Nitric Oxide–Dependent Suppression of Thioredoxin-Interacting Protein Expression Enhances Thioredoxin Activity
Objective— Cellular redox balance is regulated by enzymatic and nonenzymatic systems and freely diffusible nitric oxide (NO) promotes antioxidative mechanisms. We show the NO-dependent transcriptional regulation of the antioxidative thioredoxin system.
Methods and Results— Incubation of rat pulmonary artery smooth muscle cells (RPaSMC) with the NO donor compound S-nitroso-glutathione (GSNO, 100 μmol/L) suppressed thioredoxin-interacting protein (Txnip), an inhibitor of thioredoxin function, by 71±18% and enhanced thioredoxin reductase 2.7±0.2 fold (n=6; both P<0.001 versus control). GSNO increased thioredoxin activity (1.9±0.5-fold after 4 hours; P<0.05 versus control). Promoter deletion analysis revealed that NO suppression of Txnip transcription is mediated by cis-regulatory elements between −1777 and −1127 bp upstream of the start codon. Hyperglycemia induced Txnip promoter activity (3.9±0.2-fold; P<0.001) and abolished NO effects (−37.4±1.0% at 5.6 mmol/L glucose versus 12.4±2.1% at 22.4 mmol/L glucose; P<0.05). Immunoprecipitation experiments demonstrated that GSNO stimulation and mutation of thioredoxin at Cys69, a site of nitrosylation, had no effect on the Txnip/thioredoxin interaction.
Conclusions— NO can regulate cellular redox state by changing expression of Txnip and thioredoxin reductase. This represents a novel antioxidative mechanism of NO independent of posttranslational protein S-nitrosylation of thioredoxin.
The thioredoxin system is a ubiquitous thiol-reducing system that includes thioredoxin, thioredoxin reductase, and NADPH.1 The thioredoxin system is an essential component of cellular redox balance, and targeted deletion of the thioredoxin gene in mice leads to early embryonic lethality.2 In addition to its antioxidative function, thioredoxin mediates anti-apoptotic effects through interaction with apoptosis-signaling kinase-1 (ASK-1) mediating its ubiquitin-dependent degradation.3,4 Furthermore, thioredoxin functions as a transcriptional co-activator through interaction with transcription factors such as NF-κB and ref1.5,6 The thioredoxin system is inhibited by thioredoxin-interacting protein (Txnip), which blocks thioredoxin’s antioxidative function.7–9 Several studies have identified Txnip as a critical regulator of diverse signaling events in mammalian cells because of its direct control of thioredoxin activity.10–13 Recently, S-nitrosylation of thioredoxin at Cys69 has been identified as a posttranslational mechanism enhancing thioredoxin antioxidative and anti-apoptotic activity both in vitro14 and in vivo.15
Nitric oxide (NO) has diverse functions including vasodilator, neurotransmitter and anti-thrombotic activities.16 In the cardiovascular system, the main physiological source of NO is the endothelium, although other cell types may be induced to synthesize NO, particularly after exposure to inflammatory cytokines.17 NO relaxes smooth muscle cells and controls vascular cell proliferation, migration, and apoptosis.18 Further, NO has antioxidative properties that remain incompletely understood.
Here we report that expression of the gene encoding Txnip is robustly suppressed by NO in rat pulmonary artery smooth muscle cells (RPaSMC). NO did not affect Txnip mRNA stability. Hyperglycemia enhanced Txnip expression and abolished NO’s suppressive effects; this induction was mediated by a carbohydrate-response element which was not responsive to exogenous NO. Further, NO simultaneously induced expression of thioredoxin reductase. The net effect of these transcriptional effects was to increase thioredoxin activity. NO and mutation of thioredoxin at Cys69, a site of nitrosylation, had no effect on the ability of Txnip to interact with thioredoxin. Our findings reveal a novel NO-mediated mechanism independent of S-nitrosylation leading to enhanced thioredoxin function.
Methods and Results
Primary cultures of RPaSMC were prepared from adult Sprague-Dawley rats as previously described. Cells were exposed to S-nitroso-glutathione (GSNO) (100 μmol/L), PAPA NONOate (NOC-15) (500 μmol/L), and S-nitroso-N-acetylpenicillamine (SNAP) (100 μmol/L); 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (10 μmol/L), an inhibitor of guanylate cyclase,19 for varying durations. Transfection of 293 cells was performed at 70% confluence followed by incubation for 48 hours.
RNA was extracted from RPaSMC and mRNA expression detected using specific cDNA probes against thioredoxin, Txnip, and thioredoxin reductase.
