Thioredoxin-interacting Protein Protein Research Articles

AMPK-Dependent Degradation of TXNIP upon Energy Stress Leads to Enhanced Glucose Uptake via GLUT1

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that is induced in response to glucose elevation. It has been shown to provide a negative feedback loop to regulate glucose uptake into cells, though the biochemical mechanism of action has been obscure. Here, we report that TXNIP suppresses glucose uptake directly, by binding tothe glucose transporter GLUT1 and inducing GLUT1 internalization through clathrin-coated pits, as well as indirectly, by reducing the level of GLUT1 messenger RNA (mRNA). In addition, we show that energy stress results in the phosphorylation of TXNIP by AMP-dependent protein kinase (AMPK), leading to its rapid degradation. This suppression of TXNIP results in an acute increase in GLUT1 function and an increase in GLUT1 mRNA (hence the total protein levels) for long-term adaptation. The glucose influx through GLUT1 restores ATP-to-ADP ratios in the short run and ultimately induces TXNIP protein production to suppress glucose uptake once energy homeostasis is reestablished.

IRE1α Induces Thioredoxin-Interacting Protein to Activate the NLRP3 Inflammasome and Promote Programmed Cell Death under Irremediable ER Stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated tocause programmed cell death. We discovered thatthioredoxin-interacting protein (TXNIP) isa critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small moleculeIRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.

Redox sensitive regulation of thioredoxin interacting protein (TxnIP) gene expressions in mouse vascular smooth muscle cells

Since nitric oxide (NO) is a redox-sensitive signaling molecule, reactive oxygen species (ROS) level influences the efficiency of NO-related signaling. However, much of the knowledge has not been accumulated on how NO regulates ROS level. Here, we take the thioredoxin (Trx) system as a model of regulatory mechanism of ROS by NO. The functional availability of Trx is strictly regulated through the binding with thioredoxin interacting protein (TxnIP); TxnIP is a known regulatory factor involved in atherosclerosis and vascular endothelial cell functions. Hence, we studied effects of NO on TxnIP in mouse vascular smooth muscle cells (VSMC), where NO from vascular endothelial cells works as an intercellular signal in various degree. Following a treatment with S-nitrosoglutathione (GSNO), TxnIP mRNA level analyzed by real-time PCR method in mouse VSMC decreased 1/6 to the control (untreated) within an hour. The decrease was then followed by an increase 5-fold over control within 4–5 h. This bi-phasic response of TxnIP mRNA to GSNO was triggered by the consumption of GSNO in cells. Interestingly, the induction of TxnIP mRNA was not observed under the anaerobic condition. TxnIP proteins decreased in parallel with mRNA change. Thereby, GSNO-treated cells exhibited higher resistance to H 2 O 2 exposure (up to 30 μM) than control cells. It is probably resulting from a complementary increase of Trx and activation of peroxidase activity coupled with Trx. These results suggest NO suppresses ROS levels through the inhibition of TxnIP, which may extend the effect of NO.

Vitamin D receptor activation in a diabetic-like environment: Potential role in the activity of the endothelial pro-inflammatory and thioredoxin pathways

High blood and tissue concentrations of glucose and advanced glycation end products (AGEs) are thought to play an important role in the development of diabetic vascular complications. Thioredoxin interacting protein (TXNIP) is up-regulated in response to high levels of glucose and is an endogenous inhibitor of thioredoxin (TRX), and may play a contributory role in the occurrence of diabetic-related vascular diseases. Vitamin D inhibits endothelial proliferation and is a cardiovascular protective agent. The present study evaluated the impact of paricalcitol and calcitriol on the endothelial inflammatory and TXNIP pathways in cultured endothelial cells exposed to a diabetic-like environment. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated for 24h with 200μg/ml AGE-HSA and 250mg/dl glucose concentrations, with paricalcitol or calcitriol. IL6, IL8, NFκB (p50/p65), receptor of AGE (RAGE), TXNIP, and TRX expressions were evaluated at the levels of mRNA, protein, and TRX activity. Calcitriol and paricalcitol significantly down-regulated the markers involved in the inflammatory responses. Only paricalcitol induced a significant decrease in TXNIP mRNA and protein expressions. Neither paricalcitol nor calcitriol affected TRX reductase activity or TRX mRNA and protein expressions. Our findings indicate that in an endothelial diabetic-like environment, paricalcitol and calcitriol significantly decreased the expression of genes involved in the inflammatory pathway. In this in vitro study, it seems that the TRX antioxidant system was not involved. The different effects found between paricalcitol and calcitriol might reflect the selectivity of vitamin D receptor (VDR) activation.

Rare sugar d-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells

Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function.

