Thioredoxin-interacting Protein Protein Levels Research Articles

Blockade of thromboxane A2 signaling attenuates ethanol-induced myocardial inflammatory response in mice.

Alcohol-associated cardiomyopathy (ACM) is a cardiac muscle disease characterized by inflammation and oxidative stress. Thromboxane-prostanoid receptor (TP-R) plays an important role in the pathogenesis of cardiovascular disease. Herein, we hypothesize that TP-R mediates alcohol-induced early cardiac injury. Eight-week-old male C57BL/6 wild-type mice were fed a chronic ethanol (ET) or control diet (CON) for 10 days followed by a single binge of ethanol or maltose-dextrin through oral gavage. A cohort of ethanol-fed mice received SQ 29,548 (SQ), a TP-R antagonist. RNA sequencing, real-time PCR, and western blot analysis were performed on left ventricle to investigate alterations in genes and/or proteins mediating oxidative stress, inflammation, and cardiac remodeling. Sirius Red staining was performed to measure myocardial fibrosis. RNA-sequencing analysis of myocardium from CON and ET groups identified 142 genes that were significantly altered between the two groups. In particular, the gene expression of thioredoxin-interacting protein (TXNIP), a component of NLR family pyrin domain containing 3 (NLRP3) signaling, which mediates oxidative stress and inflammatory response, was upregulated in response to ethanol exposure. The myocardial protein levels of TP-R and thromboxane A2 synthase were increased upon alcohol exposure. Ethanol increased the levels of 4-hydroxynonenal, a marker of oxidative stress, with a concomitant increase in the protein levels of TXNIP and NLRP3, and administration of SQ attenuated these effects. Additionally, ethanol increased the protein levels of pro-inflammatory mediators, including tumor necrosis factor alpha and the NLRP3 downstream product, secretory interleukin 1 beta, and SQ blunted these effects. Finally, the Sirius red staining of the myocardium revealed an increase in collagen deposition in ethanol-fed mice which was attenuated by TP-R antagonism. This study demonstrates that ethanol promotes the NLRP3 signaling pathway within the myocardium, leading to a pro-inflammatory milieu that potentially initiates early myocardial remodeling, and TP-R antagonism attenuates this effect.

Abstract P440: Antagonism of Thromboxane-Prostanoid Receptor Attenuates Alcohol-Induced Myocardial Inflammation

Heavy alcohol use is the leading etiology of non-ischemic dilated cardiomyopathy. Alcohol-Associated Cardiomyopathy (ACM) is a specific cardiac muscle disease which is characterized by inflammation, abnormal fatty acid metabolism, and oxidative stress. TP-R, a G-protein coupled receptor, is widely expressed in the myocardium, and plays a critical role in the pathogenesis of various cardiac diseases including hypertensive heart disease and dilated cardiomyopathy. TP-R is activated by thromboxane A2 (TXA2) and 8-isoprostane. Of note, clinical studies have shown that chronic alcohol use markedly increases the level of thromboxane B2, a stable metabolite of TXA2, in plasma of alcohol use disorder patients. However, the role of TP-R signaling in the pathogenesis of ACM remains unclear. Therefore, we hypothesize that TP-R signaling mediates adverse effects of alcohol on the myocardium. To test this hypothesis, we used the chronic plus binge ethanol feeding model. C57BL/6 wild-type male mice (8-week) were fed with Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by single binge of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8). Our data indicated that mouse myocardial protein levels of thromboxane A2 synthase (TBXAS1, P <0.001), an enzyme responsible for synthesis of TXA2, was increased in response to ethanol exposure. Concomitantly, ethanol-fed mice displayed upregulated myocardial protein levels of pro-inflammatory mediators including tumor necrosis factor alpha (TNF-α, P <0.001) and secreted interleukin 1 beta (IL1β, P <0.001) compared with their controls. These adverse alterations induced by the ethanol diet were ameliorated by the pharmacological inhibition of TP-R. Interestingly, RNA-sequencing and western blotting analysis showed that expression of thioredoxin-interacting protein (TXNIP) was upregulated in response to ethanol exposure ( P <0.001). Meanwhile, mice fed with ethanol diet had the increased myocardial protein level of NLR family pyrin domain containing 3 (NLRP3, P <0.01) compared with CON mice. These ethanol-induced increases in protein levels of TXNIP ( P <0.001) and NLRP3 ( P <0.01) were attenuated due to SQ 29,548 administration. Accordingly, these findings lead us to suggest that pharmaceutical inhibiting TP-R has protective effects on attenuating ethanol-induced myocardial inflammation by inhibiting TXNIP-NLRP3-IL1β axis.

