Levels Of TXNIP Research Articles

Taohong Siwu Decoction Ameliorates Ischemic Stroke Injury Via Suppressing Pyroptosis.

Objective: Taohong Siwu decoction (THSWD) is one of the classic prescriptions for promoting blood circulation and removing blood stasis, and it has a good therapeutic effect on ischemic stroke. We sought to explore the therapeutic effects of THSWD on pyroptosis in rats with middle cerebral artery occlusion-reperfusion (MCAO/R). Methods: MCAO/R model of rats were established by suture-occluded method. MCAO/R rats were randomly divided into five groups, which were model group, nimodipine group, THSWD high, medium and low dose group (18, 9, and 4.5 g/kg, respectively), rats of sham group without thread embolus. All rats were treated by intragastric administration for 7 days. We detected the level of inflammatory factors. NLRP3 and Caspase-1 were detected by immunofluorescence. Western blot was used to detect NLRP3, Caspase-1, ASC, and GSDMD in penumbra. Also, the expression of TXNIP, HMGB1, toll-like receptors (TLR4), NF-κB, and MAPK were detected. Results: THSWD treatment improved the behavioral function and brain pathological damage. These results showed that the levels of TNF-α, TGF-β, IL-2, IL-6, IL-1β, and IL-18 were significantly reduced in THSWD treatment groups. THSWD could significantly decrease the expression levels of NLRP3, Caspase-1, Caspase-1 p10, ASC, TXNIP, GSDMD, HMGB1, TLR4/NFκB, p38 MAPK, and JNK in penumbra. Conclusion: Our results showed that THSWD could reduce the activation level of NLRP3 inflammatory corpuscle, down-regulate GSDMD, and inhibit pyroptosis in MCAO/R rats. These may be affected by inhibiting HMGB1/TLR4/NFκB, MAPK signaling pathways.

LncRNA-antisense non-coding RNA in the INK4 locus promotes pyroptosis via miR-497/thioredoxin-interacting protein axis in diabetic nephropathy

AimsDiabetic nephropathy (DN) is the most frequent complication of diabetes and causes millions of deaths each year. Finding novel therapy to DN is urgent, which requires a good understanding of the pathogenesis. Aims are to investigate the molecular mechanisms of DN by focusing on ANRIL/miR-497/TXNIP axis. Main methodsKidney tissues were collected from diagnosed DN patients. High glucose (HG) treatment of human renal tubular epithelial cell cells (HK-2) was used as the cell model of DN. qRT-PCR and Western blotting were performed to measure levels of ANRIL, miR-497, TXNIP, IL-1β, IL-18, caspase-1, and NLRP3. LDH leakage and cell viability were determined with commercial LDH activity kit and MTT assay. ELISA was employed to examine secreted IL-1β and IL-18 levels. Flow cytometry was used to examine caspase-1 activity. Dual luciferase assay was performed to validate interactions of ANRIL/miR-497 and miR-497/TXNIP. Key findingsANRIL and TXNIP were elevated in DN kidney tissues and HG-treated HK-2 cells while miR-497 was reduced. ANRIL bound miR-497 while miR-497 directly targeted TXNIP. Knockdown of ANRIL suppressed HG-induced LDH leakage, TXNIP/NLRP3/caspase-1 activation, and increases of IL-1β and IL-18 secreted levels. miR-497 knockdown or TXNIP overexpression reversed the effects of ANRIL knockdown on LDH leakage and pyroptosis-related signaling. miR-497 mimics inhibited caspase-1-dependent pyroptosis while co-overexpression of TXNIP blocked its effects in HG-treated HK-2 cells. SignificanceANRIL promotes pyroptosis and kidney injury in DN via acting as miR-497 sponge to disinhibit TXNIP expression. These results shed light on the mechanisms of DN and provide targets for therapy development.

Relationships between diabetic nephropathy and insulin resistance, inflammation, Trx, Txnip, CysC and serum complement levels.