Thioredoxin and thioredoxin-interacting protein as prognostic markers for gastric cancer recurrence

To evaluate the potential of thioredoxin (TXN) and thioredoxin-interacting protein (TXNIP) expression as biomarkers for predicting gastric cancer recurrence. TXN and TXNIP expression levels were acquired from gene expression microarray data for 65 human gastric cancer tissues. We determined whether each gene expression level was associated with cancer recurrence and investigated the relationship between the two genes. For validation, the expression levels of TXN and TXNIP were measured by quantitative real-time reverse transcription polymerase chain reaction in 68 independent stage III gastric cancer patients. The correlation between gene expression and cancer prognosis was evaluated. Immunohistochemical staining was performed to investigate the protein expression levels of TXN and TXNIP and to characterize the expression patterns of each protein. TXN was a prognosis-related gene (P = 0.009), whereas TXNIP, a TXN inhibitor, demonstrated a negative correlation with TXN in the gene expression microarray data. In the 68 stage III patients, the expression levels of both TXN and TXNIP had a statistically significant effect on recurrence-free survival (RFS, P = 0.008 and P = 0.036, respectively). The low TXN and high TXNIP expression group exhibited a better prognosis than the other groups, and the high TXN and low TXNIP expression group exhibited a poorer prognosis (P < 0.001 for RFS and P = 0.001 for overall survival). More than half of the patients in the simultaneously high TXN and low TXNIP expression group experienced a recurrence within 1 year after curative surgery, and the 5-year survival rate of the patients in this group was 29%, compared with 89% in the low TXN and high TXNIP expression group. The TXN protein was overexpressed in 65% of the gastric cancer tissues, whereas the TXNIP protein was underexpressed in 85% of the cancer cells. In a correlation analysis, TXN and TXNIP were highly correlated with many oncogenes and tumor suppressors as well as with genes related to energy, protein synthesis and autophagy. TXN and TXNIP are promising prognostic markers for gastric cancer, and performing personalized adjuvant treatment based on TXN and TXNIP expression levels would be an effective practice in the treatment of gastric cancer.

The gene encoding thioredoxin-interacting protein (TXNIP) is a frequent virus integration site in virus-induced mouse leukemia and is overexpressed in a subset of AML patients

Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML.

Diabetes induces and calcium channel blockers prevent cardiac expression of proapoptotic thioredoxin-interacting protein

Cardiomyocyte apoptosis is a critical process in the pathogenesis of ischemic and diabetic cardiomyopathy, but the mechanisms are not fully understood. Thioredoxin-interacting protein (TXNIP) has recently been shown to have deleterious effects in the cardiovascular system and we therefore investigated whether it may also play a role in diabetes-associated cardiomyocyte apoptosis. In fact, TXNIP expression was increased in H9C2 cardiomyocytes incubated at high glucose, and cardiac expression of TXNIP and cleaved caspase-3 were also elevated in vivo in streptozotocin- and obesity-induced diabetic mice. Together, these findings not only suggest that TXNIP is involved in diabetic cardiomyopathy but also that it may represent a novel therapeutic target. Surprisingly, testing putative TXNIP modulators revealed that calcium channel blockers reduce cardiomyocyte TXNIP transcription and protein levels in a dose-dependent manner. Oral administration of verapamil for 3 wk also reduced cardiac TXNIP expression in mice even in the face of severe diabetes, and these reduced TXNIP levels were associated with decreased apoptosis. To determine whether lack of TXNIP can mimic the verapamil-induced decrease in apoptosis, we used TXNIP-deficient HcB-19 mice, harboring a natural nonsense mutation in the TXNIP gene. Interestingly, we found significantly reduced cleaved caspase-3 levels in HcB-19 hearts, suggesting that TXNIP plays a critical role in cardiac apoptosis and that the verapamil effects were mediated by TXNIP reduction. Thus our results suggest that TXNIP reduction is a powerful target to enhance cardiomyocyte survival and that agents such as calcium channel blockers may be useful in trying to achieve this goal and prevent diabetic cardiomyopathy.