Molecular Regulation of Transforming Growth Factor-β1-induced Thioredoxin-interacting Protein Ubiquitination and Proteasomal Degradation in Lung Fibroblasts: Implication in Pulmonary Fibrosis.

Thioredoxin-interacting protein (TXNIP) plays a critical role in regulation of cellular redox reactions and inflammatory responses by interacting with thioredoxin (TRX) or the inflammasome. The role of TXNIP in lung fibrosis and molecular regulation of its stability have not been well studied. Therefore, here we investigated the molecular regulation of TXNIP stability and its role in TGF-β1-mediated signaling in lung fibroblasts. TXNIP protein levels were significantly decreased in lung tissues from bleomycin-challenged mice. Overexpression of TXNIP attenuated transforming growth factor-β1 (TGF-β1)-induced phosphorylation of Smad2/3 and fibronectin expression in lung fibroblasts, suggesting that decrease in TXNIP may contribute to the pathogenesis of lung fibrosis. Further, we observed that TGF-β1 lowered TXNIP protein levels, while TXNIP mRNA levels were unaltered by TGF-β1 exposure. TGF-β1 induced TXNIP degradation via the ubiquitin-proteasome system. A serine residue mutant (TNXIP-S308A) was resistant to TGF-β1-induced degradation. Furthermore, downregulationof ubiquitin-specific protease-13 (USP13) promoted the TGF-β1-induced TXNIP ubiquitination and degradation. Mechanistic studies revealed that USP13 targeted and deubiquitinated TXNIP. The results of this study revealed that the decrease of TXNIP in lungs apparently contributes to the pathogenesis of pulmonary fibrosis and that USP13 can target TXNP for deubiquitination and regulate its stability.

Fluorinated curcumin derivative (Shiga-Y6) modulates the level of thioredoxin-interacting protein (TXNIP) in a mouse model of diabetes

Thioredoxin interacting protein (TXNIP) has emerged as a significant regulator of β-cell mass and loss, rendering it an attractive target for treating diabetes. We previously showed that Shiga-Y6, a fluorinated curcumin derivative, inhibited TXNIP mRNA and protein expression in vitro, raising the question of whether the same effect could be translated in vivo. Herein, we examined the effect of Shiga-Y6 on TNXIP levels and explored its therapeutic potential in a mouse model of diabetes, Akita mice. We intraperitoneally injected Shiga-Y6 (SY6; 30 mg/kg of body weight) or vehicle into 8-week-old Akita mice for 28 consecutive days. On day 29, the mice were euthanized, following which the serum levels of glucose, insulin, and glucagon were measured using ELISA, the expression of TXNIP in pancreatic tissue lysates was determined using western blotting, and the level of β-cell apoptosis was assessed using the TUNEL assay. TXNIP levels in the pancreatic tissue of Akita mice were significantly elevated compared with wild-type (WT) mice. Shiga-Y6 administration for 28 days significantly lowered those levels compared with Akita mice that received vehicle to a level comparable to WT mice. In immunohistochemical analysis, both α- to β-cell ratio and the number of apoptotic β-cells were significantly reduced in SY6-treated Akita mice, compared with vehicle-treated Akita mice. Findings from the present study suggest a potential of Shiga-Y6 as an antidiabetic agent through lowering TXNIP protein levels and ameliorating pancreatic β-cells apoptosis.

