A transcriptional regulatory mechanism of genes in the tricarboxylic acid cycle in the heart.

The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through the coactivation of coupled transcription factors, such as estrogen-related receptor (ERR), nuclear respiratory factor 1 (Nrf1), GA-binding protein-a (Gabpa), and Yin Yang 1 (YY1). We hypothesized that PGC-1α plays an essential role in the transcription of TCA cycle genes. Thus, utilizing localization peaks of PGC-1α to TCA cycle gene promoters would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for the transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful in understanding how those genes are dysregulated in pathological conditions.

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia.

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.

Nampt Potentiates Antioxidant Defense in Diabetic Cardiomyopathy.

[Figure: see text].

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.

Proteomic analysis of mitochondrial biogenesis in cardiomyocytes differentiated from human induced pluripotent stem cells.

Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a twofold change at time points up to 120 days relative to 12 days were subjected to ingenuity pathway analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1-α), the insulin receptor (IR), and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease-a regulator of mitochondrial proteostasis, energetics, and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1, and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.

Perm1 regulates cardiac energetics as a downstream target of the histone methyltransferase Smyd1.

The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.

Yes-Associated Protein (YAP) Facilitates Pressure Overload-Induced Dysfunction in the Diabetic Heart.

Patients with diabetes are more prone to developing heart failure in the presence of high blood pressure than those without diabetes. Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is persistently activated in diabetic hearts, and YAP plays an essential role in mediating the exacerbation of heart failure in response to pressure overload in the hearts of mice fed a high-fat diet. YAP induced dedifferentiation of cardiomyocytes through activation of transcriptional enhancer factor 1 (TEAD1), a transcription factor. Thus, YAP and TEAD1 are promising therapeutic targets for diabetic patients with high blood pressure to prevent the development of heart failure.

Both gain and loss of Nampt function promote pressure overload-induced heart failure.

The heart requires high-energy production, but metabolic ability declines in the failing heart. Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD) synthesis. NAD is directly involved in various metabolic processes and may indirectly regulate metabolic gene expression through sirtuin 1 (Sirt1), an NAD-dependent protein deacetylase. However, how Nampt regulates cardiac function and metabolism in the failing heart is poorly understood. Here we show that pressure-overload (PO)-induced heart failure is exacerbated in both systemic Nampt heterozygous knockout (Nampt+/-) mice and mice with cardiac-specific Nampt overexpression (Tg-Nampt). The NAD level declined in Nampt+/- mice under PO (wild: 377 pmol/mg tissue; Nampt+/-: 119 pmol/mg tissue; P = 0.028). In cultured cardiomyocytes, Nampt knockdown diminished mitochondrial NAD content and ATP production (relative ATP production: wild: 1; Nampt knockdown: 0.56; P = 0.0068), suggesting that downregulation of Nampt induces mitochondrial dysfunction. On the other hand, the NAD level was increased in Tg-Nampt mice at baseline but not during PO, possibly due to increased consumption of NAD by Sirt1. The expression of Sirt1 was increased in Tg-Nampt mice, in association with reduced overall protein acetylation. PO-induced downregulation of metabolic genes was exacerbated in Tg-Nampt mice. In cultured cardiomyocytes, Nampt and Sirt1 cooperatively suppressed mitochondrial proteins and ATP production, thereby promoting mitochondrial dysfunction. In addition, Nampt overexpression upregulated inflammatory cytokines, including TNF-α and monocyte chemoattractant protein-1. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, whereas Nampt overexpression is detrimental during PO, possibly due to excessive activation of Sirt1, suppression of mitochondrial function, and upregulation of proinflammatory mechanisms.NEW & NOTEWORTHY Nicotinamide phosphoribosyl-transferase (Nampt) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide synthesis. We demonstrate that pressure overload-induced heart failure is exacerbated in both systemic Nampt heterozygous knockout mice and mice with cardiac-specific Nampt overexpression. Both loss- and gain-of-function models exhibited reduced protein acetylation, suppression of metabolic genes, and mitochondrial energetic dysfunction. Thus endogenous Nampt maintains cardiac function and metabolism in the failing heart, but cardiac-specific Nampt overexpression is detrimental rather than therapeutic.

Hippo Deficiency Leads to Cardiac Dysfunction Accompanied by Cardiomyocyte Dedifferentiation During Pressure Overload.