To investigate the relationships between diabetic nephropathy (DN) and insulin resistance, inflammation, thioredoxin (Trx), thioredoxin-interacting protein (Txnip), Cystatin C (CysC) and serum complement levels. A total of 119 patients with type 2 diabetes mellitus (T2DM) treated in the Endocrinology Department of our hospital from January 2017 to December 2017 were enrolled as the experiment group, while 30 healthy volunteers were selected as the control group. The expression levels of inflammatory factors, Trx, Txnip, CysC and serum complements in every subject were detected. In addition, the type 2 diabetic nephropathy rat model was established via high-fat diet and injection of low-dose streptozotocin. Blood glucose, insulin resistance indexes and 24h-urinary albumin excretion were measured, and the histomorphological characteristics of the kidney in animals were observed. In clinical subjects, Trx level was notably lower in the simple DM group, early DN group and clinical DN group in comparison with that in the control group. The levels of Txnip and CysC in the simple DM group, early DN group and clinical DN group were remarkably higher than those in the control group. Moreover, the expression levels of TNF-α and IL-6 in the clinical DN group were significantly elevated compared with those in the simple DM group and early DN group. In addition, C1q expression in the clinical DN group was higher than that in the simple DM group and early DN group. In model rats, HOMA-IR was distinctly higher in the DM group and DN group than that in the control group. The ratio of kidney weight to body weight (KW/BW) was evidently higher in the DN group in comparison with that in the control group and DM group. Insulin resistance, inflammatory factors, and levels of Trx, Txnip, CysC and serum complement C1q are related to the progression of DM.

ITCH regulates oxidative stress induced by high glucose through thioredoxin interacting protein in cultured human lens epithelial cells.

Thioredoxin (Trx) is an important protein that controls oxidative damage in almost all eukaryotic cells. Trx interaction protein (Txnip) has been reported to negatively regulate the bioavailability of Trx and inhibit its biological function. The E3 ubiquitin ligase ITCH can specifically degrade Txnip via ubiquitination. The apoptosis of human lens epithelial cells (HLECs), which are highly sensitive to redox caused by oxidative stress, is a significant factor for the development of sugar cataract in a high-glucose environment. However, whether Trx, Txnip and ITCH contribute to the progression of sugar cataracts and the underlying mechanisms remain unknown, and thus, identifying these were the aims of the present study. The present results suggested that the expression levels of Trx, Txnip and ITCH in HLECs cultured with different glucose concentrations were detected by reverse transcription-quantitative PCR and western blotting, and the apoptotic rate of the cells was detected by flow cytometry and superoxide detection assay. The interaction between ITCH and Txnip was determined by co-localization immunofluorescence and co-immunoprecipitation. In addition, a vector and small interfering RNA of ITCH were transfected to overexpress and knockdown ITCH, respectively, to alter the expression of downstream proteins and cell apoptosis. It was found that Txnip was highly expressed in cultured HLECs in high-glucose environment, and the antioxidative function of Trx was restricted and suppressed, thus promoting apoptosis. The overexpression of ITCH increased the expression of Trx and decreased oxidative stress and apoptosis by decreasing Txnip in cultured HLECs, while downregulation of ITCH significantly decreased the expression of Trx and enhanced oxidative stress and apoptosis. Therefore, the present results indicated that ITCH could regulate the apoptosis of HLECs that were cultured in high-glucose concentration and that it may be a treatment target for sugar cataract.

Dimethyl fumarate ameliorates diabetes-associated vascular complications through ROS-TXNIP-NLRP3 inflammasome pathway

Vascular complications are a leading cause of morbidity and mortality among diabetic patients. This work aimed to investigate possible influences of dimethyl fumarate (DMF) on streptozotocin (STZ) diabetes-associated vascular complications in rats, exploring its potential to modulate ROS-TXNIP-NLRP3 inflammasome pathway.Two weeks after induction of diabetes (via a single injection of 50 mg/kg STZ, i.p.), diabetic rats were administered either DMF (25 mg/kg/day) or its vehicle for further eight weeks. Age-matched normal and DMF-administered non-diabetic rats served as controls.DMF treatment elicited a mild ameliorative effect on diabetic glycemia. DMF reduced serum TG and AGE levels and enhanced serum HDL-C concentrations in diabetic rats. Moreover, DMF significantly diminished aortic levels of ROS and MDA and restored aortic GSH, SOD and Nrf2 to near-normal levels in STZ rats. Aortic mRNA levels of TXNIP, NLRP3 and NF-κB p65 in diabetic rats were significantly reduced by DMF treatment. Serum and aortic protein levels of TXNIP and aortic contents of IL-1β, iNOS, NLRP3 and TGF-β1 were significantly lower in DMF-diabetic animals than non-treated diabetic rats. Furthermore, protein expression of TNF-α and caspase-3 in diabetic aortas was greatly attenuated by DMF administration. DMF enhanced eNOS mRNA and protein levels and increased bioavailable NO in diabetic aortas. Functionally, DMF attenuated contractile responses of diabetic aortic rings to KCl and phenylephrine and enhanced their relaxant responses to acetylcholine. DMF also mitigated diabetes-induced fibrous tissue proliferation in aortic tunica media.Collectively, these findings demonstrate that DMF offered vasculoprotective influences on diabetic aortas via attenuation of ROS-TXNIP-NLRP3 inflammasome pathway.