0155 Persistent Activation of TXNIP and NLRP3 Inflammasomes in Cortical Glia and Neurons after Mild Traumatic Brain Injury

Abstract Introduction Mild traumatic brain injuries (TBI) induce persistent dysregulated sleep, reactive oxygen species (ROS), and inflammation. Nucleotide-binding domain leucine rich family pyrin containing 3 (NLRP3) inflammasomes are protein complexes that stimulate caspase-1 to activate the somnogenic pro-inflammatory cytokines IL-1β and IL-18. ROS induce thioredoxin interacting protein (TXNIP) to activate NLRP3 inflammasomes by inhibiting thioredoxin which inhibits ROS. We previously found that mice lacking NLRP3 have increased non-rapid-eye movement (NREM) sleep and slow-wave activity (SWA) 24 h after mild TBI, but at 2 months post-injury theses values are reduced. Following up on this, we sought to determine the activity of NLRP3 inflammasomes in glia and neurons after mild TBI. Methods Two-month-old mice lacking NLRP3, and wild-type mice received a craniectomy and mild TBI via controlled cortical impact or sham. Frontal and somatosensory cortices were collected without a TBI and at 24 h and 2 months post-TBI. Microglia, astrocytes, and neurons were isolated using fluorescent-activated cell sorting, and reactive oxygen species (ROS), RNA and protein were quantified using RT-PCR and ELISAs. Significance was set at p < 0.05. Results RNA, protein, and ROS control treatment values were similar for both genotypes in cortical microglia, astrocytes, and neurons. In wild-type mice, NLRP3, IL-1β, IL-18, caspase-1, and TXNIP expression along with ROS values were significantly greater and thioredoxin expression was significantly less in cortical microglia, astrocytes, and neurons 24 h and 2 months post-TBI. In wild-type cortical microglia, astrocytes, and neurons NLRP3, IL-1β, and TXNIP expression levels were significantly greater in these cell types 2 months vs. 24 h post-TBI. Wild-type mice caspase-1 activity and NLRP3, IL-18, IL-1β, and TXNIP protein levels, were significantly greater and thioredoxin protein levels were significantly less in both cortical areas 24 h and 2 months post-TBI. Mice lacking NLRP3 showed significant increased values of TXNIP but reduced thioredoxin expression and protein levels 24 and 2 months post-TBI but no significant differences in other measures. Conclusion Our findings suggest that TBI induces persistent oxidative stress, TXNIP, and NLRP3 inflammasome activation in cortical glia and neurons that likely contributes to sleep dysregulation. Support (if any) Merit Review Award (I01BX005379)(MRZ) and SPiRE Award (I21RX003722)(GBK) Department of Veterans Affairs

TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

Neuroinflammation and oxidative stress have been implicated in the pathogenesis of Alzheimer’s disease (AD). Neuroinflammation and oxidative stress are associated with neuronal death in AD. Astrocytes are linked to neuroinflammation during AD. Astrocytes are important contributors to AD progression. Although the role of thioredoxin-interacting protein (TXNIP) has been identified in inflammation and oxidative stress, the mechanism by which TXNIP regulates inflammation and oxidative stress in astrocytes during AD remains unclear. In the present study, we found that TXNIP gene levels were elevated in cerebral cortex of patients with AD. The protein levels of TXNIP were elevated in GFAP-positive astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. Our results showed that TXNIP increased expression of genes related to pro-inflammatory reactive astrocytes and pro-inflammatory cytokines and chemokines in human astrocytes. Moreover, TXNIP increased production of pro-inflammatory cytokines and chemokines in human astrocytes. TXNIP induced activation of NK-kB signaling and over-production of mitochondrial reactive oxygen species (mtROS) in human astrocytes. TXNIP also induced mitochondrial oxidative stress by reduction of mitochondrial respiration and ATP production in human astrocytes. Furthermore, elevated TXNIP levels are correlated with caspase-3 activation of GFAP-positive astrocytes in patients with AD and mouse AD. TXNIP induced mitochondria-dependent apoptosis via caspase-9 and caspase-3 activation in human astrocytes. These results suggest that TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during AD.