RATIONALE: The Hippo pathway plays an important role in determining organ size through regulation of cell proliferation and apoptosis. Hippo inactivation and consequent activation of YAP (Yes-associated protein), a transcription cofactor, have been proposed as a strategy to promote myocardial regeneration after myocardial infarction. However, the long-term effects of Hippo deficiency on cardiac function under stress remain unknown. OBJECTIVE: We investigated the long-term effect of Hippo deficiency on cardiac function in the presence of pressure overload (PO). METHODS AND RESULTS: We used mice with cardiac-specific homozygous knockout of WW45 (WW45cKO), in which activation of Mst1 (Mammalian sterile 20-like 1) and Lats2 (large tumor suppressor kinase 2), the upstream kinases of the Hippo pathway, is effectively suppressed because of the absence of the scaffolding protein. We used male mice at 3 to 4 month of age in all animal experiments. We subjected WW45cKO mice to transverse aortic constriction for up to 12 weeks. WW45cKO mice exhibited higher levels of nuclear YAP in cardiomyocytes during PO. Unexpectedly, the progression of cardiac dysfunction induced by PO was exacerbated in WW45cKO mice, despite decreased apoptosis and activated cardiomyocyte cell cycle reentry. WW45cKO mice exhibited cardiomyocyte sarcomere disarray and upregulation of TEAD1 (transcriptional enhancer factor) target genes involved in cardiomyocyte dedifferentiation during PO. Genetic and pharmacological inactivation of the YAP-TEAD1 pathway reduced the PO-induced cardiac dysfunction in WW45cKO mice and attenuated cardiomyocyte dedifferentiation. Furthermore, the YAP-TEAD1 pathway upregulated OSM (oncostatin M) and OSM receptors, which played an essential role in mediating cardiomyocyte dedifferentiation. OSM also upregulated YAP and TEAD1 and promoted cardiomyocyte dedifferentiation, indicating the existence of a positive feedback mechanism consisting of YAP, TEAD1, and OSM. CONCLUSIONS: Although activation of YAP promotes cardiomyocyte regeneration after cardiac injury, it induces cardiomyocyte dedifferentiation and heart failure in the long-term in the presence of PO through activation of the YAP-TEAD1-OSM positive feedback mechanism.

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling.

NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2- production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling.

Identification of novel nuclear targets of human thioredoxin 1.

The dysregulation of protein oxidative post-translational modifications has been implicated in stress-related diseases. Trx1 is a key reductase that reduces specific disulfide bonds and other cysteine post-translational modifications. Although commonly in the cytoplasm, Trx1 can also modulate transcription in the nucleus. However, few Trx1 nuclear targets have been identified because of the low Trx1 abundance in the nucleus. Here, we report the large-scale proteomics identification of nuclear Trx1 targets in human neuroblastoma cells using an affinity capture strategy wherein a Trx1C35S mutant is expressed. The wild-type Trx1 contains a conserved C32XXC35 motif, and the C32 thiol initiates the reduction of a target disulfide bond by forming an intermolecular disulfide with one of the oxidized target cysteines, resulting in a transient Trx1-target protein complex. The reduction is rapidly consummated by the donation of a C35 proton to the target molecule, forming a Trx1 C32-C35 disulfide, and results in the concurrent release of the target protein containing reduced thiols. By introducing a point mutation (C35 to S35) in Trx1, we ablated the rapid dissociation of Trx1 from its reduction targets, thereby allowing the identification of 45 putative nuclear Trx1 targets. Unexpectedly, we found that PSIP1, also known as LEDGF, was sensitive to both oxidation and Trx1 reduction at Cys 204. LEDGF is a transcription activator that is vital for regulating cell survival during HIV-1 infection. Overall, this study suggests that Trx1 may play a broader role than previously believed that might include regulating transcription, RNA processing, and nuclear pore function in human cells.

Hydrogen peroxide sensing and signaling by protein kinases in the cardiovascular system.

SIGNIFICANCE: Oxidants were once principally considered perpetrators of injury and disease. However, this has become an antiquated view, with cumulative evidence showing that the oxidant hydrogen peroxide serves as a signaling molecule. Hydrogen peroxide carries vital information about the redox state of the cell and is crucial for homeostatic regulation during health and adaptation to stress. RECENT ADVANCES: In this review, we examine the contemporary concepts for how hydrogen peroxide is sensed and transduced into a biological response by introducing post-translational oxidative modifications on select proteins. Oxidant sensing and signaling by kinases are of particular importance as they integrate oxidant signals into phospho-regulated pathways. We focus on CAMKII, PKA, and PKG, kinases whose redox regulation has notable impact on cardiovascular function. CRITICAL ISSUES: In addition, we examine the mechanism for regulating intracellular hydrogen peroxide, considering the net concentrations that may accumulate. The effects of endogenously generated oxidants are often modeled by applying exogenous hydrogen peroxide to cells or tissues. Here we consider whether model systems exposed to exogenous hydrogen peroxide have relevance to systems where the oxidant is generated endogenously, and if so, what concentration can be justified in terms of relevance to health and disease. FUTURE DIRECTIONS: Improving our understanding of hydrogen peroxide signaling and the sensor proteins that it can modify will help us develop new strategies to regulate intracellular signaling to prevent disease.

