belinostat prominently inhibited PI3K-mTOR-4EBP1 signaling with a 50% suppression of phorphorylated 4EBP1 (AsPc1, BxPc3, Panc0327, Panc1005 cells). Surprisingly, belinostat profoundly blocked hypoxia signaling including the suppression of hypoxia response element reporter activity; as well as an approximately 10-fold decreased transcriptional expression of VEGF, adrenomedullin, and HIF1α at 1% compared to 20% O2 . Treatment with this HDACi decreased levels of thioredoxin mRNA associated with increased levels of its endogenous inhibitor thioredoxin binding protein-2. Also, belinostat alone and synergistically with gemcitabine significantly (P = 0.0044) decreased the size of human pancreatic tumors grown in immunodeficiency mice. Taken together, HDACi decreases growth, increases apoptosis, and is associated with blocking the AKT/mTOR pathway. Surprisingly, it blocked hypoxic growth related signals. Our studies of belinostat suggest it may be an effective drug for the treatment of pancreatic cancers when used in combination with other drugs such as gemcitabine.

Overexpression of thioredoxin-binding protein 2increases oxidation sensitivity and apoptosis in human lens epithelialcells

Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multiple physiological functions in cells and its bioavailability is negatively controlled through active-site binding to a specific thioredoxin-binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx and the effect of overexpression of TBP-2 in human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a sevenfold increase in TBP-2 and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day 7. Cell cycle analysis showed that TBP-2 OE cells arrested at the G2/M stage and that they displayed low expression of the cell cycle elements P-cdc2(Y15), cdc2, cdc25A, and cdc25C. Furthermore, TBP-2 OE cells were more sensitive to oxidation. Under H2O2 (200μM, 24h) treatment, these cells lost 80% viability and became highly apoptotic. Brief oxidative stress (200μM, 30min) to TBP-2 OE cells disrupted the Trx antiapoptotic function by dissociating the cytosolic and mitochondrial Trx–ASK binding complexes. The same H2O2-treated cells also showed activated ASK (P-ASK), increased Bax, lowered Bcl-2, cytochrome c release, and elevated caspase 3/7 activity. We conclude from these studies that high cellular levels of TBP-2 can potentially suppress Trx bioavailability and increase oxidation sensitivity. Overexpression of TBP-2 also causes slow growth by mitotic arrest and apoptosis by activating the ASK death pathway.

Histone deacetylase inhibitor suberoylanilide hydroxamic acid suppresses the pro-oncogenic effects induced by hepatitis B virus pre-S 2 mutant oncoprotein and represents a potential chemopreventive agent in high-risk chronic HBV patients

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S(2) mutant large HBV surface antigen (LHBS) in type II ground glass hepatocytes (GGHs) has been recognized as an emerging viral oncoprotein; it directly interacts with the c-Jun activation domain-binding protein 1 (JAB1) and subsequently causes hyperphosphorylation of the tumor-suppressor retinoblastoma and, consequently, leads to disturbed cell cycle progression. The interaction of the pre-S(2) mutant LHBS with JAB1 could provide a potential target for chemoprevention. In this study, we found that the preneoplastic type II GGHs showed a significant decrease of the cyclin-dependent kinase inhibitor p27(Kip1), which serves as a marker for pre-S(2) mutant-JAB1 complex formation. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) elevated expression of the tumor-suppressor thioredoxin-binding protein 2 (TBP2), which subsequently enhanced the JAB1-TBP2 interaction and abolished the pre-S(2) mutant LHBS-induced degradation of p27(Kip1), which, in turn, recovered the normal cell cycle checkpoint. The pre-S(2) mutant LHBS-induced pro-oncogenic effects: increased cell proliferation, nuclear/cytoplasmic ratio and proliferating cell nuclear antigen expression, were all greatly ameliorated after SAHA treatments, which suggested SAHA as a promising chemopreventive agent for the pre-S(2) mutant oncoprotein-induced HCC. In conclusion, this study provides the mechanism of histone deacetylase (HDAC) inhibitor in preventing the pre-S(2) mutant-induced oncogenic phenotype. The HDAC inhibitor SAHA is therefore a potential chemopreventive agent for high-risk chronic HBV patients who may develop HCC.

