Thioredoxin-interacting Protein Knockdown Research Articles

TXNIP mediated by EZH2 regulated osteogenic differentiation in hBmscs and MC3T3-E1 cells through the modulation of oxidative stress and PI3K/AKT/Nrf2 pathway

ABSTRACT Background Previous research has identified a significant role of Thioredoxin-interacting protein (TXNIP) in bone loss. The purpose of this investigation was to assess the role and the underlying molecular mechanisms of TXNIP in the osteogenic differentiation of human bone marrow stromal cells (hBMSCs) and pre-osteoblast MC3T3-E1 cells. Methods Human bone marrow stem cells (hBMSCs) and MC3T3-E1 cells were used to induce osteogenic differentiation. The expression of genes and proteins was assessed using RT-qPCR and western blot, respectively. ChIP assay was used to validate the interaction between genes. The osteogenic differentiation ability of cells was reflected using ALP staining and detection of ALP activity. The mineralization ability of cells was assessed using ARS staining. DCFCA staining was employed to evaluate the intracellular ROS level. Results Initially, downregulation of TXNIP and upregulation of EZH2 were observed during osteogenesis in hBMSCs and MC3T3-E1 cells. Additionally, it was discovered that EZH2 negatively regulates TXNIP expression in these cells. Furthermore, experiments indicated that the knockdown of TXNIP stimulated the activation of the PI3K/AKT/Nrf2 signaling pathway in hBMSCs and MC3T3- E1 cells, thus inhibiting the production of reactive oxygen species (ROS). Further functional experiments revealed that overexpression of TXNIP inhibited the osteogenic differentiation in hBMSCs and MC3T3-E1 cells by enhancing ROS produc-tion. On the other hand, knockdown of TXNIP promoted the osteogenic differentiation capacity of hBMSCs and MC3T3-E1 cells through the activation of the PI3K/AKT/Nrf2 pathway. Conclusion In conclusion, this study demonstrated that TXNIP expression, under the regulation of EZH2, plays a crucial role in the osteogenic differentiation of hBMSCs and MC3T3-E1 cells by regulating ROS production and the PI3K/AKT/Nrf2 pathway.

TXNIP knockdown ameliorates hepatic ischemia/reperfusion injury by inhibiting apoptosis and improving mitochondrial dysfunction via HIF-1α.

This study aims to investigate whether thioredoxin-interacting protein (TXNIP) regulates cell viability, cell apoptosis and mitochondrial damage in OGD/R-induced hepatocytes and to explore its underlying mechanism. AML12 cells were cultured under oxygen-glucose deprivation/reperfusion (OGD/R) conditions. TXNIP mRNA was detected using qRT-PCR, and the TXNIP protein was analyzed using western blotting. TXNIP-targeted short hairpin RNA (sh-TXNIP) lentivirus was used to infect the AML12 cells. CCK8 and TUNEL assays were applied to detect cell viability and apoptosis, respectively. DCFH-DA probe was used to determine reactive oxygen species (ROS) release level, and JC-1 probe was used to evaluate mitochondrial membrane potential (MMP). The localization of TXNIP and HIF-1α was observed using immunofluorescence. Our results showed that TXNIP markedly increased in AML12 cells treated with OGD/R. TXNIP knockdown increased cell viability and reduced cell apoptosis under OGD/R treatment. Moreover, MMP significantly increased and ROS release decreased in cells after TXNIP knockdown under OGD/R treatment. Additionally, TXNIP knockdown markedly increased the expression of HIF-1α. HIF-1α exhibited nuclear translocation following OGD/R induction, and TXNIP knockdown further promoted it. Compared with the OGD/R + sh-TXNIP group, HIF-1α agonist ML228 inhibited cell apoptosis and ROS release, and increased MMP. However, HIF-1α inhibitor PX478 had the opposite effect. In summary, TXNIP deletion ameliorated AML12 cell injury caused by OGD/R via promoting HIF-1α expression and nuclear translocation, manifested by inhibiting cell apoptosis and alleviating mitochondrial dysfunction.