Quantitative Real-Time Polymerase Chain Reaction
Txnip gene expression was analyzed by real-time polymerase chain reaction (LightCycler, Roche) using specific oligonucleotides against Txnip and β-tubulin.
RPaSMC were harvested, cellular proteins isolated, and 50 μg of protein subjected to gel electrophoresis followed by transfer to nitrocellulose membranes. The membranes were blocked 5% nonfat milk/phosphate-buffered saline and then incubated with antibodies directed against Txnip, thioredoxin reductase, or thioredoxin.
Nuclear Run-off Experiments
Nuclear run-off assays were performed as previously described.20 cDNA probes were created using oligonucleotides for Txnip, β-tubulin, and thioredoxin.
Cells were pretreated with actinomycin D before stimulation. RNA was extracted and gene expression measured as described before.21
Plasmid Construction and Transient Transfection Experiments
The human Txnip promoter region including −1777 bp upstream of the ATG start codon was cloned from human genomic DNA using primers 1 to 6 (supplemental Table I, available online at http://atvb.ahajournals.org). Transcriptional activity was assessed under stimulation with GSNO at 5.6 mmol/L and 22.4 mmol/L glucose. Further, full-length human Txnip was cloned into a mammalian expression vector (pcDNA3.1, Invitrogen). Expression plasmids for human wild-type thioredoxin or mutant thioredoxin with a serine replacing cysteine 69 (C69S) were kindly provided by Dr Judith Haendeler (Molecular Cardiology, University of Frankfurt, Germany). Equal amounts of empty expression plasmids served as control vectors.
Protein G sepharose beads were incubated with anti-Txnip antibody and equal amounts of total protein lysates were incubated with antibody-bead complexes for 2 hours rotating at 4°C. Beads were washed 3 times, resuspended, and the supernatant electrophoresed. Signals were visualized by enhanced chemiluminescence.
Thioredoxin Activity Assay
Thioredoxin activity was measured using the insulin disulfide reduction assay as previously described.12
Measurement of Oxidative Stress
Cells were incubated with 2′,7′-DCFDA for 45 minutes, washed in phosphate-buffered saline, and fluorescence measured using a fluorometer (Perkin Elmer) at 595 nm.
All experiments were performed at least 3 times and data are expressed as mean±SD. Data were analyzed by Student t test or 1-way ANOVA with post-hoc analysis. P<0.05 was considered statistically significant.
NO Reduces Txnip and Enhances Thioredoxin Reductase Gene Expression
We performed Northern analyses using total RNA prepared from RPaSMC exposed to GSNO (100 μmol/L for 1, 2, 4, 6, and 16 hours; 0, 10, 100, and 500 μmol/L for 2 hours). GSNO decreased Txnip gene expression and increased thioredoxin reductase gene expression in a time- and dose-dependent manner in RPaSMC. Txnip mRNA levels decreased rapidly within 1 hour after exposure to GSNO (−71±18%) and returned to baseline 4 hours after exposure to GSNO (Figure 1A). Txnip mRNA levels decreased in RPaSMC exposed to 100 μmol/L and 500 μmol/L of GSNO for 2 hours (Figure 1B). Thioredoxin reductase mRNA levels increased in RPaSMC within 2 hours after exposure to GSNO (+2.7±0.2-fold) and returned to baseline 16 hours after exposure to GSNO (Figure 1A). The thioredoxin reductase mRNA levels increased with 100 μmol/L and 500 μmol/L GSNO after 2 hours of exposure (Figure 1B).
NO Decreases Txnip Protein Levels, Increases Thioredoxin Reductase Levels, and Enhances Thioredoxin Activity
Incubation of RPaSMC with GSNO decreased Txnip protein levels in a time- and dose-dependent manner (Figure 1C). Further, protein levels of thioredoxin reductase increased under GSNO stimulation (Figure 1D). The suppression of Txnip was also induced by treatment of RPaSMC with the NO donor compounds NOC-15 and SNAP (Figure 1E). In contrast, endogenous thioredoxin protein levels in RPaSMC remain unchanged throughout 4 hours of GSNO incubation (Figure 1C).