Bakuchiol Attenuates Oxidative Stress and Neuron Damage by Regulating Trx1/TXNIP and the Phosphorylation of AMPK After Subarachnoid Hemorrhage in Mice.

Subarachnoid hemorrhage (SAH) is a fatal cerebrovascular condition with complex pathophysiology that reduces brain perfusion and causes cerebral functional impairments. An increasing number of studies indicate that early brain injury (EBI), which occurs within the first 72 h of SAH, plays a crucial role in the poor prognosis of SAH. Bakuchiol (Bak) has been demonstrated to have multiorgan protective effects owing to its antioxidative and anti-inflammatory properties. The present study was designed to investigate the effects of Bak on EBI after SAH and its underlying mechanisms. In this study, 428 adult male C57BL/6J mice weighing 20 to 25 g were observed to investigate the effects of Bak administration in an SAH animal model. The neurological function and brain edema were assessed. Content of MDA/3-NT/8-OHdG/superoxide anion and the activity of SOD and GSH-Px were tested. The function of the blood-brain barrier (BBB) and the protein levels of claudin-5, occludin, zonula occludens-1, and matrix metalloproteinase-9 were observed. TUNEL staining and Fluoro-Jade C staining were conducted to evaluate the death of neurons. Ultrastructural changes of the neurons were observed under the transmission electron microscope. Finally, the roles of Trx, TXNIP, and AMPK in the protective effect of Bak were investigated. The data showed that Bak administration 1) increased the survival rate and alleviated neurological functional deficits; 2) alleviated BBB disruption and brain edema; 3) attenuated oxidative stress by reducing reactive oxygen species, MDA, 3-NT, 8-OHdG, gp91phox, and 4-HNE; increased the activities of SOD and GSH-Px; and alleviated the damage to the ultrastructure of mitochondria; 4) inhibited cellular apoptosis by regulating the protein levels of Bcl-2, Bax, and cleaved caspase-3; and 5) upregulated the protein levels of Trx1 as well as the phosphorylation of AMPK and downregulated the protein levels of TXNIP. Moreover, the protective effects of Bak were partially reversed by PX-12 and compound C. To summarize, Bak attenuates EBI after SAH by alleviating BBB disruption, oxidative stress, and apoptosis via regulating Trx1/TXNIP expression and the phosphorylation of AMPK. Its powerful protective effects might make Bak a promising novel drug for the treatment of EBI after SAH.

Curcumin relieves paraquat‑induced lung injury through inhibiting the thioredoxin interacting protein/NLR pyrin domain containing 3‑mediated inflammatory pathway.

When paraquat (PQ) enters the human body, it increases oxidative stress and inflammation, ultimately resulting in acute lung injury (ALI). Curcumin, a naturally occurring compound, has been reported to ameliorate PQ-induced ALI; however, the underlying molecular mechanisms remain unclear. In the present study, normal lung fibroblasts (WI-38VA13) were treated with 10 µmol/l PQ for 48 h, followed by a further 48 h incubation with 300 µmol/l curcumin. Cells were then harvested to determine their viability. Flow cytometry was performed to analyze the levels of reactive oxygen species (ROS) and the rate of apoptosis. The levels of apoptotic proteins and activation of the thioredoxin interacting protein/NLR pyrin domain containing 3 (TXNIP/NLRP3) axis were measured via reverse transcription-quantitative polymerase chain reaction and western blot analyses. Proinflammatory cytokine levels were examined using enzyme-linked immunosorbent assays. Finally, the expression levels of Notch1, extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated-ERK1/2 were evaluated via western blotting. Following treatment with curcumin, PQ-induced increases in ROS levels and apoptosis were significantly attenuated, and Bcl-2 expression levels were upregulated, whereas those of Bax were downregulated. It was also observed that curcumin treatment downregulated the expression levels of TXNIP, NLRP3, interleukin (IL)-1β and IL-18, and downstream caspase-1 compared with PQ treatment alone. Curcumin significantly attenuated the upregulation of Notch1 without affecting ERK1/2 phosphorylation. The present findings suggested that the inhibitory effects of curcumin on TXINP1 may inhibit activation of the NLRP3 inflammasome, subsequently suppressing the upregulation of proinflammatory cytokines and ultimately improving PQ-induced ALI.

Role of TXNIP/NLRP3 in sepsis-induced myocardial dysfunction.