Competitive adsorption of microRNA-532-3p by circular RNA SOD2 activates Thioredoxin Interacting Protein/NLR family pyrin domain containing 3 pathway and promotes pyroptosis of non-alcoholic fatty hepatocytes

ObjectiveThere is a growing body of evidence indicating that pyroptosis, a programmed cell death mechanism, plays a crucial role in the exacerbation of inflammation and fibrosis in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Circular RNAs (circRNAs), functioning as vital regulators within NAFLD, have been shown to mediate the process of cell pyroptosis. This study aims to elucidate the roles and mechanisms of circRNAs in NAFLD.MethodsUtilizing a high-fat diet (HFD)-induced rat model for in vivo experimentation and hepatocytes treated with palmitic acid (PA) for in vitro models, we identified circular RNA SOD2 (circSOD2) as our circRNA of interest through analysis with the circMine database. The expression levels of associated genes and pyroptosis-related proteins were determined using quantitative real-time polymerase chain reaction and Western blotting, alongside immunohistochemistry. Serum liver function markers, cellular inflammatory cytokines, malondialdehyde, lactate dehydrogenase levels, and mitochondrial membrane potential, were assessed using enzyme-linked immunosorbent assay, standard assay kits, or JC-1 staining. Flow cytometry was employed to detect pyroptotic cells, and lipid deposition in liver tissues was observed via Oil Red O staining. The interactions between miR-532-3p/circSOD2 and miR-532-3p/Thioredoxin Interacting Protein (TXNIP) were validated through dual-luciferase reporter assays and RNA immunoprecipitation experiments.ResultsOur findings demonstrate that, in both in vivo and in vitro NAFLD models, there was an upregulation of circSOD2 and TXNIP, alongside a downregulation of miR-532-3p. Mechanistically, miR-532-3p directly bound to the 3'-UTR of TXNIP, thereby mediating inflammation and cell pyroptosis through targeting the TXNIP/NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling pathway. circSOD2 directly interacted with miR-532-3p, relieving the suppression on the TXNIP/NLRP3 signaling pathway. Functionally, the knockdown of circSOD2 or TXNIP improved hepatocyte pyroptosis; the deletion of miR-532-3p reversed the effects of circSOD2 knockdown, and the deletion of TXNIP reversed the effects of circSOD2 overexpression. Furthermore, the knockdown of circSOD2 significantly mitigated the progression of NAFLD in vivo.ConclusioncircSOD2 competitively sponges miR-532-3p to activate the TXNIP/NLRP3 inflammasome signaling pathway, promoting pyroptosis in NAFLD.

TXNIP knockdown protects rats against bupivacaine-induced spinal neurotoxicity via the inhibition of oxidative stress and apoptosis

Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting protein (TXNIP) is a member of the α-arrestin protein superfamily that binds covalently to thioredoxin (TRX) to inhibit its function, leading to increased oxidative stress and activation of apoptosis. The role of TXNIP in BUP-induced oxidative stress and apoptosis remains to be elucidated. In this context, the present study aimed to explore the effects of TXNIP knockdown on BUP-induced oxidative stress and apoptosis in the spinal cord of rats and in PC12 cells through the transfection of adeno-associated virus-TXNIP short hairpin RNA (AAV-TXNIP shRNA) and siRNA-TXNIP, respectively. In vivo, a rat model of spinal neurotoxicity was established by intrathecally injecting rats with BUP. The BUP + TXNIP shRNA and the BUP + Control shRNA groups of rats were injected with an AAV carrying the TXNIP shRNA and the Control shRNA, respectively, into the subarachnoid space four weeks prior to BUP treatment. The Basso, Beattie & Bresnahan (BBB) locomotor rating score, % MPE of TFL, H&E staining, and Nissl staining analyses were conducted. In vitro, 0.8 mM BUP was determined by CCK-8 assay to establish a cytotoxicity model in PC12 cells. Transfection with siRNA-TXNIP was carried out to suppress TXNIP expression prior to exposing PC12 cells to BUP. The results revealed that BUP effectively induced neurological behavioral dysfunction and neuronal damage and death in the spinal cord of the rats. Similarly, BUP triggered cytotoxicity and apoptosis in PC12 cells. In addition, treated with BUP both in vitro and in vivo exhibited upregulated TXNIP expression and increased oxidative stress and apoptosis. Interestingly, TXNIP knockdown in the spinal cord of rats through transfection of AAV-TXNIP shRNA exerted a protective effect against BUP-induced spinal neurotoxicity by ameliorating behavioral and histological outcomes and promoting the survival of spinal cord neurons. Similarly, transfection with siRNA-TXNIP mitigated BUP-induced cytotoxicity in PC12 cells. In addition, TXNIP knockdown mitigated the upregulation of ROS, MDA, Bax, and cleaved caspase-3 and restored the downregulation of GSH, SOD, CAT, GPX4, and Bcl2 induced upon BUP exposure. These findings suggested that TXNIP knockdown protected against BUP-induced spinal neurotoxicity by suppressing oxidative stress and apoptosis. In summary, TXNIP could be a central signaling hub that positively regulates oxidative stress and apoptosis during neuronal damage, which renders TXNIP a promising target for treatment strategies against BUP-induced spinal neurotoxicity.

