Hyperglycemia Promotes Oxidative Stress through Inhibition of Thioredoxin Function by Thioredoxin-interacting Protein

Increased intracellular reactive oxygen species (ROS) contribute to vascular disease and pro-atherosclerotic effects of diabetes mellitus may be mediated by oxidative stress. Several ROS-scavenging systems tightly control cellular redox balance; however, their role in hyperglycemia-induced oxidative stress is unclear. A ubiquitous antioxidative mechanism for regulating cellular redox balance is thioredoxin, a highly conserved thiol reductase that interacts with an endogenous inhibitor, thioredoxin-interacting protein (Txnip). Here we show that hyperglycemia inhibits thioredoxin ROS-scavenging function through p38 MAPK-mediated induction of Txnip. Overexpression of Txnip increased oxidative stress, while Txnip gene silencing restored thioredoxin activity in hyperglycemia. Diabetic animals exhibited increased vascular expression of Txnip and reduced thioredoxin activity, which normalized with insulin treatment. These results provide evidence for the impairment of a major ROS-scavenging system in hyperglycemia. These studies implicate reduced thioredoxin activity through interaction with Txnip as an important mechanism for vascular oxidative stress in diabetes mellitus. Increased intracellular reactive oxygen species (ROS) contribute to vascular disease and pro-atherosclerotic effects of diabetes mellitus may be mediated by oxidative stress. Several ROS-scavenging systems tightly control cellular redox balance; however, their role in hyperglycemia-induced oxidative stress is unclear. A ubiquitous antioxidative mechanism for regulating cellular redox balance is thioredoxin, a highly conserved thiol reductase that interacts with an endogenous inhibitor, thioredoxin-interacting protein (Txnip). Here we show that hyperglycemia inhibits thioredoxin ROS-scavenging function through p38 MAPK-mediated induction of Txnip. Overexpression of Txnip increased oxidative stress, while Txnip gene silencing restored thioredoxin activity in hyperglycemia. Diabetic animals exhibited increased vascular expression of Txnip and reduced thioredoxin activity, which normalized with insulin treatment. These results provide evidence for the impairment of a major ROS-scavenging system in hyperglycemia. These studies implicate reduced thioredoxin activity through interaction with Txnip as an important mechanism for vascular oxidative stress in diabetes mellitus. Vascular complications such as coronary disease, peripheral artery disease, and stroke are major determinants of morbidity and mortality of patients with diabetes mellitus (1Rosen P. Nawroth P.P. King G. Moller W. Tritschler H.J. Packer L. Diabetes Metab. Res. Rev. 2001; 17: 189-212Crossref PubMed Scopus (813) Google Scholar). Atherosclerosis accelerates in the diabetic state (1Rosen P. Nawroth P.P. King G. Moller W. Tritschler H.J. Packer L. Diabetes Metab. Res. Rev. 2001; 17: 189-212Crossref PubMed Scopus (813) Google Scholar, 2Park L. Raman K.G. Lee K.J. Lu Y. Ferran Jr., L.J. Chow W.S. Stern D. Schmidt A.M. Nat. Med. 1998; 4: 1025-1031Crossref PubMed Scopus (1023) Google Scholar, 3Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7065) Google Scholar). Increased formation of reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; Txnip, thioredoxin-interacting protein; SMC, smooth muscle cell; PDGF, platelet-derived growth factor; MAPK, mitogen-activated protein kinase; RNAi, small interfering RNA; DCFDA, 2′,7′-dichlorodihydrofluorecein diacetate; DHE, dihydroethidium bromide; STZ, streptozotocin; Ad, adenovirus; GFP, green fluorescent protein; PI, phosphatidylinositol; PKC, protein kinase C; Trx, thioredoxin. contributes to endothelial dysfunction, vessel wall thickening, and lesion formation, thereby playing a crucial role in the progressive deterioration of vascular function and structure (4Suh Y.A. Arnold R.S. Lassegue B. Shi J. Xu X. Sorescu D. Chung A.B. Griendling K.K. Lambeth J.D. Nature. 