To examine the mechanisms by which NO regulates Txnip and thioredoxin reductase gene expression, we evaluated the role of soluble guanylate cyclase. RPaSMC were exposed to 100 μmol/L of GSNO for 2 hours (Txnip) and 4 hours (thioredoxin reductase) in the presence or absence of the soluble guanylate cyclase inhibitor ODQ at a concentration previously shown to inhibit guanylate cyclase in RPaSMC (10 μmol/L).19 Pretreatment with ODQ did not inhibit the GSNO-mediated changes in Txnip or thioredoxin reductase gene expression (Figure 1F). ODQ itself had no effect on gene expression of Txnip or thioredoxin reductase.
The changes in gene and protein expression of Txnip and thioredoxin reductase predict that GSNO will increase thioredoxin activity in RPaSMC. Consistent with previous findings in endothelial cells,14 GSNO increased thioredoxin activity in RPaSMC. Incubation with 100 μmol/L GSNO increased thioredoxin activity levels 1.6±0.3-fold at 1 hour (P<0.02 versus unstimulated cells), 1.7±0.2-fold at 2 hours (P<0.01 versus unstimulated cells), and 1.9±0.5-fold after 4 hours of stimulation (P<0.05 versus unstimulated cells) (Figure 1G). Further, GSNO increased thioredoxin activity 1.5±0.5-fold at 10 μmol/L (p=NS), 1.9±0.5-fold at 100 μmol/L (P<0.05 versus unstimulated cells), and 1.9±0.2-fold at 500 μmol/L (P<0.001 versus unstimulated cells) of GSNO stimulation (Figure 1H). The increase of thioredoxin activity after 4 hours of GSNO stimulation was reflected in a decrease by 53±15% in levels of hydrogen peroxide as measured by DCFDA fluorescence (P<0.05 versus unstimulated cells).
NO Suppresses Txnip mRNA Expression Without Changing Its Stability
To determine whether GSNO reduces Txnip mRNA accumulation by decreasing the rate of synthesis or by increasing the rate of degradation, RPaSMCs were pretreated with actinomycin D (5 μg/mL) to inhibit transcriptional activity and then exposed to 100 μmol/L GSNO for several time intervals. The half-life of Txnip mRNA was not affected by GSNO stimulation (1.0±0.2 hour versus 0.8±0.2 hour; P=NS) (Figure 2A). Using nuclear run-off experiments, we observed that de novo synthesis of Txnip mRNA was reduced in cells exposed to GSNO (Figure 2B). Assessment of specific radioactive count activity revealed a reduction of de novo Txnip mRNA levels from 0.28±0.03 to 0.19±0.05 (n=3; P<0.05), whereas levels of de novo thioredoxin mRNA remained stable (0.4±0.02 versus 0.39±0.15; n=3; P=NS).
NO Effects on Txnip Promoter Activity
While several studies have previously investigated the transcriptional regulation of thioredoxin reductase,22,23 little is known about the specific transcriptional regulation of Txnip expression by NO. To investigate the mechanisms underlying NO’s suppressive effects on Txnip expression, RPaSMCs were transfected with a series of constructs of the human Txnip promoter driving expression of firefly luciferase (Figure 3A). Basal expression of those constructs reveals that the Txnip promoter was active in RPaSMC’s (Figure 3B). Incubation of RPaSMCs transfected with the Txnip promoter constructs in the presence of GSNO strongly suppressed luciferase activity only in cells transfected with the full-length (−1777) Txnip promoter (−42±12% of unstimulated cells; P<0.001) (Figure 3C). This finding suggests the presence of an NO-responsive cis-regulatory element in the Txnip promoter between −1777 and −1127 bp upstream of the ATG codon.
High Glucose Prevents NO Effects on Txnip Promoter Activity
Increased glucose levels induce Txnip gene expression both in vitro and in vivo.21,24,25 We, therefore, tested whether high glucose (22.4 mmol/L) induces Txnip promoter activity in RPaSMC’s. Transfection of RPaSMC with full-length Txnip promoter constructs followed by incubation in high glucose (22.4 mmol/L) or low glucose (5.6 mmol/L) revealed a strong induction of Txnip promoter activity in RPaSMC’s under hyperglycemic conditions (3.8±0.3 fold of unstimulated cells; P<0.001) (Figure 3D). Incubation of transfected cells with GSNO reduced Txnip promoter activity at 5.6 mmol/L glucose (-45±6% versus unstimulated cells; P<0.05) but had no effect at 22.4 mmol/L demonstrating that NO’s effects are restricted to normoglycemic conditions.