Thioredoxin binding protein-2 (TBP-2)/ Txnip/ VDUP1 with multifunctional biostress signal regulatory activities

Thioredoxin binding protein-2 (TBP-2)/ Txnip/ VDUP1 with multifunctional biostress signal regulatory activities

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

BackgroundTransforming 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 FindingsIn 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/SignificanceOur 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.

The importance of adding EDTA for the nanopore analysis of proteins

Nanopore analysis is a promising technique for studying the conformation of proteins and protein/protein interactions. Two proteins (bacterial thioredoxin and maltose binding protein) were subjected to nanopore analysis with α-hemolysin. Two types of events were observed; bumping events with a blockade current less than -40 pA and intercalation events with blockade currents between -40 pA and -100 pA. In potassium phosphate buffer, pH 7.8, both proteins gave intercalation events but the frequency of these events was significantly reduced in TRIS or HEPES buffers especially in the presence of 0.01 mM divalent metal ions. The frequency of events was restored by the addition of EDTA. For maltose binding protein, the frequency of intercalation events was also decreased in the presence of maltose but not lactose to which it does not bind. It is proposed that the events with large blockade currents represent transient intercalation of a loop or end of the protein into the pore and that divalent metal ions inhibit this process. The results demonstrate that the choice of buffer and the effects of metal ion contamination are important considerations in nanopore analysis.

Thioredoxin interacting protein: redox dependent and independent regulatory mechanisms.

The thioredoxin-interacting protein (TXNIP, also termed VDUP1 for vitamin D upregulated protein or TBP2 for thioredoxin-binding protein) was originally discovered by virtue of its strong regulation by vitamin D. Recently, TXNIP has been found to regulate the cellular reduction-oxidation (redox) state by binding to and inhibiting thioredoxin (TRX) in a redox-dependent fashion. Studies of the Hcb-19 mouse, TXNIP nonsense mutated mouse, demonstrate redox-mediated roles in lipid and glucose metabolism, cardiac function, inflammation, and carcinogenesis. Exciting recent data indicate important roles for TXNIP in redox independent signaling. Specifically, sequence analysis revealed that TXNIP is a member of the classical visual/β-arrestin superfamily, and is one of the six members of the arrestin domain-containing (ARRDC, or α-arrestin) family. Although the function of α-arrestins is not well known, recent studies suggest roles in endocytosis and protein ubiquitination through PPxY motifs in their C-terminal tails. Importantly, the ability of TXNIP to inhibit glucose uptake was found to be independent of TRX binding. Further investigation showed that several metabolic functions of TXNIP were due to the arrestin domains, thus further supporting the importance of redox independent functions of TXNIP. Since TXNIP transcription and protein stability are highly regulated by multiple tissue-specific stimuli, it appears that TXNIP should be a good therapeutic target for metabolic diseases.

Treatment with Auranofin Induces Oxidative and Lethal Endoplasmic Reticulum (ER) Stress Exerting Single Agent Activity Against Primary CLL Cells