1999; 401: 79-82Crossref PubMed Scopus (1280) Google Scholar, 5Griendling K.K. Sorescu D. Lassegue B. Ushio-Fukai M. Arterioscler. Thromb. Vasc. Biol. 2002; 20: 2175-2183Crossref Scopus (829) Google Scholar). While low levels of ROS participate in important cellular signaling mechanisms (6Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar), increased formation of ROS results in cytotoxic oxidative stress (5Griendling K.K. Sorescu D. Lassegue B. Ushio-Fukai M. Arterioscler. Thromb. Vasc. Biol. 2002; 20: 2175-2183Crossref Scopus (829) Google Scholar). Several antioxidative systems tightly regulate cellular redox balance and control formation and reduction of ROS (5Griendling K.K. Sorescu D. Lassegue B. Ushio-Fukai M. Arterioscler. Thromb. Vasc. Biol. 2002; 20: 2175-2183Crossref Scopus (829) Google Scholar, 7Prieto-Alamo M.J. Jurado J. Gallardo-Madueno R. Monje-Casas F. Holmgren A. Pueyo C. J. Biol. Chem. 2000; 275: 13398-13405Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 8Nordberg J. Arner E.S. Free Radic. Biol. Med. 2001; 31: 1287-1312Crossref PubMed Scopus (2201) Google Scholar). Previously, oxidative stress in diabetes mellitus has been linked to enhanced production of superoxide anion by mitochondria (9Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3672) Google Scholar) and through protein kinase C-dependent activation of membranous NADPH oxidase (10Inoguchi T. Li P. Umeda F. Yu H.Y. Kakimoto M. Imamura M. Aoki T. Etoh T. Hashimoto T. Naruse M. Sano H. Utsumi H. Nawata H. Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1273) Google Scholar). However, the intracellular redox balance is maintained by ROS-scavenging systems, and the two major intracellular thiol-reducing mechanisms are the interacting glutathione and thioredoxin systems (8Nordberg J. Arner E.S. Free Radic. Biol. Med. 2001; 31: 1287-1312Crossref PubMed Scopus (2201) Google Scholar, 11Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 12Griendling K.K. Alexander R.W. Circulation. 1997; 96: 3264-3265PubMed Google Scholar). Thioredoxin reduces ROS through reversible oxidation of thioredoxin at two cysteine residues (Cys-32 and Cys-35); thioredoxin is then reduced by thioredoxin reductase and NAPDH (11Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Thioredoxin-interacting protein (Txnip), the endogenous inhibitor of thioredoxin also known as vitamin D3 up-regulated protein-1 (VDUP-1) (13Wang Y. De Keulenaer G.W. Lee R.T. J. Biol. Chem. 2002; 277: 26496-26500Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Schulze P.C. De Keulenaer G.W. Yoshioka J. Kassik K.A. Lee R.T. Circ. Res. 2002; 91: 689-695Crossref PubMed Scopus (132) Google Scholar) or thioredoxin-binding protein-2 (TBP-2) (15Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar), inhibits thioredoxin antioxidative function by binding to its redox-active cysteine residues (15Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar, 16Junn E. Han S.H. Im J.Y. Yang Y. Cho E.W. Um H.D. Kim D.K. Lee K.W. Han P.L. Rhee S.G. Choi I. J. Immunol. 2000; 164: 6287-6295Crossref PubMed Scopus (401) Google Scholar). We show here the inhibition of the thioredoxin ROS-scavenging system through induction of Txnip in hyperglycemia. In contrast to previous reports describing an increased formation of reactive oxygen species in hyperglycemia, our study demonstrates the functional inhibition of a major cytoplasmic antioxidant system in hyperglycemia. Moreover, the inhibition of thioredoxin function in hyperglycemia through increased interaction with its inhibitor Txnip forms the molecular basis of increased levels of the freely diffusible molecule hydrogen peroxide contributing to oxidative stress. This molecular interaction might play a central role in increasing