The protective role of the transmembrane thioredoxin-related protein TMX in inflammatory liver injury.

AIMS: Accumulating evidence indicates that oxidative stress is associated with inflammation, and the cellular redox status can determine the sensitivity and the final outcome in response to inflammatory stimuli. To control the redox balance, mammalian cells contain a variety of oxidoreductases belonging to the thioredoxin superfamily. The large number of these enzymes suggests a complex mechanism of redox regulation in mammals, but the precise function of each family member awaits further investigations. RESULTS: We generated mice deficient in transmembrane thioredoxin-related protein (TMX), a transmembrane oxidoreductase in the endoplasmic reticulum (ER). When exposed to lipopolysaccharide (LPS) and d-(+)-galactosamine (GalN) to induce inflammatory liver injury, mutant mice were highly susceptible to the toxicants and developed severe liver damage. LPS-induced production of inflammatory mediators was equivalent in both wild-type and TMX(-/-) mice, whereas neutralization of the proinflammatory cytokine tumor necrosis factor-α suppressed the toxic effects of LPS/GalN in the mutant mice. Liver transcriptional profiles revealed enhanced activation of the p53-signaling pathway in the TMX(-/-) mice after LPS/GalN treatment. Furthermore, TMX deficiency also caused increased sensitivity to thioacetamide, which exerts its hepatotoxicity through the generation of reactive oxygen species. INNOVATION: The present study is the first to address the role of the oxidoreductase TMX in inflammatory liver injury. The phenotype of mice deficient in TMX suggests a functional link between redox regulation in the ER and susceptibility to oxidative tissue damage. CONCLUSION: We conclude that TMX plays a major role in host defense under the type of inflammatory conditions associated with oxidative stress.

Suppression of ERR targets by a PPARα/Sirt1 complex in the failing heart.

Heart failure is a leading cause of death worldwide. Estrogen-related receptors (ERRs) are a nuclear receptor subfamily that facilitates the transcription of contractile and nucleus-encoded mitochondrial genes in the heart. Impaired expression of these ERR target genes is frequently observed in human heart failure patients. However, the responsible molecular mechanism is not well-understood. Recently, we have shown that PPARα forms a protein complex with Sirt1, which is involved in the downregulation of ERR targets through direct interaction with the ERR response element (ERRE) in the failing heart. Here, we provide additional lines of evidence supporting the pathological involvement of the PPARα/Sirt1 complex in heart failure. Pressure overload-induced left ventricular (LV) hypertrophy was attenuated in mice with heterozygous knockout of either PPARα (PPARα (+/-) ) or Sirt1 (Sirt1 (+/-) ), whereas cardiac-specific PPARα and Sirt1 bigenic mice showed LV hypertrophy accompanied by a high mortality rate even without pressure overload. Microarray analyses indicated that nuclear-encoded mitochondrial genes were largely downregulated and mitochondrial morphological abnormalities were observed in PPARα/Sirt1 bigenic mice. Those downregulated mitochondrial genes frequently harbor the ERRE in the promoter regions. Artificial and physiological PPARα ligands suppressed reporter genes driven by the ERREs. PPARα bound to and recruited Sirt1 to the genomic flanking region of the ERREs in the heart. Pressure overload downregulated many ERR targets, which were partly normalized by PPARα (+/-) and Sirt1 (+/-) mice. These results suggest that PPARα and Sirt1 downregulate ERR target gene expression through direct interaction with the ERRE in the failing heart.

Effect of CD4+ T-lymphocyte count on hospital outcome of elective general thoracic surgery patients with human immunodeficiency virus.

PURPOSE: Traditionally, the chief surgical indicator for human immunodeficiency virus (HIV)-infected patients was the CD4-positive T-lymphocyte count; however, there is no current consensus. Reports published after 2006 indicated that HIV-infected patients had a higher incidence of postoperative pneumonia and higher 12-month mortality rates. In addition, CD4 counts had no relation to the in-hospital outcome. Therefore, we retrospectively examined all of the previous patients who underwent operations in our department on the basis of these findings. METHODS: Regardless of the initiation of highly active anti-retroviral therapy (HAART), we retrospectively reviewed 10 general thoracic surgeries performed in our department according to the CD4 cell count, HIV-ribonucleic acid (RNA) viral load, time of HAART initiation, operating time, amount of blood, postoperative course, and period of observation. RESULTS: There was no incidence of postoperative pneumonia or wound infection. There were also no complications during the perioperative period. One patient died 7 months after surgery. CONCLUSION: Our retrospective study demonstrates that the indicator for elective general thoracic surgery is not the CD4-positive T-lymphocyte count and that the initiation of HAART may reduce the 12-month mortality rates. In HIV-positive patients, regardless of the CD4-positive T-lymphocyte count, surgeons can operate in the same manner as they would with HIV-negative patients.