Myocardial injury is one of the main symptoms of sepsis. However, the mechanisms underlying sepsis-induced myocardial dysfunction remain unclear. In the present study, the concentration of cardiac troponin T (CTnT) in serum was measured using an enzyme-linked immunosorbent assay kit. The levels of interleukin (IL)-1β and IL-18 were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and the level of malondialdehyde (MDA) was determined using a corresponding kit. Myocardial pathology was analyzed via hematoxylin and eosin staining. RT-qPCR analysis and western blotting and/or immunohistochemistry were used to quantify the expression levels of thioredoxin-interacting protein (TNXIP), NOD-like receptor pyrin domain containing 3 (NLRP3), cleaved caspase-1, caspase-1, catalase and manganese-superoxide dismutase (MnSOD). The viability of cells was determined using a cell counting kit-8. Apoptosis and reactive oxygen species (ROS) were examined using flow cytometry. Models of sepsis-induced myocardial injury were successfully established; evidence included increases in the levels of CTnT, IL-1β, IL-18 and MDA and myocardial tissue damage in vivo, and decreased cell viability and improvements in IL-1β and IL-18 in vitro. The levels of TXNIP, NLRP3 and cleaved caspase-1 were upregulated in the sepsis models. Small interfering RNA targeting TNXNIP (siTXNIP) increased cell viability, reduced the apoptotic rate and attenuated the release of IL-1β and IL-18. The levels of TXNIP, NLRP3 and cleaved caspase-1 and production of ROS were suppressed by siTXNIP, accompanied by increases in catalase and MnSOD. TXNIP/NLRP3 serves an important role in the development of sepsis-induced myocardial damage.

Effects of saxagliptin on adipose tissue inflammation and vascular function in overweight and obese people: a placebo-controlled study.

To test the effect of the dipeptidyl peptidase-4 inhibitor saxagliptin on adipose tissue inflammation and microvascular function, and whole-body postprandial endothelial function. A randomized, double-blind, placebo-controlled, parallel study was conducted between June 2013 and November 2016 in 44 overweight or obese people without diabetes (saxagliptin, n=28; placebo, n=16). Subcutaneous abdominal adipose tissue biopsies, a 4-h fat-enriched meal test and peripheral arterial tonometry for measurement of endothelial function were performed at baseline and after 6 weeks of treatment with saxagliptin (5 mg/day) or matching placebo. Forty participants were analysed (saxagliptin, n=26; placebo, n=14). Secretion of interleukin-8 from adipose tissue explants was reduced after saxagliptin (median fold-change from baseline: 0.8 saxagliptin vs 3.3 placebo; P=0.02). Adipose tissue expression of thioredoxin-inhibitory protein (TxNIP) was lower after saxagliptin (0.75 vs 1.0; P=0.02), while there were no significant differences in adipose tissue secretion of interleukin-1b, interleukin-6 or macrophage chemoattractant protein 1 (MCP-1), adipose tissue macrophage content, adipose tissue mRNA levels of mcp1, cd36, cd68, il6, il8, txnip and adpq, and activation of adipose tissue inflammatory pathways [extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF- κB)] or insulin-induced vasodilation of adipose tissue arterioles. Postprandial plasma glucose was slightly lower (by an estimated 0.3 mmol/l; P=0.01), while postprandial insulin, triglyceride levels and endothelial function were unchanged after saxagliptin. The effect of saxagliptin on adipose tissue inflammation was relatively modest, with many inflammatory markers unchanged. We also found no evidence that saxagliptin therapy improved adipose tissue arteriole vasodilation or postprandial endothelial function.

TXNIP knockdown alleviates hepatocyte ischemia reperfusion injury through preventing p38/JNK pathway activation

Hepatic ischemia and reperfusion (I/R) injury is a major cause of liver damage during liver transplantation, resection surgery, shock, and trauma. It has been reported that TXNIP expression was upregulated in a rat model of hepatic I/R injury. However, the role of TXNIP in the hepatic I/R injury is little known. In our study, we investigated the biological role of TXNIP and its potential molecular mechanism in the human hepatic cell line (HL7702 cells). Using oxygen-glucose deprivation and reoxygenation (OGD/R) to create a cell model of hepatic I/R injury, we found that the mRNA and protein expression levels of TXNIP were upregulated in HL7702 cells exposed to OGD/R. TXNIP overexpression remarkably promoted OGD/R-induced cell apoptosis and lactate dehydrogenase (LDH) release, both of which were significantly decreased by TXNIP knockdown. The production of malondialdehyde (MDA) was also increased by TXNIP overexpression, but was reduced by TXNIP knockdown. Moreover, TXNIP overexpression significantly upregulated the phosphorylation of p38 and JNK, which was remarkably inhibited by TXNIP knockdown. Additionally, p38-specific inhibitor SB203580 abrogated the effect of TXNIP overexpression on OGD/R-induced cell injury. Taken together, these results indicated that TXNIP knockdown alleviated hepatocyte I/R injury through preventing p38/JNK pathway activation. Thus, TXNIP might offer a novel potential therapeutic target for the treatment of hepatic I/R injury.