Knockdown of TXNIP alleviates gestational diabetes mellitus by activating autophagy to regulate cell proliferation and apoptosis in high glucose-treated trophoblasts

Dysregulated thioredoxin-interacting protein (TXNIP) has been observed in women with gestational diabetes mellitus (GDM), but the specific role of TXNIP in GDM and the underlying mechanism remain unclear. HTR-8/SVneo cells were treated with high glucose to mimic the injured trophoblasts of GDM. In vitro, TXNIP knockdown was performed by siRNA. RTqPCR was performed to determine the expression of the corresponding genes. Cell proliferation and apoptosis were measured using CCK-8, EdU and Annexin V/PI assays. The autophagosome number was assessed using transmission electron microscopy. The expression of the autophagy substrate sequestosome 1 (P62) was evaluated by immunofluorescence. Autophagy-related proteins, including P62, light chain 3 (LC3)-I, and LC3-II, were analysed by Western blotting. HTR-8/Svneo cells treated with high glucose demonstrated reduced proliferation, increased apoptosis, decreased autophagosome formation and overall decreased autophagy. However, knockdown of TXNIP reversed the effects of HG on HTR-8/Svneo cells. However, the effect of TXNIP knockdown on HG-treated HTR-8/Svneo cells was inhibited by 3-methyladenine (3-MA) (widely used as an inhibitor of autophagy). We concluded that knockdown of TXNIP has the potential to enhance the activity of high glucose-treated human trophoblasts through autophagic activation, thereby improving pregnancy outcomes in patients with GDM.

PIAS3 promotes ferroptosis by regulating TXNIP via TGF-β signaling pathway in hepatocellular carcinoma

Ferroptosis has been suggested to play a potential role in cancer therapy as an iron-dependent programmed cell death mechanism distinct from other forms. Hepatocellular carcinoma (HCC) remains a great threat, with high mortality and limited therapeutic options. The induction of ferroptosis has emerged as a novel and promising therapeutic strategy for HCC. In the present study, we identified protein inhibitor of activated STAT3 (PIAS3) as a driver of ferroptosis in HCC using TMT-based quantitative proteomics and ferroptosis-related functional assays. Mechanistically, thioredoxin-interacting protein (TXNIP) was confirmed to be PIAS3 in promoting ferroptotic cell death, based on RNA-seq analysis. Knockdown of TXNIP degrades ferroptotic susceptibility caused by PIAS3-overexpression, whereas transfection-forced reexpression of TXNIP restores sensitivity to ferroptosis in PIAS3-downregulated cells. PIAS3 interacts with SMAD2/3 to activate transforming growth factor (TGF)-β signaling, leading to increased TXNIP expression. Our study revealed the critical role of PIAS3 in ferroptosis and a novel actionable axis-PIAS3/TGF-β/TXNIP that could govern ferroptotic sensitivity, paving the path for using ferroptosis as an efficient approach in HCC therapies.

Upregulation of TXNIP contributes to granulosa cell dysfunction in polycystic ovary syndrome via activation of the NLRP3 inflammasome

Polycystic ovary syndrome (PCOS) is a complex endocrine disease. Thioredoxin-interacting protein (TXNIP) promotes oxidative stress and triggers inflammation. Herein, we investigated the role and potential mechanism of TXNIP in PCOS. In a mouse model of dehydroepiandrosterone (DHEA)-induced PCOS, we found that TXNIP was upregulated in the ovaries, especially in granulosa cells (GCs). TXNIP was also upregulated in testosterone (T)-treated GCs in vitro. Knockdown of TXNIP by lentivirus-constructed shRNA attenuated T-induced GC injury and oxidative stress, as well as inflammation and the NLRP3 inflammasome. The mechanism by which TXNIP promotes inflammation may involve TXNIP dissociation from the TXNIP-TRX complex and binding to NLRP3 to form the inflammasome. Additionally, we verified that knockdown of TXNIP ameliorated ovarian injury and inflammation in mice with DHEA-induced PCOS in vivo. Collectively, we demonstrated that TXNIP is involved in GC inflammation by promoting NLRP3 inflammasome activation in PCOS.

Thioredoxin-Interacting Protein Inhibited Vascular Endothelial Cell-Induced HREC Angiogenesis Treatment of Diabetic Retinopathy.