Abstract 929Auranofin (AF) (Ridaura®) is an oral, FDA-approved, lipophilic, gold-containing compound used for the treatment of rheumatoid arthritis, based upon its anti-inflammatory and immunosuppressive effects. In the present studies, we determined for the first time the lethal activity of AF (250 to 1000 nM) and its underlying mechanism(s) in CD19+ primary CLL cells, including those with del(17p13.1). We demonstrate that treatment with AF induced 20 to 40% increase in reactive oxygen species (ROS) levels (assessed by flow cytometry), decrease in thioredoxin reductase (TRR) activity (mean reduction of 45%) without alteration in the expression of TRR, as determined in the AF treated cell lysates of CLL cells. AF treatment also increased the levels of thioredoxin binding protein 2. These findings were associated with induction of nuclear factor erythroid 2-related factor 2 (NRF2) activity marked by substantial increase in hemeoxygenase-1 (HO-1) levels. Thus, AF treatment perturbed the redox status in CLL cells. Consistent with increased levels of intracellular ROS, AF treatment also induced the markers of ER stress response including the induction of GRP78 and a sustained increase in the levels of the pro-apoptotic transcription factor CHOP (CAAT/enhancer binding protein homologous protein). This was associated with induction of the levels of the pro-death BH3 only domain protein BIM, which is a known mediator of lethal ER stress. Exposure to 1000 nM AF induced significantly more apoptosis (range 40 to 60%) in primary CLL cells, as compared to CD19+ normal B cells and CD34+ human cord blood and bone marrow progenitor cells (< 15% apoptosis) (p < 0.01). AF treatment induced apoptosis to a similar extent in primary CLL cells with del(17p13.1) as compared to other genetic abnormalities. Based on the observations that AF treatment induced oxidative and ER stress, we determined whether exposure to AF also increased the intracellular levels of misfolded and polyubiquitylated proteins, which would disrupt the cytosolic complex consisting of the heat shock protein (hsp) 90, heat shock factor (hsf) 1, histone deacetylase 6 (HDAC6) and p97/VCP, resulting in hyperacetylation and inhibition of the chaperone function of hsp90. Indeed, treatment with AF disrupted the association between hsp90 and HDAC6, resulting in hyperacetylation of hsp90 and depletion of the levels of HDAC6 in CLL cells. AF mediated hyperacetylation of hsp90 and α-tubulin was associated with proteasomal degradation of several of the CLL-relevant hsp90 client proteins, including ZAP70, c-Raf and AKT. Co-treatment of CLL cells with AF and the proteasome inhibitor carfilzomib (20 nM) significantly restored the levels of ZAP70, c-Raf, AKT and HDAC6. Finally, relatively lower levels of AF (250 nM) combined with the pan-HDAC inhibitor panobinostat (20 nM) or carfilzomib (5 nM) induced more cell death of primary CLL cells than each agent alone. These findings indicate that treatment of primary CLL cells with AF results in oxidative and ER stress. AF treatment also promotes the depletion of pro-survival CLL-relevant hsp90 client proteins. Collectively, our data creates a strong rationale for determining the in vivo activity of AF in patients with CLL including those with del(17p13.1). Disclosures:Reyes:Millennium, Sanofi Aventis: Consultancy.

Molecular mechanism of impaired metabolism-secretion coupling in diabetic pancreatic β cells

In pancreatic β cells, glucose stimulates insulin secretion according to metabolism-secretion coupling. We investigated the mechanism of impaired glucose-induced insulin secretion from β cells in various conditions. Cytosolic alkalization and a decreased supply of electron donors, including nicotinamide adenine dinucleotide, reduced (NADH), to the respiratory chain due to impaired glycolysis, shuttles, and the Krebs cycle reduce hyperpolarlization of mitochondrial inner membrane and adenosine triphosphate (ATP) production, causing decreased glucose-induced insulin release. Ouabain, a sodium/potassium–adenosine triphosphatase (Na+/K+ ATPase) inhibitor, reduces carbohydrate metabolism in the Krebs cycle by generating reactive oxygen species (ROS) to impair metabolism-secretion coupling; endogenous ROS generation by ouabain involves activation of Src, a nonreceptor tyrosine kinase. In type 2 diabetes, the insulin secretory response of β cells is selectively impaired to glucose. In the Goto-Kakizaki (GK) rat, a model of nonobese type 2 diabetes mellitus, Src is endogenously activated in islets, and glucose-induced insulin secretion is selectively impaired due to deficient ATP production derived from impaired glucose metabolism, which is caused by overproduction of ROS produced by Src activation. In addition, exendin-4, a glucagon-like peptide-1 receptor agonist, decreases Src activation and ROS production and ameliorates impaired ATP production in GK islets. Moreover, thioredoxin-binding protein-2 is overexpressed in islets from ob/ob mice, a model of obese type 2 diabetes mellitus, which causes overexpression of uncoupling protein-2, reduced mitochondrial ATP production, and decreased glucose-induced insulin secretion. Further studies are necessary to relate these mechanisms to β-cell dysfunction in patients with type 2 diabetes.

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus

Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus.