Perilla aldehyde attenuates CUMS-induced depressive-like behaviors via regulating TXNIP/TRX/NLRP3 pathway in rats

BackgroundCurrent evidence supports that inflammatory reaction in the hippocampus is a potential cause of major depressive disorder (MDD). Perilla aldehyde (PAH), a major constituent from Perilla frutescens, has been reported to have anti-inflammatory and anti-oxidant activity. The aim of this study is to explore the antidepressant-like effect and the underlying mechanism of PAH on the rats model induced by chronic unpredictable mild stress (CUMS). MethodsCUMS rats model was built to tested their depressive-like behaviors. The levels of pro-inflammatory cytokines were tested. Proteins were analyzed by Western blot and Immunohistochemistry. ResultsWe found that treatment with PAH (20, 40 mg/kg) and fluoxetine (FLU, 10 mg/kg) significantly improved the sucrose consumption, immobility time in forced swim test (FST), as well as locomotor activity in open-field test (OFT). The levels of pro-inflammatory cytokines in hippocampus were also suppressed effectively by PAH and FLU administration. Western blot analysis showed the up-regulated levels of TXNIP, NLRP3, Cleaved caspase-1 and p-NF-κB p65 in the hippocampus in rats exposed to CUMS paradigm, while different degrees of down-regulation in their expression were detected after PAH (20, 40 mg/kg) and FLU (10 mg/kg) treatment respectively. The results from histopathological examination further demonstrated that PAH (20, 40 mg/kg) and FLU (10 mg/kg) treatment reversed the alteration of TRX, NLRP3 and Cleaved caspase-1 induced by CUMS procedure. ConclusionsOur results demonstrated that PAH exhibited antidepressant-like effect in CUMS-induced rats model of depression, which might be mediated by TXNIP/TRX/NLRP3 pathway.

Pro-inflammatory mechanism of lectin from Pinellia pedatisecta on macrophage

To investigate the mechanism of lectin from Pinellia pedatisecta(PPL) on macrophage-induced inflammation and its association with inflammatory corpuscles NLRP3. Lectin from P. pedatisecta was isolated and purified by gel chromatography, and its purity was analyzed by using SDS-PAGE gel electrophoresis. ELISA was used to investigate the effect of PPL on inflammatory cytokines released by macrophages, with IL-1β as indicators;and fluorescence probe DCFH-DA fluorometer was used to determine changes in active oxygen ROS of macrophages after application of lectin from P. pedatisecta.RAW264.7 cells were pre-treated with ROS inhibitor N-acetylcysteine (NAC) to investigate the effect on ROS and the release of inflammatory factor IL-1β from macrophages to research the relationship between them. The protein levels of NLRP3, Caspase-1 p20, ASC and TXNIP were determined by Western blot.The results showed that isolated and purified PPL could reach electrophoretic purity; PPL stimulated macrophages and induced the excessive release of ROS, leading to strong oxidative stress reaction, and the levels of intracellular inflammatory factorsIL-1β were significantly increased. NAC could inhibit PPL-induced ROS excessive production and significantly reduce the release of IL-1β. In addition, PPL could induce the increase in protein expression levels of Caspase-1 p20, NLRP3 and ASC, and significantly reduce TXNIP expression. The results showed that PPL could cause a strong oxidative stress response by stimulating macrophages, activate inflammatory corpuscles NLRP3, and result in large amount of IL-1β release. That is, PPL could lead to inflammatory cascade reaction by promoting the maturation and secretion of IL-1β through ROS-TXNIP-NLRP3-IL-1β signaling pathway.

TXNIP/TRX/NF-κB and MAPK/NF-κB pathways involved in the cardiotoxicity induced by Venenum Bufonis in rats.