Glucose stimulates Txnip expression in pancreatic β-cells through a carbohydrate response element (ChRE) (400 bp upstream of the ATG codon).24 To test whether this regulation also occurs in RPaSMCs, we transfected cells with promoter constructs containing the ChRE site (−400) and constructs with a mutated ChRE site (−400ΔChRE). Hyperglycemia induced a 2-fold induction of Txnip promoter activity in cells transfected with wild-type constructs. Mutation of the ChRE site completely abolished glucose’s induction of Txnip promoter activity (supplemental Figure I, available online at http://atvb.ahajournals.org).
GSNO Stimulation Does Not Affect the Thioredoxin/Txnip Interaction
It has been shown previously that S-nitrosylation of thioredoxin at Cys69 by NO enhances thioredoxin activity.14 Because Txnip may bind to thioredoxin at its reactive cysteine residues of thioredoxin,7–9 we tested whether NO modulates Txnip/thioredoxin binding. For these experiments, we used 293 cells because they have a higher transfection efficiency than do RPaSMC. Whereas 293 cells have very low levels of Txnip at baseline, transfection of cells with an expression plasmid resulted in robust protein expression of Txnip. To assess the role of thioredoxin independent from its S-nitrosylation at Cys69 by NO, wild-type thioredoxin and C69S-mutated thioredoxin, which is resistant to S-nitrosylation,14,15 were overexpressed in 293 cells. Cells were stimulated with 100 μmol/L GSNO for 2 hours. Immunoprecipitation of Txnip followed by Western analysis for Xpress–thioredoxin by anti-Xpress antibodies revealed no differences of Txnip/thioredoxin binding in the presence or absence of NO (Figure 4). These data show that the binding of Txnip to thioredoxin is NO-independent and that the regulation of Txnip levels by NO represents an additional regulatory mechanism by which NO modulates thioredoxin function.
In the current study, we provide evidence that exogenous administration of NO results in enhanced activity of thioredoxin through transcriptional regulation of Txnip, the endogenous inhibitor of thioredoxin, and thioredoxin reductase. NO suppresses expression of Txnip and induces expression of thioredoxin reductase through redox-dependent mechanisms independent of soluble guanylate cyclase. Promoter analysis revealed that cis-regulatory elements >1127 bp upstream of the ATG codon mediate NO’s effects on Txnip transcription. Hyperglycemia induced Txnip promoter activity through a carbohydrate-response element, which is not affected by NO stimulation. The interaction of thioredoxin with Txnip was not affected by stimulation with NO excluding S-nitrosylation of thioredoxin as a factor regulating the Txnip/thioredoxin interaction. These findings reveal a novel mechanism by which NO regulates thioredoxin activity.
The thioredoxin system is a major thiol reducing system of the cell and has antioxidative and anti-apoptotic functions mediated by reactive cysteines (Cys32 and Cys35).1 Thioredoxin forms a disulfide bond on oxidation and in turn is reduced by thioredoxin reductase and NADPH. Thioredoxin also binds to transcription factors as well as ASK-1 targeting the latter for ubiquitination and degradation.4,5,26 Recently, Txnip has been described as an endogenous inhibitor of thioredoxin.7–9 The interaction of thioredoxin with Txnip can lead to increased levels of reactive oxygen species.8,21 This mechanism may play a role in the pathogenesis of vascular oxidative stress in diabetes mellitus since hyperglycemia directly induces Txnip expression in vascular smooth muscle cells.21,25
The current study reveals a time- and concentration-dependent increase in thioredoxin activity in response to exogenous administration of NO in RPaSMC. Increased thioredoxin activity is accompanied by changes in expression of 2 central regulators of thioredoxin function: thioredoxin reductase and the thioredoxin inhibitor Txnip. The transcriptional regulation of these molecules is independent of the NO donor compound applied and was observed in cells stimulated with GSNO, NOC-15, and SNAP. Intriguingly, our results demonstrate a concentration-dependent regulation of these effects up to 500 μmol/L of GSNO which is consistent with the NO-dependent increase in thioredoxin activity.
NO signaling targets several transcription factors, ion channels, G-proteins, protein tyrosine kinases, Janus kinases, mitogen-activated protein kinases, and caspases.16 NO signals in part via activation of the soluble guanylate cyclase leading to increased cGMP production. cGMP effector proteins include cGMP-dependent protein kinase, cyclic nucleotide-regulated ion channels, and phosphodiesterases, which hydrolyze cGMP and/or cAMP.27 Our experiments show that the transcriptional regulation of Txnip and thioredoxin reductase by NO are independent of soluble guanylate cyclase.