MiR-223 Enhances the Neuroprotection of Estradiol Against Oxidative Stress Injury by Inhibiting the FOXO3/TXNIP Axis.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder characterized by complex pathogenesis, of which oxidative stress has long been regarded as a major mechanism. Previously, the protective effects of estradiol on SH-SY5Y cells against Aβ42-induced injuries were demonstrated. In this study, the protection of SH-SY5Y cells by estradiol from H2O2-caused oxidative stress injury and Alzheimer's mice was further confirmed. H2O2 downregulated, whereas estradiol upregulated miR-223 expression. miR-223 overexpression promoted cell viability, inhibited cell apoptosis, reduced ROS levels, enhanced Superoxide Dismutase (SOD) activity, and decreased malondialdehyde (MDA) content. However, miR-223 inhibition exerted opposite effects. miR-223 directly targeted forkhead box O3 (FOXO3) and inhibited FOXO3 expression. H2O2 increased, whereas estradiol decreased thioredoxin interacting protein (TXNIP) levels; FOXO3 positively regulated TXNIP protein levels. In SH-SY5Y cells, FOXO3 overexpression increased, whereas FOXO3 knockdown reduced the cell apoptosis and ROS levels. FOXO3 bound to TXNIP promoter region and activated TXNIP transcription, whereas the activation could be partially inhibited by estradiol. Collectively, the FOXO3/TXNIP axis is downstream of miR-223. miR-223 enhances the neuroprotection of estradiol against oxidative stress injury through the FOXO3/TXNIP axis.

Imbalance in thioredoxin system activates NLRP3 inflammasome pathway in epicardial adipose tissue of patients with coronary artery disease

Atherosclerosis is the leading cause of death worldwide and has in part an inflammatory basis. Since epicardial adipose tissue (EAT) is in close contact with coronary arteries we hypothesized that an imbalance in thioredoxin-1 (TRX-1) and thioredoxin interacting protein (TXNIP) in EAT, activates NLRP3 inflammasome and promotes production of IL-1β, leading to the development of atherosclerosis. Thirty-eight patients with coronary artery disease (CAD) and thirty patients with no clinical signs of atherosclerosis who underwent open-heart surgery for valve replacement were classified as CAD and control groups, respectively. Biopsy samples from EAT were collected and expression of TXNIP, TRX-1, NLRP3 and IL-1β genes were assessed using qRT-PCR. Tissue protein levels of TXNIP and TRX-1 were determined by Western blotting while ELISA was applied to measure IL-1β. Haematoxylin and eosin staining was used for histological examination. mRNA and protein levels of TXNIP in EAT were significantly higher in patients with CAD compared with control group, whereas CAD patients showed lower TRX-1 gene and protein expression. In addition, in CAD patients the NLRP3 gene expression was almost doubled and IL-1β significantly increased at the both mRNA and protein levels. Enhancment in NLRP3 gene expression and TXNIP protein levels were accompanied with the increase in IL-1β protein level whereas TRX-1 protein content showed a negative correlation with IL-1β level. Concurrent increase in TXNIP, NLRP3, and IL-1β suggests possible involvement of thioredoxin system in the activation of NLRP3 inflammasome, production of IL-1β, and the presence of inflammation in CAD patients.

Oxidized low-density lipoprotein contributes to injury of endothelial cells via the circ_0090231/miR-9-5p/TXNIP axis.

Atherosclerosis (AS) has been reported to induce severe clinical complications. Circular RNA (circRNA) circ_0090231 was found to be aberrantly overexpressed in oxidized low-density lipoprotein (ox-LDL)-induced endothelial cells. This study was designed to explore the role and mechanism of circ_0090231 in ox-LDL-triggered endothelial cell injury in AS. Circ_0090231, microRNA-9-5p (miR-9-5p), and thioredoxin interacting protein (TXNIP) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, angiogenesis, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), tube formation, and flow cytometry assay. Bcl-2, Bax, and TXNIP protein levels were gauged by western blot assay. Malondialdehyde (MDA), lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activity were determined by special kits. Tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6) levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits. The binding relationship between miR-9-5p and circ_0090231 or TXNIP was predicted by starBase, and then verified by a dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Circ_0090231 and TXNIP were increased, and miR-9-5p was decreased in ox-LDL-treated HUVECs. Moreover, circ_0090231 knockdown mitigated ox-LDL-induced HUVEC injury by boosting angiogenesis, oxidative stress, and inflammation, and hindering apoptosis. The mechanical analysis revealed that circ_0090231 acted as a sponge of miR-9-5p to regulate TXNIP expression. Circ_0090231 could attenuate ox-LDL-mediated HUVEC damage by the miR-9-5p/TXNIP axis, providing a promising therapeutic strategy for AS treatment.