Venenum Bufonis (VB) is a widely used traditional medicine with serious cardiotoxic effects. The inflammatory response has been studied to clarify the mechanism of the cardiotoxicity induced by VB for the first time. In the present study, Sprague Dawley (SD) rats, were administered VB (100, 200, and 400 mg/kg) intragastrically, experienced disturbed ECGs (lowered heart rate and elevated ST-segment), increased levels of serum indicators (creatine kinase (CK), creatine kinase isoenzyme-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST)) and serum interleukin (IL-6, IL-1β, TNF-α) at 2 h, 4 h, 6 h, 8 h, 24 h, and 48 h, which reflected that an inflammatory response, together with cardiotoxicity, were involved in VB-treated rats. In addition, the elevated serum level of MDA and the down-regulated SOD, CAT, GSH, and GPx levels indicated the appearance of oxidative stress in the VB-treated group. Furthermore, based on the enhanced expression levels of TXNIP, p-NF-κBp65, p-IκBα, p-IKKα, p-IKKβ, p-ERK, p-JNK, and p-P38 and the obvious myocardial degeneration, it is proposed that VB-induced cardiotoxicity may promote an inflammatory response through the TXNIP/TRX/NF-κB and MAPK/NF-κB pathways. The observed inflammatory mechanism induced by VB may provide a theoretical reference for the toxic effects and clinical application of VB.

Genome-Wide shRNA Screen Implicates Mitogen Activated Protein Kinase (MAPK) Pathway In Glucocorticoid Resistance In Acute Lymphoblastic Leukemia

Resistance to glucocorticoids (GC) is a hallmark of relapsed acute lymphoblastic leukemia (ALL) and is a predictor of outcome at diagnosis. In spite of the importance of GC in the treatment of ALL and other hematological malignancies the molecular mechanisms that lead to effective eradication of leukemic cells is incompletely understood. To address this problem we have performed a functional screen for genes involved in prednisolone resistance in ALL cell line (Reh) and correlated these results with our previously published results using an integrated genomic analysis to discover genes (pathways) altered at relapse (Hogan et al 2011).Cells were infected with a pooled whole genome shRNA library that contained approximately 80,000 shRNAs targeting 18,000 genes. Deep sequencing was used to identify shRNAs enriched or depleted upon treatment with prednisolone. Three computational methods including; bioinformatics for next generation sequencing analysis (BiNGS), redundancy & fold change analysis (RFC) and strict standardized mean difference (SSMD) were applied to the sequencing data in efforts to obtain the most robust set of candidate genes for validation (Porter et. al, Leukemia 2012, Zhang XD, J Biomol Screen, 2007).Through our primary screen a total of 263 genes were identified to modulate prednisolone sensitivity in ALL. Upon knockdown, 142 genes increased the sensitivity of the cells to prednisolone and 121 genes increased resistance to prednisolone. Five genes overlapped with genes previously identified to be altered at relapse compared to matched diagnosis samples including SLC6A18, AARSD1, MIER3, CDC42BPB, and YAP1. We hypothesize that genes that are altered at relapse in ALL and identified through functional genomics screening to modulate chemosensitivity in vitro, are likely drivers of chemoresistance and eventual relapse.We also performed gene ontology (GO) analysis using DAVID Bioinformatics to identify pathways that may be responsible for altered resistance to prednisolone. This analysis strongly implicated the mitogen-activated kinase (MAPK) pathway. The MAPK was also identified as a pathway with increased activity at relapse through our integrative genomics analysis(Hogan et al 2011). One gene of particular interest was MAP2K4 which encodes for MEK4, an upstream kinase involved in JNK phosphorylation and c-Jun activation. Knockdown of MAP2K4 by shRNA in B-precursor ALL cell lines (Reh and RS4;11) results in statistically significant (p-value < 0.5) increased sensitivity to prednisolone induced apoptosis at a range of prednisolone concentrations but not to other chemotherapy tested (etoposide, doxorubicin, and 6-thioguanine).604. Molecular Pharmacology, Drug Resistance: Poster I Upon treatment with prednisolone MAP2K4 knockdown cells have increased levels of prednisolone responsive genes GILZ (1.4-2.4 fold in Reh, 2.1-3.8 fold in RS4;11) and TXNIP (1.8-5.7 fold in Reh, 2.5-2.6 in RS4;11). Increased sensitivity to prednisolone and increased levels of prednisolone responsive genes was associated with decreased levels of p-JNK that has been previously implicated in regulating glucocorticoid signaling through phosphorylation of the glucocorticoid receptor (GR) at S226 (Roatsky et. al, PNAS 1998, Itoh et. al, Mol. Endo. 2002).s Overall this data suggests that decreased levels of MAP2K4 results in increased sensitivity to GC by increasing GC signaling and implicates MEK4 as novel drug target in ALL. Disclosures:No relevant conflicts of interest to declare.