levels of reactive oxygen species by reduced flow through the enzymatic pathways of ROS scavenging. Cell Culture—Human aortic smooth muscle cells (SMCs) were isolated from surgical specimens and cultured in Dulbecco's modified essential medium with 10% fetal calf serum. Cells were starved for 48 h in insulin-transferrin medium prior to experiments. Stimulation experiments were performed using glucose (Sigma), insulin (100 nm, Sigma), insulin-like growth factor-1 (IGF-1) (100 ng/ml, Upstate Biotechnology), PDGF-BB (4 ng/ml, Upstate Biotechnology). The following inhibitors were used: wortmannin (100 nm, Sigma), PD169316 (100 nm, Calbiochem), U0126 (10 μm, Calbiochem), GF109203X (5 μm, Calbiochem), and pertussis toxin (500 ng/ml, Sigma). Northern and Western Analyses—For the detection of mRNA transcripts by Northern analysis, specific cDNA probes were synthesized using the following oligonucleotides: thioredoxin, 5′-AGCAGCCAAGATGGTGAAGCAGA-3′ and 5′-GCTCCAGAAAATTCACCCACC-3′; Txnip, 5′-TCTGCCAAAAAGGAGAAGAAAG-3′ and 5′-GGCGTACATAAAGATAGGGCTG-3′. Total RNA was isolated and loaded to a 1% agarose formaldehyde gel. After transfer, the membranes were incubated with radioactive labeled probes, and specific binding was visualized with autoradiography. Western analysis was performed using a specific monoclonal anti-Txnip antibody that was raised against full-length human Txnip. Thioredoxin was detected by a monoclonal anti-thioredoxin antibody (Medical Biological Laboratories International). After incubation with horseradish peroxidase-conjugated secondary antibody, specific bands were visualized by enzymatic chemiluminescence reaction (PerkinElmer Life Sciences). Total Akt, phospho-Akt, total p38 MAPK, and phospho-p38 MAPK were detected using specific antibodies (Cell Signaling). Real Time PCR—Txnip gene expression was analyzed by real time PCR (LightCycler, Roche Applied Science) using specific oligonucleotides: human Txnip, 5′-TGGTGGATGTCAATACCCCT-3′ (sense) and 5′-ATTGGCAAGGTAAGTGTGGC-3′ (antisense); human β-tubulin, 5′-TCTGTTCGCTCAGGTCCTTT-3′ (sense) and 5′-TTCATGATGCGATCAGGGTA-3′ (antisense); rat Txnip, 5′-CAAGTTCGGCTTTGAGCTTC-3′ (sense) and 5′-GCCATTGGCAAGGTAAGTGT-3′ (antisense); rat β-tubulin, 5′-CATCCAGGAGCTCTTCAAGC-3′ (sense) and 5′-CGCCTTAGGCCTCTTCTTCT-3′ (antisense); human thioredoxin, 5′-AGCAGCCAAGATGGTGAAGCAGA-3 (sense) and 5′-GCTCCAGAAAATTCACCCACC-3′ (antisense); rat thioredoxin, 5′-GCTGATCGAGAGCAAGGAAG-3 (sense) and 5′-TCAAGGAACACCACATTGGA-3′ (antisense). Adenoviral Vectors for Gene Transfer—Adenoviral vectors for overexpression of TRX (AdTRX) and Txnip (AdTxnip) were used as described elsewhere (14Schulze P.C. De Keulenaer G.W. Yoshioka J. Kassik K.A. Lee R.T. Circ. Res. 2002; 91: 689-695Crossref PubMed Scopus (132) Google Scholar). Gene Silencing by Small Interfering RNA (RNAi)—Double-stranded RNAi (Qiagen) for selective silencing of Txnip (rACAGACUUCGGAGUACCUGdTT) was transfected into cells (FuGENE Reagent, Roche Applied Science). After transfection, cells were incubated with glucose for Txnip expression. Scrambled RNAi was used as control. Measurement of Oxidative Stress—Cells were incubated with 2′,7′-dichlorodihydrofluorecein diacetate (DCFDA) for 45 min, washed in phosphate-buffered saline, and fluorescence intensity measured using a fluorometer (PerkinElmer Life Science) at 595 nm. Tissue levels of ROS were detected on frozen sections by incubation with dihydroethidium bromide (DHE) for 30 min at 37 °C followed by fluorescence microscopy. Thioredoxin Activity Assay—Thioredoxin activity was measured using the insulin disulfide reduction assay as described elsewhere (11Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). In brief, 50 μg of cellular protein extracts were incubated at 37 °C for 15 min with activation buffer to reduce thioredoxin. After addition of reaction buffer, the reaction was started with 