Endoplasmic reticulum stress related factor IRE1α regulates TXNIP/NLRP3-mediated pyroptosis in diabetic nephropathy

The nod-like receptor protein-3 (NLRP3)-mediated pyroptosis is involved in kidney diseases. Thioredoxin interacting protein (TXNIP) directly interacts with NLRP3. This study aimed to probe the mechanism of TXNIP and NLRP3 pathway in diabetic nephropathy (DN). Marker detection and histological staining indicated that in DN rats, the renal function was destroyed, and the TXNIP/NLRP3 axis was activated to induce inflammatory generation and pyroptosis. The protein levels of TXNIP, NLRP3 inflammatory components and endoplasmic reticulum stress (ERS)-related factors (ATF4, CHOP and IRE1α) were measured. DN rats were injected with LV-TXNIP-shRNA or IRE1α RNase specific inhibitor (STF-083010) to examine ERS- and pyroptosis-related proteins, and renal injury. Silencing TXNIP inhibited the NLRP3 axis and reduced renal damage in DN rats. ERS was activated in DN rats, and miR-200a expression was degraded by IRE1α. miR-200a bound to TXNIP. NRK-52E cells were induced by high glucose (HG) to simulate DN in vitro. The damage and pyroptosis of NRK-52E cells were analyzed. After inhibiting IRE1α, miR-200a expression increased and TXNIP expression decreased. miR-200a inhibition in HG-induced NRK-52E cells partially reversed the reduced pyroptosis by STF-083010. Overall, IRE1α upregulates miR-200a degradation in DN rats, and stimulates the TXINP/NLRP3 pathway-mediated pyroptosis and renal damage.

Parallel TXNIP‐ and TLR2‐ Dependent Pathways Synergistically Mediate Saturated Fatty Acid‐Induced Apoptosis in Cardiomyocytes

BACKGROUNDSaturated fatty acids (SFA) induce apoptosis in many cell types in vitro, and high SFA diet increases apoptotic cardiomyocytes, but mechanism remains unclear. We had previously demonstrated that p38α mitogen activated protein kinase (MAPK)–dependent signaling mediates cardiomyocyte apoptosis induced by palmitic acid (PA), a SFA. More recently, direct binding of thioredoxin interacting protein (TXNIP) and p38 MAPK has been demonstrated. SFA has been shown to bind to and activate Toll‐like receptor‐2 (TLR2), as well as the TLR4 accessory protein, MD2, triggering inflammation, oxidative stress, and cell death. We hypothesized that parallel TXNIP‐, and TLR‐ dependent pathways, both triggered by SFA, synergistically mediate apoptosis in cardiomyocytes.METHODS AND RESULTSA human cardiomyocyte line, AC16, was used in the study. TXNIP was inhibited by pre‐treating (24hours) cells with SBI‐477, a chemical inhibitor of MondoA/ChREBP. TXNIP knockdown was achieved with siRNA. TXNIP was overexpressed by transfecting with TXNIP plasmid, then 48 h later treated with TLR2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS), for an additional 2–3 days. TXNIP expression was determined by Western blotting of whole cell lysates. Apoptosis was measured by flow cytometry using Annexin V/PI staining.PA increased TXNIP protein levels dose‐dependently (1.5‐ and 2.3‐fold increase at 150 uM and 300 uM PA respectively) (ANOVA p<0.01, n=3). PA did not affect MondoA and ChREBP protein expression but increased nuclear translocation 2‐fold. TXNIP inhibition reduced PA‐induced apoptosis by 79% (p<0.05; n=3). TXNIP knockdown reduced PA‐induced cardiomyocyte apoptosis by 61% (p<0.01; n=7).TXNIP overexpression for 24 h increased apoptosis 1.8‐fold compared to control plasmid (PCDNA), but this was attenuated by siRNA knockdown of p38α or c‐fos, 59% and 84% respectively. TXNIP overexpression for 4–5 days markedly increased apoptosis rate to 4.0‐fold. The combined stressors of transfection with control plasmid and TLR ligand increased cell death rate 3.2 and 4.6‐fold for PGN and LPS respectively, compared to PCDNA + Vehicle. TXNIP overexpression followed by PGN increased apoptosis rate 8‐fold, whereas LPS reduced it to 2.7‐fold.CONCLUSIONSThis study in a human cardiomyocyte line demonstrated that (1) PA increased TXNIP levels, (2) TXNIP inhibition or knockdown attenuated PA‐induced apoptosis, (3) TXNIP overexpression increased apoptosis, (4) TXNIP overexpression combined with a TLR2 ligand, PGN, synergistically amplified the apoptosis rate, whereas LPS, a TLR4 ligand did not. These findings suggest that TXNIP‐dependent pathway synergistically combine with TLR2‐dependent signaling, but not TLR4‐, to amplify apoptosis, and suggest a mechanism by which elevated plasma SFA levels may lead to development of lipotoxic cardiomyopathy.Support or Funding InformationThis work has been funded by VA VISN 18 New Investigator Grant (to C.O.), CSR&D 5I01CX000598 grant (to P.R.), and VA Merit Grant BLRD I01BX007080 (to R.M.). The study was supported by VA Employment and Carl T. Hayden Medical Research Foundation. K.T. is supported by the M. Lowell Edwards Endowment.