Diabetic retinopathy is the most common reason for blindness among employed adults worldwide. Hyperglycemia and the overaccumulation of vascular endothelial growth factor (VEGF) lead to diabetic retinopathy, pathological angiogenesis in diabetic retinopathy, and consequent visual impairment. Expression levels of thioredoxin-interacting protein (TXNIP) substantially increase in retinal endothelial cells in diabetic circumstances. The part of TXNIP in retinal angiogenesis combined with diabetes remains unclear. This study examined the effect of reduced TXNIP expression levels and determined how it affects diabetic retinal angiogenesis. Display of human retinal vascular endothelial cells (HRECs) to moderately high glucose (MHG) encouraged tube formation and cell migration, not cell proliferation. In response to MHG conditions, in HRECs, TXNIP knockdown inhibited the production of reactive oxygen species (ROS), cell migration, tube formation, and the Akt/mTOR activation pathway. In addition, gene silencing of TXNIP decreased the VEGF-triggered angiogenic response in HRECs by preventing activation of both VEGF receptor 2 and the downstream components of the Akt/mTOR pathway signaling. Furthermore, TXNIP knockout mice reduced VEGF-induced or VEGF- and MHG-triggered ex vivo retinal angiogenesis compared to wild-type mice. Finally, our findings revealed that TXNIP knockdown suppressed VEGF- and MHG-triggered angiogenic responses in HRECs and mouse retinas, indicating that TXNIP is a promising therapeutic window against the proliferation of diabetic patients' retinopathy.

Thioredoxin-interacting protein deficiency protects against severe acute pancreatitis by suppressing apoptosis signal-regulating kinase 1

Acute pancreatitis is a common acute inflammatory abdominal disease. When acute pancreatitis progresses to severe acute pancreatitis (SAP), it can lead to systemic inflammation and even multiple organ failure. Thioredoxin-interacting protein (TXNIP) is an important protein involved in redox reactions of the inflammatory response. However, the specific role of TXNIP in SAP remains unclear. In this study, we investigated the role of thioredoxin interacting protein (TXNIP) in acute pancreatitis when induced by high doses of arginine. We found that pancreatic damage and the inflammatory response associated with acute pancreatitis were largely restrained in TXNIP knock-out mice but were enhanced in mice overexpressing TXNIP. Interestingly, the phosphorylation of p38, JNK, and ASK1 diminished in TXNIP-KO mice with pancreatitis in comparison with wild-type mice. The role of oxidative stress in SAP was explored in two models: TXNIP and AVV-TXNIP. TXNIP knockdown or the inhibition of ASK1 by gs-4997 abrogated the increase in p-p38, p-JNK, and p-ASK1 in AR42J cells incubated with L-Arg. The administration of gs-4997 to mice with pancreatitis largely reduced the upregulation of IL-6, IL-1β, TNF-α, and MCP-1. Systemic inflammatory reactions and injury in the lungs and kidneys were assessed in TXNIP-KO and AVV-TXNIP mice with expected outcomes. In conclusion, TXNIP is a novel mediator of SAP and exerts action by regulating inflammatory responses and oxidative stress via the ASK1-dependent activation of the JNK/p38 pathways. Thus, targeting TXNIP may represent a promising approach to protect against SAP.

TXNIP Promotes Human NK Cell Development but Is Dispensable for NK Cell Functionality.

The ability of natural killer (NK) cells to kill tumor cells without prior sensitization makes them a rising player in immunotherapy. Increased understanding of the development and functioning of NK cells will improve their clinical utilization. As opposed to murine NK cell development, human NK cell development is still less understood. Here, we studied the role of thioredoxin-interacting protein (TXNIP) in human NK cell differentiation by stable TXNIP knockdown or overexpression in cord blood hematopoietic stem cells, followed by in vitro NK cell differentiation. TXNIP overexpression only had marginal effects, indicating that endogenous TXNIP levels are sufficient in this process. TXNIP knockdown, however, reduced proliferation of early differentiation stages and greatly decreased NK cell numbers. Transcriptome analysis and experimental confirmation showed that reduced protein synthesis upon TXNIP knockdown likely caused this low proliferation. Contrary to its profound effects on the early differentiation stages, TXNIP knockdown led to limited alterations in NK cell phenotype, and it had no effect on NK cell cytotoxicity or cytokine production. Thus, TXNIP promotes human NK cell differentiation by affecting protein synthesis and proliferation of early NK cell differentiation stages, but it is redundant for functional NK cell maturation.

Reactive Oxygen Species Contributes to Type 2 Diabetic Neuropathic Pain via the Thioredoxin-Interacting Protein-NOD-Like Receptor Protein 3- N -Methyl-D-Aspartic Acid Receptor 2B Pathway.