Thioredoxin-binding protein-2 (TBP-2/VDUP1/TXNIP) regulates T-cell sensitivity to glucocorticoid during HTLV-I-induced transformation

Although glucocorticoid (GC) is widely used for treating hematopoietic malignancies including adult T-cell leukemia (ATL), the mechanism by which leukemic cells become resistant to GC in the clinical course remains unclear. Using a series of T-cell lines infected with human T lymphotropic virus type-I (HTLV-I), the causative virus of ATL, we have dissected the transformation from interleukin (IL)-2-dependent to -independent growth stage. The transformation associates the loss of thioredoxin-binding protein-2 (TBP-2), a tumor suppressor and regulator of lipid metabolism. Here we show that TBP-2 is responsible for GC-induced apoptosis in ATL cells. In the IL-2-dependent stage, dexamethasone induced TBP-2 expression and apoptosis, both of which were blocked by GC receptor (GR) antagonist RU486. Knockdown of TBP-2 consistently reduced the amount of GC-induced apoptosis. In IL-2-independent stage, however, expression of GR and TBP-2 was suppressed and GC failed to induce apoptosis. Forced expression of GR led the cells to mild sensitivity to GC, which was also accomplished by treatment with suberoylanilide hydroxamic acid, a TBP-2 inducer. A transfection experiment showed that TBP-2 expression induced apoptosis in IL-2-independent ATL cells. Thus, TBP-2 is likely to be one of the key molecules for GC-induced apoptosis and a potential target for treating the advanced stage of ATL.

Thioredoxin binding protein-2 mediates metabolic adaptation in response to lipopolysaccharide in vivo

Endotoxin triggers a reorganization of the energy metabolic pathway, including the promotion of fatty acid utilization to adapt to a high energy demand during endotoxemia. However, the factors responsible for the metabolic adaptation and characteristic pathologies resulting from defective utilization fatty acids during endotoxin response have not been fully clarified. The thioredoxin binding protein-2 (TBP-2) knockout (TBP-2) mouse is an animal model of fatty acid oxidation disorder. The aim of this study was to determine whether and how TBP-2 is involved in metabolic regulation in a lipopolysaccharide (LPS)-induced endotoxemia model in mice. Prospective animal trial. Research laboratory. TBP-2 and wild control mice. TBP-2 and wild control mice were intraperitoneally injected with LPS. Mortality, serum levels of markers of hepatorenal injuries, cytokines, insulin, glucose and lipid derivatives, and the hepatic signaling pathway regulating gluconeogenesis were investigated. Following the administration of LPS, TBP-2 mice showed a predisposition for death without any significant elevation of inflammatory cytokines, compared to the wild mice. LPS-challenged TBP-2 mice showed fat deposition in the liver and kidney, organ injuries, glycogen depletion, and elevation of serum lipid derivatives such as free fatty acids, triglyceride and cholesterol. Hyperinsulinemia and hypoglycemia were observed in TBP-2 mice after LPS injection. Death due to the LPS administration was prevented by supplementation of glucose. Phosphorylation of Akt and FoxO1, an inhibitory pathway of gluconeogenesis in the liver of LPS-challenged TBP-2 mice was demonstrated, suggesting the enhancement of insulin signaling. TBP-2 is involved in metabolic control during LPS-induced endotoxemia. After the LPS challenge, TBP-2 mice showed several characteristic aspects, such as hepatorenal injuries, and dysregulation of the lipid and glucose metabolisms. Furthermore, hypoglycemia promoted by hyperinsulinemia may be a critical risk factor for mortality in circumstances in which fatty acid utilization is impaired during endotoxemia.

Disruption of TBP-2 ameliorates insulin sensitivity and secretion without affecting obesity

Type 2 diabetes mellitus (T2DM) is characterized by defects in both insulin sensitivity and glucose-stimulated insulin secretion (GSIS) and is often accompanied by obesity. In this study, we show that disruption of thioredoxin binding protein-2 (TBP-2, also called Txnip) in obese mice (ob/ob) dramatically improves hyperglycaemia and glucose intolerance, without affecting obesity or adipocytokine concentrations. TBP-2-deficient ob/ob mice exhibited enhanced insulin sensitivity with activated insulin receptor substrate-1/Akt signalling in skeletal muscle and GSIS in islets compared with ob/ob mice. The elevation of uncoupling protein-2 (UCP-2) expression in ob/ob islets was downregulated by TBP-2 deficiency. TBP-2 overexpression suppressed glucose-induced adenosine triphosphate production, Ca2+ influx and GSIS. In β-cells, TBP-2 enhanced the expression level and transcriptional activity of UCP-2 by recruitment of peroxisome proliferator-activated receptor-γ co-activator-1α to the UCP-2 promoter. Thus, TBP-2 is a key regulatory molecule of both insulin sensitivity and GSIS in diabetes, raising the possibility that inhibition of TBP-2 may be a novel therapeutic approach for T2DM.

Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli

Large scale production of recombinant human flotillin-2 (reggie-1) is desirable for structural and biochemical studies. However, as the major lipid rafts specific hydrophobic protein, flotillin-2 was difficult to be expressed as soluble and functional form in prokaryotic system. In this study, we first cloned and expressed human flotillin-2 in Escherichia coli with five different fusion tags: poly-histidine, glutathione S-transferase (GST), thioredoxin (TRX), N-Utilization substance (NusA) and maltose binding protein (MBP). We screened the expression level and solubility of the five flotillin-2 fusion proteins, the best MBP tagged flotillin-2 was then large scale produced. The optimized purification procedure included two steps of chromatography: Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 36.0 mg soluble and functional recombinant flotillin-2 from 1 L of culture medium with purity above 97%. The activity of recombinant flotillin-2 was verified by pull-down assay with flotillin-1, showing that the purified recombinant flotillin-2 can specifically interact with flotillin-1. The circular dichroism (CD) spectroscopy showed that recombinant flotillin-2 had a very stable secondary structure dominated by α-helix, β-turn and random structure.

Anti-oxidative, anti-cancer and anti-inflammatory actions by thioredoxin 1 and thioredoxin-binding protein-2

Thioredoxin 1 (Trx 1) is a 12-kDa protein with redox-active dithiol in the active site –Cys–Gly–Pro–Cys– that is ubiquitously present in the human body. Trx 1 is a defensive protein induced by various stresses and has anti-oxidative, anti-apoptotic and anti-inflammatory effects. The anti-oxidative effect of Trx 1 is mediated by the dithiol–disulfide exchange in the active site. Trx 1 is able to interact with certain molecules, one of which is thioredoxin-binding protein-2 (TBP-2)/Vitamin D3 upregulated protein 1 (VDUP1)/thioredoxin interacting protein (TXNIP). TBP-2 was originally identified as a negative regulator of Trx 1 and acts as a cell growth suppressor and a regulator in lipid/glucose metabolism. Trx 1 and TBP-2 play crucial roles in pathophysiological mechanisms in metabolic disorders, cancer and inflammation. Here we discuss pharmacological aspects of Trx 1 and TBP-2 in these diseases and propose potential therapeutic approaches for intractable oxidative stress-related disorders.

Differential roles of Annexin A1 (ANXA1/lipocortin-1/lipomodulin) and thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP) in glucocorticoid signaling of HTLV-I-transformed T cells

Glucocorticoid (GC) is widely used for therapeutic purposes in immunological and hematological disorders. Annexin A1 (ANXA1/lipocortin-1/lipomodulin), a GC-inducible molecule, was regarded as a vital anti-inflammatory mediator of GC. Thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP), a regulator of redox reactions, cell growth and lipid metabolism, was also reportedly induced by GC. HTLV-I infected T cells undergo the transition from the IL-2 dependent to IL-2 independent growth during the long-term culture in vitro. We found that these T cells responded to GC with growth arrest and apoptosis in the IL-2 dependent growth stage, whereas they failed to respond to GC after their growth had shifted into the IL-2 independent stage. Here we employed these T cell lines and studied the roles of ANXA1 and TBP-2 in mediating GC-induced apoptosis. In GC-sensitive T cells, ANXA1 expression was negligible and unaffected by GC treatment, whereas TBP-2 was expressed and induced by GC treatment. In GC-resistant T cells, however, ANXA1 was highly expressed regardless of GC treatment and promoted cellular proliferation. In contrast, TBP-2 expression was lost and could not mediate the GC-induced apoptosis. In conclusion, these results suggest that TBP-2, but not ANXA1, is directly involved in the switching of GC sensitivity and GC resistance in HTLV-I infected T cell lines, whereas ANXA1 may be a biomarker indicative of the advanced stage of the transformation.