Icariin Ameliorates Palmitate-Induced Insulin Resistance Through Reducing Thioredoxin-Interacting Protein (TXNIP) and Suppressing ER Stress in C2C12 Myotubes.

Both thioredoxin-interacting protein (TXNIP) and endoplasmic reticulum (ER) stress are implicated in skeletal muscle insulin resistance. Icariin has been found to mimic insulin action in normal skeletal muscle C2C12 cells and display anti-diabetic properties in diet-induced obese mice. However, the underlying molecular mechanism remains to be well-established. Herein, we tested the hypothesis that the protective effects of icariin on free fatty acid-induced insulin resistance were attributed to its regulation on TXNIP protein levels and ER stress in skeletal muscle cells. We found that TXNIP mediated the saturated fatty acid palmitate (PA)-induced insulin resistance in C2C12 myotubes. Icariin treatment significantly restored PA-reduced proteasome activity resulting in reduction of TXNIP protein and suppression of ER stress, as well as improvement of insulin sensitivity. Proteasome inhibition by its specific inhibitor MG132 obviously abolished the inhibitory effect of icariin on PA-induced insulin resistance. In addition, MG132 supplementation markedly abrogated the impacts of icariin on ER stress and TXNIP-mediated downstream events such as inflammation and STAT3 phosphorylation. These results clearly indicate that icariin improves PA-induced skeletal muscle insulin resistance through a proteasome-dependent mechanism, by which icariin downregulats TXNIP levels and inhibits ER stress.

Evaluation on the effect of hydrogen sulfide on the NLRP3 signaling pathway and its involvement in the pathogenesis of atherosclerosis.

As a common disease, the incidence of atherosclerosis (AS) in the world is high. Therefore, we aimed to evaluate the involvement of hydrogen sulfide (H 2 S)/cystathionine γ-lyase (CSE) in the pathogenesis of AS as well as their possible signaling pathways. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis were used to detect the effect of CSE on the expression of inflammatory cytokines, ie, H 2 S, thioredoxin-interacting protein (TXNIP), NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, and interleukin (IL)-1β. In addition, immunohistochemistry and Western blot analysis were performed to detect the levels of TXNIP, NLRP3, ASC, caspase-1, IL-1β, and IL-18 among different groups. Knockdown of CSE by the transfection of CSE small interfering RNA upregulated the levels of two inflammatory cytokines, ie, IL-1β and IL-18. In addition, the downregulation of CSE promoted the expression of TXNIP, NLRP3, ASC, caspase-1, and IL-1β in THP-1 cells. Meanwhile, treating the cells with sodium hydrosulfide (NaHS) inhibited the productions of IL-1β and IL-18. Furthermore, upregulation of H 2 S synthesis by treating the cells with NaHS also reduced the protein levels of TXNIP, NLRP3, ASC, caspase-1, and IL-1β. Finally, the protein levels of TXNIP and NLRP3 in the AS group were much higher than those in the AS + H 2 S group, which in turn was higher than the sham group. In addition, the AS group displayed the highest protein levels of TXNIP, NLRP3, ASC, caspase-1, IL-1β, and IL-18, while the levels of these proteins in the AS + H 2 S group were higher than those in the sham group. In summary, the present finding suggested a possible linkage between H 2 S metabolism and AS through the H 2 S/CSE-TXNIP-NLRP3-IL-18/IL-1β-nitric oxide (NO) signaling pathway.