Insulin counteracts glucotoxic effects by suppressing thioredoxin-interacting protein production in INS-1E beta cells and in Psammomys obesus pancreatic islets

In type 2 diabetes, glucose toxicity leads to beta cell apoptosis with decreased beta cell mass as a consequence. Thioredoxin-interacting protein (TXNIP) is a critical mediator of glucose-induced beta cell apoptosis. Since hyperglycaemia leads to elevated serum insulin, we hypothesised that insulin is involved in the regulation of TXNIP protein levels in beta cells. We studied the production of TXNIP in INS-1E beta cells and in islets of Psammomys obesus, an animal model of type 2 diabetes, in response to glucose and different modulators of insulin secretion. TXNIP production was markedly augmented in islets from diabetic P. obesus and in beta cells exposed to high glucose concentration. In contrast, adding insulin to the culture medium or stimulating insulin secretion with different secretagogues suppressed TXNIP. Inhibition of glucose and fatty acid-stimulated insulin secretion with diazoxide increased TXNIP production in beta cells. Nitric oxide (NO), a repressor of TXNIP, enhanced insulin signal transduction, whereas inhibition of NO synthase abolished its activation, suggesting that TXNIP inhibition by NO is mediated by stimulation of insulin signalling. Treatment of beta cells chronically exposed to high glucose with insulin reduced beta cell apoptosis. Txnip knockdown mimicking the effect of insulin prevented glucose-induced beta cell apoptosis. Insulin is a potent repressor of TXNIP, operating a negative feedback loop that restrains the stimulation of TXNIP by chronic hyperglycaemia. Repression of TXNIP by insulin is probably an important compensatory mechanism protecting beta cells from oxidative damage and apoptosis in type 2 diabetes.

Expression, Localization, and Function of the Thioredoxin System in Diabetic Nephropathy

Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site- and cell-specific manner.

Identification of Thioredoxin-Interacting Protein 1 as a Hypoxia-Inducible Factor 1α-Induced Gene in Pancreatic Cancer

To investigate the expression of thioredoxin-interacting protein (TXNIP) during hypoxia and its dependency on hypoxia-inducible factor 1alpha (HIF-1alpha) in pancreatic cancer cell lines. MiaPaCa-2 pancreatic cancer cells were transiently transfected with siRNA to HIF-1alpha and TXNIP protein measured after growth in normoxia or hypoxia. In addition, HIF-1alpha dependency was assessed by transiently transfecting MiaPaCa-2 pancreatic cancer cells with HIF-1alpha with a mutated oxygen degradation domain resulting in stable HIF-1alpha expression in normoxic conditions. Panc-1 pancreatic cancer cells with low endogenous TXNIP expression were stably transfected with TXNIP, and cell survival and response to platinum cancer agents were tested. Quantitative immunohistochemistry was utilized to measure the expression of TXNIP and thioredoxin 1 in human pancreatic cancer tissues. Thioredoxin-interacting protein was induced during hypoxia in pancreatic cancer cells in a HIF-1alpha-dependent manner. Overexpression of TXNIP in the Panc-1 cells resulted in a higher basal apoptosis and increased sensitivity to cisplatin and oxaliplatin. A negative correlation was observed between TXNIP and thioredoxin 1 expression in human pancreatic cancer tissues. Thioredoxin-interacting protein, a putative tumor suppressor gene, is induced in response to hypoxia in a HIF-1alpha-dependent manner in pancreatic cancer cells, resulting in increased apoptosis and increased sensitivity to platinum anticancer therapy. Increased TXNIP may be a mechanism to counterbalance the prosurvival effects of HIF-1alpha.

Thioredoxin-Interacting Protein

In diabetes, glucose toxicity affects different organ systems, including pancreatic islets where it leads to beta-cell apoptosis, but the mechanisms are not fully understood. Recently, we identified thioredoxin-interacting protein (TXNIP) as a proapoptotic beta-cell factor that is induced by glucose, raising the possibility that TXNIP may play a role in beta-cell glucose toxicity. To assess the effects of glucose on TXNIP expression and apoptosis and define the role of TXNIP, we used INS-1 beta-cells; primary mouse islets; obese, diabetic BTBR.ob mice; and a unique mouse model of TXNIP deficiency (HcB-19) that harbors a natural nonsense mutation in the TXNIP gene. Incubation of INS-1 cells at 25 mmol/l glucose for 24 h led to an 18-fold increase in TXNIP protein, as assessed by immunoblotting. This was accompanied by increased apoptosis, as demonstrated by a 12-fold induction of cleaved caspase-3. Overexpression of TXNIP revealed that TXNIP induces the intrinsic mitochondrial pathway of apoptosis. Islets of diabetic BTBR.ob mice also demonstrated increased TXNIP and apoptosis as did isolated wild-type islets incubated at high glucose. In contrast, TXNIP-deficient HcB-19 islets were protected against glucose-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and caspase-3, indicating that TXNIP is a required causal link between glucose toxicity and beta-cell death. These findings shed new light onto the molecular mechanisms of beta-cell glucose toxicity and apoptosis, demonstrate that TXNIP induction plays a critical role in this vicious cycle, and suggest that inhibition of TXNIP may represent a novel approach to reduce glucotoxic beta-cell loss.