BACKGROUND:The number of patients with diabetic neuropathic pain (DNP) continues to increase, but available treatments are limited. This study aimed to examine the influence of reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NOD–like receptor protein 3 (NLRP3)-N-methyl-D-aspartic acid receptor 2B (NR2B) pathway on type 2 DNP.METHODS:Male Sprague-Dawley rats were fed with a high-fat and high-sugar diet for 8 weeks. Then, rats were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg) to induce type 2 diabetes mellitus in rats. Diabetic rats with <85% of their basic levels in mechanical withdrawal threshold and thermal withdrawal latency were classified as DNP rats on day 14 after STZ injection. DNP rats were treated with ROS scavenger N-tert-Butyl-α-phenylnitrone (PBN, 100 mg·kg–1·d–1) or TXNIP small interfering ribonucleic acid (10 μg/d) once daily for 14 days. The level of ROS, protein levels of NLRP3, TXNIP, cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1β (IL-1β), NR2B phosphorylation at Tyr1472 (p-NR2B), total NR2B (t-NR2B), and distribution of NLRP3 in the spinal cord were examined. In vitro experiments, BV2 cells and PC12 cells were individually cultured and cocultured in a high-glucose environment (35 mmol/L D-glucose). The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, and IL-1β in BV2 cells, and p-NR2B, t-NR2B in PC12 cells were detected. The level of ROS was detected by the flow cytometry approach. The protein levels were detected by the Western blot technique. The location of NLRP3 was observed by immunofluorescent staining. The interaction between TXNIP and NLRP3 was detected by coimmunoprecipitation assay.RESULTS:The level of spinal ROS increased in DNP rats. The mechanical allodynia and thermal hyperalgesia of DNP rats were alleviated after systemic administration of PBN. This administration decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1β, and p-NR2B and the coupling of TXNIP to NLRP3 in spinal cords of DNP rats. Furthermore, knockdown of spinal TXNIP alleviated nociceptive hypersensitivity and decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1β, and p-NR2B in DNP rats. The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, IL-1β, the coupling of TXNIP to NLRP3, and the IL-1β secretion increased in BV2 cells, and the protein expression of p-NR2B increased in cocultured PC12 cells in a high-glucose environment. All of these in vitro effects were significantly blocked after treatment of PBN.CONCLUSIONS:Our findings suggest that spinal ROS can contribute to type 2 DNP through TXNIP-NLRP3-NR2B pathway.

ITCH facilitates proteasomal degradation of TXNIP in hypoxia- induced lung cancer cells.

BackgroundLung cancer (LC) is one of the most common cancers and a leading cause of cancer‐related deaths worldwide. In many pathological conditions, particularly in the tumor microenvironment, cells and tissues frequently exist in a hypoxic state.Here, we evaluated Itchy E3 ubiquitin protein ligase (ITCH) expression in LC cells following hypoxia treatment.MethodsLC cell lines were treated with hypoxic condition. Cell migration, invasion, inflammation, reactive oxygen species (ROS) production, and apoptosis of LC cells were determined by wound healing assay, Transwell invasive assay, ELISA, DCFH‐DA staining, and flow cytometry, respectively. qPCR and WB were used to determine the expression of ITCH and TXNIP. Co‐IP was performed to assess the interaction between ITCH and TXNIP.ResultsITCH expression was downregulated in LC cells under hypoxic conditions. Next, LC cells were subjected to hypoxic conditions and changes in cell viability and metastasis were determined. Hypoxic conditions resulted in increased migration and invasion abilities of LC cells. Intracellular reactive oxygen species (ROS) production, inflammation, and apoptosis were also promoted by hypoxia. We found that ITCH overexpression led to the proteasomal degradation of thioredoxin‐interacting protein (TXNIP), whereas the expression of the ITCH C830A mutant did not affect TXNIP levels in LC cells. The gain‐of‐function experiment demonstrated that migration, invasion, ROS generation, inflammation, and apoptosis of hypoxia‐conditioned LC cells were ameliorated by ITCH overexpression, whereas the ITCH C830A mutant did not cause any changes in these phenotypes. Furthermore, the contribution of TXNIP knockdown and ITCH overexpression to the hypoxia‐induced features in LC cells with ITCH C830A was found to be similar.ConclusionOur results suggest a novel mechanism underlying the changes in ITCH‐mediated malignant phenotypes of hypoxia‐conditioned LC cells via TXNIP.

Thioredoxin-Interacting Protein (TXNIP) Knockdown Protects against Sepsis-Induced Brain Injury and Cognitive Decline in Mice by Suppressing Oxidative Stress and Neuroinflammation.