Abstract B48: Oncogenic Ras reprograms metabolism by suppressing TXNIP translation

Abstract Salt Lake City, UT. Background: Oncogenic Ras promotes aerobic glycolysis and glutaminolysis to supply central carbon metabolism. Ras-transformed cells also upregulate autophagy and internalization of extracellular proteins and lipids to fuel their enhanced metabolic needs. Ras-mediated metabolic reprogramming generates sufficient ATP and macromolecule precursors to sustain rapid growth and proliferation. Thioredoxin interacting protein (TXNIP) is a potent inhibitor of glucose uptake and aerobic glycolysis. It also promotes β-oxidation, apoptosis, and mediates cell cycle arrest. Consistent with these anti-proliferative functions, TXNIP is strongly downregulated in a variety of human cancers. Further, low TXNIP expression predicts poor clinical outcomes in breast cancer patients. TXNIP transcription and translation are tightly regulated in response to various intracellular and extracellular cues. Our lab previously discovered that TXNIP is acutely downregulated during the G0/G1 transition. Activation of the PI3K and MAPK pathways downregulate TXNIP transcription and translation, respectively. Interestingly, constitutively active RasG12V also suppresses TXNIP transcription and translation. We hypothesize that TXNIP coordinates cell growth by functioning as a metabolic checkpoint. In transformed cells, persistent engagement of growth factor signaling pathways suppresses TXNIP to allow metabolic reprogramming for unchecked cell growth. Understanding the mechanism(s) by which TXNIP expression is inhibited by oncogenic signaling will shed light on novel therapeutic targets for Ras-driven cancers. Results: In this study, we investigated the mechanism by which constitutively active RasG12V (Ras henceforth) suppresses TXNIP expression in mouse embryonic fibroblasts. Our results demonstrated that Ras is a potent suppressor of TXNIP mRNA and protein levels. By expressing TXNIP under a constitutive promoter, we discovered that Ras downregulates TXNIP protein level independent of its mRNA downregulation. Further, that repression resulted from inhibition of protein synthesis, rather than increased protein turnover. Polysome profiling experiments revealed that Ras inhibits translation elongation of TXNIP mRNA. Consistent with this result, the TXNIP coding region (CDS) was sufficient for Ras to stall translation elongation. Ras did not suppress TXNIP translation when it was encoded by a message with optimized codon usage. This finding suggests that poor codon fitness of the TXNIP CDS is required for its translational inhibition by Ras. Conclusions: Our studies established that oncogenic Ras represses TXNIP expression by inhibiting translation elongation of the TXNIP mRNA. Given the prevalence of Ras activation and TXNIP downregulation in cancers, our findings suggest a novel route by which Ras-driven cancers rewire their metabolism to support their aggressive growth. Because Ras activation upregulates global translation, our experiments suggest that the translation elongation machinery discriminates among mRNAs based on codon usage. Specifically, in non-transformed cells, the translation machinery can translate a spectrum of mRNAs with different codon adaptiveness. By contrast, Ras transformation places an increased demand on the translational machinery, resulting in a bias towards mRNAs with optimal codon usage over mRNAs like TXNIP with relatively poor codon usage. Citation Format: Zhizhou Ye, Donald E. Ayer. Oncogenic Ras reprograms metabolism by suppressing TXNIP translation. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B48.

High Glucose and Lipopolysaccharide Prime NLRP3 Inflammasome via ROS/TXNIP Pathway in Mesangial Cells.

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1β was observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1β were significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1β inflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy.

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.