Several studies have investigated the transcriptional regulation of thioredoxin reductase by NO. Park et al showed induction of thioredoxin reductase expression by NO and peroxynitrite and the inhibition of this mechanism by NAC.22 Recently, Sakurai et al demonstrated the induction of thioredoxin reductase by cadmium and identified an antioxidant response element in the thioredoxin reductase promoter to be responsible for this regulation.23 They also identified the transcriptional factor Nrf2 as a mediator of thioredoxin reductase induction in response to cadmium incubation. These findings are consistent with our current study demonstrating the redox-dependent activation of thioredoxin reductase expression by NO. We, therefore, concentrated our studies on the regulation of Txnip expression because less is known about the underlying transcriptional mechanisms controlling Txnip mRNA expression.
Previously, hyperglycemia has been identified as a strong inducer of Txnip expression by several groups.21,24,25,28 This induction is mediated by a carbohydrate-response element (also called USF-1 binding site) 400 bp 5′ to the start codon. Promoter deletion analysis in the current study revealed that cis-regulatory elements -1127 bp upstream of the start codon mediate NO’s effects on Txnip expression. Notably, GSNO stimulation does not affect the induction of Txnip promoter activity by hyperglycemia, whereas hyperglycemia completely abolishes NO’s effects on Txnip promoter activity. Therefore, the induction of Txnip by glucose and the suppression of Txnip by NO are most likely regulated through different transcriptional elements, which form a molecular balance regulating the expression levels of Txnip in RPaSMC.
The posttranslational modification of proteins by S-nitrosylation accounts for various biological effects of NO,29 and proteomic screening for S-nitrosylated proteins has revealed numerous protein targets which remain to be functionally characterized.15,30 Direct activation of thioredoxin’s antioxidative and antiapoptotic activity by NO has been linked to S-nitrosylation of thioredoxin at Cys69 both in vitro and in vivo.14, 15 As demonstrated by Haendeler et al, S-nitrosylation of thioredoxin at Cys69 but not Cys32 or Cys35 in response to NO increases thioredoxin’s activity and anti-apoptotic effects in endothelial cells.14 Further, infusion of S-nitrosylated thioredoxin has been shown to reduce myocardial ischemia/reperfusion injury15 and to ameliorate myosin-induced myocarditis.15 Notably, while S-nitrosylation reduces caspase activity and inhibits apoptosis,31 S-nitrosylation of Ras increases its activity32 indicating both enhancement or inhibition of cellular pathways caused by protein S-nitrosylation. The current study tested the effect of NO and the resulting S-nitrosylation of thioredoxin on the interaction between thioredoxin and Txnip. These immunoprecipitation experiments were performed in HEK293 cells because of the higher efficiency of transfection compared with RPaSMCs. Since HEK293 cells do not produce NO endogenously, NO was administered exogenously to the cells to study these effects. Mutation of the thioredoxin molecule with a substitution of Cys69 to Ser had no effect on the ability of Txnip to interact with thioredoxin. Therefore, NO-induced protein S-nitrosylation of thioredoxin does not inhibit the interaction with Txnip. We conclude that our findings reveal a novel mechanism by which NO activates the thioredoxin system through reduction of Txnip and enhanced expression of thioredoxin reductase.
An important function of thioredoxin is its effects as a transcriptional co-activator. On nuclear translocation, thioredoxin interacts directly with NF-κB and ref1.5,6 As previously reported, this mechanism is redox-dependent and can be blocked by antioxidants and overexpression of Txnip.10,12 Additional experiments have revealed a redox-dependent translocation of thioredoxin in response to incubation with GSNO that accompanies the GSNO-induced suppression of Txnip (data not shown). Intriguingly, a recent study has demonstrated nuclear localization of Txnip suggesting that it may also have a nuclear function.33 This suggests a role for thioredoxin and interaction with Txnip in the transcriptional events mediated by NO.
In conclusion, we have demonstrated a novel NO-dependent mechanism of enhanced thioredoxin activity through suppression of Txnip and increased expression of thioredoxin reductase. Our findings emphasize pivotal transcriptional effects downstream of NO signaling that contribute to the anti-apoptotic and antioxidative defense of the cell.
Sources of Funding
This work was supported, in part, by grants from the Deutsche Akademie der Naturforscher - Leopoldina (BMBF-LPD) (9901/8-41 to P.C.S.) and from the NIH (PO1 HL64858 to R.T.L.).
Current affiliation for P.C.S. is Department of Medicine, Boston University Medical Center, Boston, Mass.
↵*P.C.S. and H.L. contributed equally to this work.
Original received September 28, 2005; final version accepted September 13, 2006.
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