Sepsis-associated encephalopathy (SAE) is linked to increased morbidity and mortality rates in patients with sepsis. Increased cytokine production and neuronal apoptosis are implicated in the pathogenesis of the SAE. Neuroinflammation plays a major role in sepsis-induced brain injury. Thioredoxin-interacting protein (TXNIP), an inhibitor of thioredoxin, is associated with oxidative stress and inflammation. However, whether the TXNIP is involved in the sepsis-induced brain injury and the underlying mechanism is yet to be elucidated. Therefore, the present study was aimed at elucidating the effects of TXNIP knockdown on sepsis-induced brain injury and cognitive decline in mice. Lipopolysaccharide (LPS) was injected intraperitoneally to induce sepsis brain injury in mice. The virus-carrying control or TXNIP shRNA was injected into the lateral ventricle of the brain 4 weeks before the LPS treatment. The histological changes in the hippocampal tissues, encephaledema, and cognitive function were detected, respectively. Also, the 7-day survival rate was recorded. Furthermore, the alterations in microglial activity, oxidative response, proinflammatory factors, apoptosis, protein levels (TXNIP and NLRP3 inflammasome), and apoptosis were examined in the hippocampal tissues. The results demonstrated that the TXNIP and NLRP3 inflammasome expression levels were increased at 6, 12, and 24 h post-LPS injection. TXNIP knockdown dramatically ameliorated the 7-day survival rate, cognitive decline, brain damage, neuronal apoptosis, and the brain water content, inhibited the activation of microglia, downregulated the NLRP3/caspase-1 signaling pathway, and reduced the oxidative stress and the neuroinflammatory cytokine levels at 24 h post-LPS injection. These results suggested a crucial effect of TXNIP knockdown on the mechanism of brain injury and cognitive decline in sepsis mice via suppressing oxidative stress and neuroinflammation. Thus, TXNIP might be a potential therapeutic target for SAE patients.

Thioredoxin-interacting protein regulates keratinocyte differentiation: Implication of its role in psoriasis.

Thioredoxin-interacting protein (TXNIP), also known as Vitamin-D upregulated protein-1 (VDUP-1), interacts with thioredoxin to regulate redox responses and participates in diverse disorders including metabolic, cardiovascular, inflammatory and malignant diseases. Psoriasis is characterized by chronic skin inflammation and an aberrant pattern of keratinocyte differentiation. Clinically, psoriasis is associated with various cardiometabolic comorbidities but studies on TXNIP's biological role in skin disorders are limited. In this study, we investigated TXNIP expression in psoriasis and its regulation in normal human epidermal keratinocytes (NHEKs), and then explored how TXNIP regulated skin keratinocyte differentiation to determine its role in psoriasis pathogenesis. Our immunohistochemical study demonstrated extensive TXNIP expression in the upper and lower epidermis of psoriasis compared to predominant TXNIP expression in the basal layer of normal skin. 1, 25-dihydroxyvitamin D3 suppressed but TGF-α and EGF enhanced TXNIP expression in NHEKs. An inducer of keratinocyte differentiation, phorbol 12-myristate 13-acetate (PMA), also diminished TXNIP expression, which was reversed by PKC-δ knockdown. TXNIP knockdown reduced PMA-induced involucrin and transglutaminse-1 expression, and increased p63 expression in NHEKs but did not significantly affect cell proliferation. H2 O2 -induced ROS production and EGFR phosphorylation decreased in NHEKs with TXNIP knockdown. Furthermore, PMA-induced PKC-δ phosphorylation, TGF-α, and EGF-triggered EGFR phosphorylation were attenuated by TXNIP knockdown. Our results unraveled the regulation and function of TXNIP expression in skin keratinocytes and the cross-regulation between TXNIP and EGFR signaling. These findings imply a role of TXNIP in psoriasis and provide insight into the possible impact of TXNIP regulators on the skin or psoriasis.

N-WASP Attenuates Cell Proliferation and Migration through ERK2-Dependent Enhanced Expression of TXNIP.