Redox regulatory mechanism of transnitrosylation by thioredoxin.

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys(73) only after the formation of a Cys(32)-Cys(35) disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1(C32S/C35S) (a mutant Trx1 with both Cys(32) and Cys(35) replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys(173) and Cys(83) and protects it from H(2)O(2)-induced overoxidation. Functionally, we found that Cys(73)-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation.

Thioredoxin-binding protein-2 deficiency enhances methionine-choline deficient diet-induced hepatic steatosis but inhibits steatohepatitis in mice.

In nonalcoholic fatty liver disease, oxidative stress is believed to play a crucial role as a second-hit for the progression of simple steatosis to steatohepatitis. Thioredoxin (TRX) is a potent antioxidant molecule that exerts anti-apoptotic and anti-inflammatory functions. TRX-binding protein-2 (TBP-2) is an endogenous negative regulator of TRX. Deficiency of TBP-2 in mice causes hyperlipidemia, hepatic steatosis, hypoglycemia, and bleeding tendency, resembling Reye syndrome in a fasting/glucose-deficient state. The aim of this study was to investigate the role of TBP-2 in the development of nonalcoholic steatohepatitis (NASH). TBP-2-deficient (TBP-2(-/-)) and wild-type (WT) mice were fed either a normal or methionine-choline-deficient (MCD) diet for up to 10 weeks. Compared with WT mice, TBP-2(-/-) mice showed severe simple steatosis rather than steatohepatitis. However, oxidative stress determined by lipid peroxidation and DNA damage, neutrophil infiltration, and hepatic fibrosis were attenuated in TBP-2(-/-) mice. PCR analysis showed the expressions of fibrosis-inducing and inflammatory cytokine-related genes were less in TBP-2(-/-) mice. Moreover, leptin, SREBP1c, PPARgamma, and adipogenesis-lipogenesis-related genes were upregulated in TBP-2(-/-) mice. These results strongly suggested that TBP-2 might be involved in pathogenesis of NASH in WT mice, and inhibitors of TBP-2 could be useful in the prevention or treatment of NASH.

The role of redox modulation of class II histone deacetylases in mediating pathological cardiac hypertrophy.

Many biological functions in cells are regulated by the effects of the redox state on cellular signaling pathways. In the heart, pathological hypertrophy caused by a wide variety of stimuli is commonly mediated by nucleo-cytoplasmic translocation of class II histone deacetylases (HDACs) and subsequent de-suppression of transcription factors, including nuclear factor of activated T-cells and MEF2. One of the primary triggers of class II HDAC nuclear export is phosphorylation by HDAC kinases activated by hypertrophic stimuli. However, oxidative modification of conserved cysteine residues can also potentially induce nuclear export of class II HDACs. Thioredoxin 1 (Trx1), a 12 kDa anti-oxidant, inhibits pathological hypertrophy through reduction of cysteine residues in class II HDACs. In this review, we discuss the role of posttranslational modification of class II HDACs in mediating cardiac hypertrophy and the molecular mechanism by which Trx1 inhibits pathological cardiac hypertrophy.

Dendritic cells derived from TBP-2-deficient mice are defective in inducing T cell responses.

Thioredoxin-binding protein-2 (TBP-2), also known as vitamin D3-up-regulated protein 1 (VDUP1), was identified as an endogenous molecule interacting with thioredoxin (TRX). Here, we show that dendritic cells (DC) derived from TBP-2-deficient mice are defective in the function of T cell activation. To compare TBP-2(-/-) DC function with wild-type (WT) DC, we stimulated DC with lipopolysaccharide (LPS). Although TBP-2(-/-) DC and WT DC expressed comparable levels of MHC class II and costimulatory molecules such as CD40, CD80 and CD86, the IL-12p40, IL-12p70 and IL-6 productions of TBP-2(-/-) DC were attenuated. In a mixed leukocyte reaction (MLR), the concentrations of IL-2, IFN-gamma, IL-4 and IL-10 in the culture supernatant of MLR with TBP-2(-/-) DC were significantly lower than those in the cultures with WT DC. In MLR also, as with LPS stimulation, IL-12p40 and IL-12p70 production from TBP-2(-/-) DC was less than that from WT DC. Proliferation of T cells cultured with TBP-2(-/-) DC was poorer than that with WT DC. In vivo delayed-type hypersensitivity responses in TBP-2(-/-) mice immunized with ovalbumin were significantly reduced compared to WT mice. These results indicate that TBP-2 plays a crucial role in DC to induce T cell responses.