The roles of thioredoxin and thioredoxin-binding protein-2 in endometriosis

Oxidative stress is considered to be involved in the establishment and development of endometriosis. Thioredoxin (TRX) is an endogenous redox regulator that protects cells against oxidative stress, and TRX-binding protein-2 (TBP-2) is a negative regulator of TRX in the biological function and expression. The aim of this study was to investigate the roles of TRX and TBP-2 in the pathophysiology of endometriosis. A total of 35 patients with histologically confirmed endometriosis and 31 patients without endometriosis participated in this study. Real-time polymerase chain reaction was used to quantify TRX and TBP-2 mRNA levels, and immunohistochemistry (IHC) was used to assess TRX and TBP-2 protein localization in the endometrium. Serum and peritoneal fluid levels of TRX and TBP-2 were measured using a specific commercial ELISA. There were no significant differences in TRX mRNA levels in the endometrium of patients with endometriosis and the control groups. However, TBP-2 mRNA levels in the endometrium were lower, and the TRX to TBP-2 ratio was higher in patients with endometriosis than in the control group. In particular, the TRX to TBP-2 ratio was significantly higher during late secretory and menstrual phase in patients with endometriosis compared with the control group. IHC studies also showed the decreased TBP-2 immunoreactivity in patients with endometriosis compared with the control group. There was no correlation between TRX and TBP-2 mRNA levels in patients with endometriosis, whereas TRX mRNA levels were positively correlated with TBP-2 mRNA levels in the control group. There were no significant differences between the two groups in TRX and TBP-2 levels in serum or peritoneal fluid. Aberrant expression of TRX and TBP-2 in the endometrium may be associated with the establishment of endometriosis.

Thioredoxin Binding Protein-2 Inhibits Excessive Fetal Hypoglycemia During Maternal Starvation by Suppressing Insulin Secretion in Mice

Glucose is a major fuel for fetal development. Fetal blood glucose level is mainly dependent on maternal blood glucose concentration, though it is also regulated by fetal insulin level. Thioredoxin binding protein-2 (TBP-2), which is identical to vitamin D3 up-regulated protein (VDUP1) and thioredoxin interacting protein (Txnip), was recently reported to be a key transcriptional factor controlling glucose metabolism. Here, we elucidated the functions of TBP-2 in maintaining blood glucose homeostasis during the fetal period. TBP-2(+/-) female mice were mated with TBP-2(+/-) male mice; beginning 16.5-d post coitum, pregnant mice were fed or fasted for 24 h. Under conditions of maternal starvation, the blood glucose levels of TBP-2(-/-) fetuses were significantly lower than those of TBP-2(+/+) fetuses, corresponding to the elevated plasma insulin levels of TBP-2(-/-) fetuses compared with those of TBP-2(+/+) fetuses. There was no difference between TBP-2(+/+) and TBP-2(-/-) fetuses in terms of their pancreatic beta-cell masses or the expression of placental glucose transporters under conditions of either maternal feeding or fasting. Thus, during maternal fasting, fetal TBP-2 suppresses excessive insulin secretion to maintain the fetus's glucose levels, implying that TBP-2 is a critical molecule in mediating fetal glucose homeostasis depending on nutrient availability.

Involvement of ETS1 in thioredoxin-binding protein 2 transcription induced by a synthetic retinoid CD437 in human osteosarcoma cells

CD437, a synthetic retinoid, has a potent antitumor activity, in which an RAR-independent mechanism may be involved. Our previous study showed that CD437 transcriptionally upregulates the expression of thioredoxin-binding protein 2 (TBP2), leading to c-Jun N-terminal kinase 1 (JNK1)-mediated apoptosis. In the present study, we addressed the mechanism, by which CD437 induces TBP2 mRNA expression. CD437 efficiently caused the cell death of human osteosarcoma cells via apoptosis. CD437 also induced JNK1 activation through the upregulation of TBP2 mRNA, in consistent with our previous observation. A luciferase reporter assay for TBP2 promoter activation suggested that CD437-regulated TBP2 mRNA transcription requires the region between −400 and −300, which contains multiple possible ETS-binding sites. Finally, we demonstrated CD437-dependent recruitment of ETS1 transcription factor to this region by chromatin immunoprecipitation assay. These data suggest that ETS1 is involved in CD437-induced TBP2 mRNA expression in human osteosarcoma MG-63 cells.