Simple SummaryNeural Wiskott–Aldrich Syndrome Protein (N-WASP) regulates actin cytoskeleton remodeling and can, it has been suggested, suppress several cancers. In this study, HSC-5 cells, a mammalian cell line with reduced N-WASP expression, were used to generate control cells and HSC-5 cells with increased N-WASP expression that is comparable to that of normal keratinocytes. The two cell lines were used to elucidate the regulation of cell proliferation and migration by N-WASP. Our findings suggest that N-WASP increases ERK2-dependent phosphorylation of FOXO1 and increases TXNIP expression, which reduces cell proliferation and migration. This study is the first to propose an antiproliferative role of N-WASP, which is mediated via ERK2, and it suggests new avenues for cancer therapeutic research and treatment.Neural Wiskott–Aldrich Syndrome Protein (N-WASP) regulates actin cytoskeleton remodeling. It has been known that reduced N-WASP expression in breast and colorectal cancers is associated with poor prognosis. Here, we found reduced N-WASP expression in squamous cell carcinoma (SCC) patient samples. The SCC cell line HSC-5 with reduced N-WASP expression was used to generate HSC-5CN (control) and HSC-5NW (N-WASP overexpression) cells. HSC-5NW cells had reduced cell proliferation and migration compared to HSC-5CN cells. HSC-5NW cells had increased phospho-ERK2 (extracellular signal-regulated kinase 2), phosphorylated Forkhead box protein class O1 (FOXO1) and reduced nuclear FOXO1 staining compared to HSC-5CN cells. Proteasome inhibition stabilized total FOXO1, however, not nuclear staining, suggesting that FOXO1 could be degraded in the cytoplasm. Inhibition of ERK2 enhanced nuclear FOXO1 levels and restored cell proliferation and migration of HSC-5NW to those of HSC-5CN cells, suggesting that ERK2 regulates FOXO1 activity. The expression of thioredoxin-interacting protein (TXNIP), a FOXO1 target that inhibits thioredoxin and glucose uptake, was higher in HSC-5NW cells than in HSC-5CN cells. Knockdown of TXNIP in HSC-5NW cells restored cell proliferation and migration to those of HSC-5CN cells. Thus, we propose that N-WASP regulates cell proliferation and migration via an N-WASP-ERK2-FOXO1-TXNIP pathway.

Regulation of influenza A virus infection by Lnc-PINK1-2:5.

Influenza virus causes approximately 291,000 to 646,000 human deaths worldwide annually. It is also a disease of zoonotic importance, affecting animals such as pigs, horses, and birds. Even though vaccination is being used to prevent influenza virus infection, there are limited options available to treat the disease. Long noncoding RNAs (lncRNAs) are RNA molecules with more than 200 nucleotides that do not translate into proteins. They play important roles in the physiological and pathological processes. In this study, we identified a novel transcript, Lnc‐PINK1‐2:5 that was upregulated by influenza virus. This lncRNA was predominantly located in the nucleus and was not affected by type I interferons. Overexpression of Lnc‐PINK1‐2:5 reduced the influenza viral mRNA and protein levels in cells as well as titres in culture media. Knockdown of Lnc‐PINK1‐2:5 using CRISPR interference enhanced the virus replication. Antiviral activity of Lnc‐PINK1‐2:5 was independent of influenza virus strains. RNA sequencing analysis revealed that Lnc‐PINK1‐2:5 upregulated thioredoxin interacting protein (TXNIP) during influenza virus infection. Overexpression of TXNIP reduced influenza virus infection, suggesting that TXNIP is an antiviral gene. Knockdown of TXNIP abolished the Lnc‐PINK1‐2:5‐mediated increase in influenza virus infection. In conclusion, the newly identified Lnc‐PINK1‐2:5 isoform is an anti‐influenza lncRNA acting through the upregulation of TXNIP gene expression.

YAP/miR-524-5p axis negatively regulates TXNIP expression to promote chondrosarcoma cell growth

Chondrosarcoma (CHS) is the second most common bone malignant tumor and currently has limited treatment options. We have recently demonstrated that thioredoxin interacting protein (TXNIP) plays a crucial role in the oncogenesis of bone sarcoma, yet its implication in CHS is underdetermined. In the present study, we first found that knockdown of TXNIP promotes the proliferation of CHS cell largely through increasing their glycolytic metabolism, which is well-known as Warburg effect for providing energy. Consistent with our previous report that YAP is fundamental for CHS cell growth, herein we revealed that YAP functioned as an upstream molecule of TXNIP, and that YAP negatively regulated TXNIP mRNA and protein expression both in vitro and in vivo. Mechanistically, although knockdown of YAP upregulated both the nuclear and cytoplasmic TXNIP expression, we did not observe any obvious interaction between YAP and TXNIP; instead, miRNA-524-5p was demonstrated to be required for YAP-regulated TXNIP expression and thus controlling CHS cell growth. Together, our study reveals that TXNIP is a tumor suppressor in terms of CHS, and that the YAP/miRNA-524-5p/TXNIP signaling axis may provide a novel clue for CHS targeted therapy.

TXNIP Links Anticipatory Unfolded Protein Response to Estrogen Reprogramming Glucose Metabolism in Breast Cancer Cells.