Thioredoxin Interacting Protein Is a Potential Regulator of Glucose and Energy Homeostasis in Endogenous Cushing's Syndrome

Recent studies have described bone as an endocrine organ regulating glucose metabolism, with insulin signaling regulating osteocalcin secretion and osteocalcin regulating β cell function. We have previously demonstrated increased bone expression of TXNIP in patients with endogenous Cushing's syndrome (CS), and we hypothesized that TXNIP could contribute to the dysregulated glucose metabolism in CS. We studied 33 CS patients and 29 matched controls, with bone biopsies from nine patients, before and after surgical treatment. In vitro, the effect of silencing TXNIP (siTXNIP) in osteoblasts, including its effect on human islet cells, was examined. Our major findings were: (i) The high mRNA levels of TXNIP in bone from CS patients were significantly associated with high levels of glucose and insulin, increased insulin resistance, and decreased insulin sensitivity in these patients. (ii) Silencing TXNIP in osteoblasts enhanced their OC response to insulin and glucose and down-regulated interleukin (IL)-8 levels in these cells. (iii) Conditional media from siTXNIP-treated osteoblasts promoted insulin content and anti-inflammatory responses in human islet cells. We recently demonstrated that the thioredoxin/TXNIP axis may mediate some detrimental effects of glucocorticoid excess on bone tissue in CS. Here we show that alterations in this axis also may affect glucose metabolism in these patients.

New causal relationship between PLA2g6, store‐operated Ca2+ entry, refilling of Ca2+ stores and ER stress in mouse embryonic fibroblasts.

Store‐operated Ca2+ entry (SOCE) is essential for maintenance and timely refilling of Ca2+ stores in the endoplasmic reticulum (ER). Here we used molecular, imaging and genetic approaches to unmask new determinants of this process and their role in ER stress and the unfolded protein response (UPR). Analysis of endogenous SOCE in primary embryonic fibroblasts (MEF) from a new knockout mouse model revealed that constitutive genetic impairment of interaction of specific plasma membrane‐associated phospholipase A2 (PLA2g6) with STIM1 (ER‐resident Ca2+ sensor) leads to impairment of endogenous SOCE. Such deficiency resulted in sustained depletion of intracellular Ca2+ stores, and more than 50% reduction in ionomycin‐induced Ca2+ release from the stores. We found that PLA2g6 and SOCE‐deficient MEF cells experience significant ER stress: qRT‐PCR analysis of ER stress markers showed a 2–4 fold increase in mRNA expression levels of BiP, CHOP, and TXNIP. Discovery of a new causal relationship between PLA2g6, SOCE, filling state of ER Ca2+ stores and the UPR unveiled a novel mechanism of ER stress, and identified new molecular targets that can be used for prevention of ER stress‐related pathologies.

Lactic Acidosis Triggers Starvation Response with Paradoxical Induction of TXNIP through MondoA

Although lactic acidosis is a prominent feature of solid tumors, we still have limited understanding of the mechanisms by which lactic acidosis influences metabolic phenotypes of cancer cells. We compared global transcriptional responses of breast cancer cells in response to three distinct tumor microenvironmental stresses: lactic acidosis, glucose deprivation, and hypoxia. We found that lactic acidosis and glucose deprivation trigger highly similar transcriptional responses, each inducing features of starvation response. In contrast to their comparable effects on gene expression, lactic acidosis and glucose deprivation have opposing effects on glucose uptake. This divergence of metabolic responses in the context of highly similar transcriptional responses allows the identification of a small subset of genes that are regulated in opposite directions by these two conditions. Among these selected genes, TXNIP and its paralogue ARRDC4 are both induced under lactic acidosis and repressed with glucose deprivation. This induction of TXNIP under lactic acidosis is caused by the activation of the glucose-sensing helix-loop-helix transcriptional complex MondoA:Mlx, which is usually triggered upon glucose exposure. Therefore, the upregulation of TXNIP significantly contributes to inhibition of tumor glycolytic phenotypes under lactic acidosis. Expression levels of TXNIP and ARRDC4 in human cancers are also highly correlated with predicted lactic acidosis pathway activities and associated with favorable clinical outcomes. Lactic acidosis triggers features of starvation response while activating the glucose-sensing MondoA-TXNIP pathways and contributing to the “anti-Warburg” metabolic effects and anti-tumor properties of cancer cells. These results stem from integrative analysis of transcriptome and metabolic response data under various tumor microenvironmental stresses and open new paths to explore how these stresses influence phenotypic and metabolic adaptations in human cancers.