Estrogen and estrogen receptor (ER) play a fundamental role in breast cancer. To support the rapid proliferation of ER+ breast cancer cells, estrogen increases glucose uptake and reprograms glucose metabolism. Meanwhile, estrogen/ER activates the anticipatory unfolded protein response (UPR) preparing cancer cells for the increased protein production required for subsequent cell proliferation. Here, we report that thioredoxin-interacting protein (TXNIP) is an important regulator of glucose metabolism in ER+ breast cancer cells, and estrogen/ER increases glucose uptake and reprograms glucose metabolism via activating anticipatory UPR and subsequently repressing TXNIP expression. In 2 widely used ER+ breast cancer cell lines, MCF7 and T47D, we showed that MCF7 cells express high TXNIP levels and exhibit mitochondrial oxidative phosphorylation (OXPHOS) phenotype, while T47D cells express low TXNIP levels and display aerobic glycolysis (Warburg effect) phenotype. Knockdown of TXNIP promoted glucose uptake and Warburg effect, while forced overexpression of TXNIP inhibited glucose uptake and Warburg effect. We further showed that estrogen represses TXNIP expression and activates UPR sensor inositol-requiring enzyme 1 (IRE1) via ER in the breast cancer cells, and IRE1 activity is required for estrogen suppression of TXNIP expression and estrogen-induced cell proliferation. Our study suggests that TXNIP is involved in estrogen-induced glucose uptake and metabolic reprogramming in ER+ breast cancer cells and links anticipatory UPR to estrogen reprogramming glucose metabolism.

Role of Thioredoxin-Interacting Protein in Diabetic Fatty Kidney Induced by Advanced Glycation End-Products.

Advanced glycation end-products (AGEs) have been identified as the etiological factors associated with the fatty kidney. Thioredoxin-interacting protein (TXNIP) might be a mediator involved in AGE-induced fatty kidney. This study focused on investigating how TXNIP affected the AGE-mediated renal lipid deposition. In an in vivo experiment, the db/db mice injected with the lentiviral vector encoding shRNA targeting TXNIP were given the AIN-76 basal or the high-AGE diet. TXNIP-targeting siRNA-transfected human renal proximal tubular epithelial (HK-2) cells were exposed to AGE-BSA in a study in vitro. The results showed that the silencing of TXNIP reduced tubular lipid droplets and intracellular cholesterol content, as well as upregulated Insig-1 and downregulated HMGCoAR, LDLr, nSREBP-2, and SCAP in the kidneys of the db/db mice, the high-AGE-diet-fed db/db mice, and AGE-BSA-treated HK-2 cells. Furthermore, AGE-BSA enhanced SCAP-SREBP-2 complex formation while promoting their transportation to the Golgi apparatus. However, these could be inhibited by TXNIP silencing in the HK-2 cells. The above findings indicated that TXNIP knockdown mitigated the accumulation of renal tubular lipids in diabetes through the regulation of SCAP, thereby inhibiting the SCAP-SREBP-2 signaling pathway, resulting in reduced cholesterol uptake and synthesis. Therefore, TXNIP might be a potential therapeutic target to treat a diabetic fatty kidney.

ChREBP deficiency alleviates apoptosis by inhibiting TXNIP/oxidative stress in diabetic nephropathy

AimsIn the present study, we investigated the effect of carbohydrate responsive element binding protein (ChREBP) on the TXNIP/oxidative stress and apoptosis in diabetic nephropathy. MethodsChREBP−/− mice (8-week old) were produced using the CRISPR/Cas9 gene editing approach. Diabetes was induced in C57BL/6 mice with streptozotocin. HK-2 cells was transfected with plasmid containing either ChREBP shRNA or TXNIP siRNA. ResultsRenal expression of ChREBP and thioredoxin-interacting protein (TXNIP) was increased in patients with type 2 diabetes mellitus (T2DM) and diabetic mice. ChREBP deficiency improved renal function, apoptosis as well as endoplasmic reticulum (ER) stress in diabetic mice. In addition, ChREBP deficiency prevented expression levels of TXNIP and NADPH oxidase 4 (Nox4), 8-hydroxydeoxyguanosine (8-OHdG) and heme oxygenase-1 (HO-1) in diabetic kidneys. The increased urinary 8-OHdG level induced by diabetes was also attenuated in ChREBP deficiency mice. Similarly, HG was shown to induce ChREBP expression and nuclear translocation in HK-2 cells. HG-induced apoptosis was inhibited by transfection of ChREBP shRNA plasmid. Moreover, we found that knockdown of ChREBP suppressed HG-induced TXNIP and Nox4 expression, reactive oxygen species (ROS) generation and ER stress in HK-2 cells. Furthermore, TXNIP knockdown effectively abrogated HG-induced apoptosis in HK-2 cells. ConclusionsThese results suggest that ChREBP deficiency prevents diabetes-induced apoptosis via inhibiting oxidative stress and ER stress, highlighting ChREBP as a potential therapy target for diabetic nephropathy.