Abstract 1027: Thioredoxin-interacting protein-reactive oxygen species (TXNIP/ROS) relationship defines a stress response gene signature in patients with CLL

Abstract Thioredoxin-interacting protein (TXNIP) binds and inactivates thioredoxin (TRX) leading to increased reactive oxygen species (ROS) production and apoptosis in many cell types. TXNIP expression has never been evaluated in CLL. Since it has been previously shown that patients with CLL have high levels of ROS compared to normal individuals and that TRX has a protective function in CLL, our goal was to measure the levels of TXNIP and determine whether there is any correlation with ROS. Furthermore, we also studied whether this correlation was associated with any particular stress response gene signature. Venous blood samples were obtained from consecutive patients with definite diagnosis of CLL during routine clinic visits over a 12-week period. Patients were selected independently from demographics, stage of disease or prior treatment. Seventeen patient samples were analyzed for this study, 8 under observation, 6 receiving fludarabine-based regimen and 3 receiving other regimens. Gene expression levels were measured by semiquantitative PCR and expressed as relative intensity. ROS was measured by DCFDA fluorescence and expressed as mean fluorescence. TRX in plasma was assessed by ELISA. Statistical analysis included student t-test (p values) and Pearson's correlation, (ρ values), with ρ ≥ 0.7 considered high positive correlation and ρ ≤ −0.7 strong inverse correlation. TXNIP levels (avg. 0.82 ± 0.18, range 0.45-1.15) and ROS (avg. 7967 ± 2515, range 2340-12340) were found to be highly positively correlated (ρ = 0.92). Neither TRX message nor protein in serum correlated with TXNIP or ROS. TXNIP expression was generally higher in our observation group than treated (0.89 ± 0.18 vs 0.76 ± 0.15) although not statistically significant. Four samples with highest levels of TXNIP (3 obs, 1 treat; avg TXNIP 1.06 ± 0.06) and 4 with lowest TXNIP (3 treat, 1 obs; avg TXNIP 0.59 ± 0.1) were further evaluated by using a stress response PCR assay. We found 50 genes with differing expression pattern between the groups. We chose BAX, GSK3-B and Bcxl to further analyze in all samples. Whereas BAX, Gsk3-B and Bclx all showed an inverse correlation with TXNIP expression (BAX: ρ = −0.67, GSK3-B ρ = −0.66, Bclx: ρ = −0.64), Bcl-2 showed no correlation. We then grouped these genes into treated vs observation for further analysis. BAX (0.45 ± 0.18 vs 0.26 ± 0.06, p &amp;lt; 0.05), GSK-3B (0.55 ± 0.17 vs 0.38 ± 0.17, p &amp;lt; 0.05) and Bclx gene expression levels (0.46 ± 0.21 vs 0.27 ± 0.05, p &amp;lt; 0.03) were all significantly higher in the treated vs observation group only. In conclusion, we have measured the expression of TXNIP in 17 CLL patients and found that TXNIP expression significantly correlates with ROS in the circulating CLL cells. By analyzing genes that may be associated with TXNIP levels in these cells we have identified BAX, GSK-3B and Bclx as genes of interest in a platform for further analysis in patients with CLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1027.

Estradiol regulates the thioredoxin antioxidant system in the mouse uterus.

The rodent uterus responds to acute estradiol (E2) treatment with a series of well characterized physiological responses. In a recent screen for genes involved in this response, we found that several genes in the thioredoxin (Txn) pathway were rapidly modified after E2 treatment in the mouse uterus. Txn is a 12-kDa protein with multiple roles in the cell, including protection against oxidative stress and apoptosis, regulation of transcription factor activity, and regulation of cellular proliferation. Txn in combination with Txn reductase (Txnrd) and Txn-interacting protein (Txnip) constitute the mammalian Txn pathway. This pathway exists in multiple locations in the cell, including the cytosol and mitochondria. To analyze the levels of Txn, Txnrd, and Txnip in the uterus, we treated ovariectomized adult mice with a time course of E2 and analyzed mRNA levels by real-time PCR. E2 rapidly decreased the expression of Txnip, but increased the levels of cytosolic Txn1 and Txnrd1 as well as mitochondrial Txn2. Using the ER antagonist, ICI 182,780, and mice lacking functional estrogen receptor alpha (ERalpha), we demonstrate that these E2-mediated changes require ERalpha, but not ERbeta. The repression of Txnip by E2 was also demonstrated in vitro in MCF-7 human breast cancer cells. This repression was blocked by treatment with the histone deacetylase inhibitor, trichostatin A, suggesting that repression by E2 may involve regulation of histone acetylation. We conclude that the rapid E2-mediated activation of the Txn pathway is an important step in the response of the mammalian uterus to estrogen.