5 μl of bovine thioredoxin reductase (American Diagnostica Inc., Greenwich, CT) or 5 μl of water to controls and samples incubated for 20 min at 37 °C. The reaction was terminated by adding 250 μl of stopping buffer followed by absorption measurement at 412 nm. In Vivo Model of Diabetes Mellitus—Streptozotocin (STZ, 60 mg/kg) was injected intraperitoneally in adult Sprague-Dawley rats. The animals were either sacrificed 4 weeks after injection of STZ or with insulin (10 animals as Vascular were isolated from the of the with the and of the on and at the Diabetes and Txnip were detected by incubation with antibody followed by incubation with secondary antibody binding was visualized by in experiments were performed at and are as The were analyzed by analysis of with analysis was used for the analysis of of two was of Txnip in Oxidative increased oxidative stress contributes to the of vascular complications in we the of thioredoxin antioxidative function in hyperglycemia. aortic incubated with increasing of glucose a reduction of thioredoxin activity Thioredoxin be through interaction with hydrogen peroxide (11Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar), which be measured by the and (6Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar). of with glucose increased intracellular levels of ROS as by fluorescence in a (10 glucose and incubation the expression of thioredoxin mRNA or protein levels However, incubation with glucose expression of Txnip in cells with glucose after cultured for 48 h with glucose and cells cultured in medium with insulin on the of Txnip that glucose and Txnip expression studies that the hyperglycemia-induced in Txnip protein levels enhanced the protein interaction The induction of Txnip by glucose was also in and cells such as rat human endothelial cells cells and of thioredoxin through Txnip to be a ubiquitous Overexpression of Txnip using gene in increased cellular levels of ROS at glucose and at glucose In overexpression of thioredoxin the in ROS at and glucose These are with previous reports the functional effects of gene of Txnip and thioredoxin (13Wang Y. De Keulenaer G.W. Lee R.T. J. Biol. Chem. 2002; 277: 26496-26500Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 14Schulze P.C. De Keulenaer G.W. Yoshioka J. Kassik K.A. Lee R.T. Circ. Res. 2002; 91: 689-695Crossref PubMed Scopus (132) Google Scholar). Overexpression of Txnip results in increased protein levels of Txnip and functional inhibition of thioredoxin activity by In gene of thioredoxin thioredoxin protein levels and thioredoxin activity by (14Schulze P.C. De Keulenaer G.W. Yoshioka J. Kassik K.A. Lee R.T. Circ. Res. 2002; 91: 689-695Crossref PubMed Scopus (132) Google Scholar). 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However, the of full-length thioredoxin and participate in the of ROS M. S. J. Cell PubMed Scopus Google Scholar). The endogenous thioredoxin inhibitor Txnip to the of thioredoxin and inhibits thioredoxin activity (15Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). Txnip with thioredoxin-binding such as E. Han S.H. Im J.Y. Yang Y. Cho E.W. Um H.D. Kim D.K. Lee K.W. Han P.L. Rhee S.G. Choi I. J. Immunol. 2000; 164: 6287-6295Crossref PubMed Scopus (401) Google Scholar) and inhibits the of through Y. W. Circ. Res. 2002; PubMed Scopus Google Scholar), thereby cells to (13Wang Y. De Keulenaer G.W. Lee R.T. J. Biol. Chem. 2002; 277: 26496-26500Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In Txnip the of thioredoxin (14Schulze P.C. De Keulenaer G.W. Yoshioka J. Kassik K.A. Lee R.T. Circ. Res. 2002; 91: 689-695Crossref PubMed Scopus (132) Google Scholar) and Txnip has been described as an intracellular protein that interacts with thioredoxin at its redox-active cysteine residues (15Nishiyama A. Matsui M. Iwata S. Hirota K. Masutani H. Nakamura H. Takagi Y. Sono H. Gon Y. Yodoi J. J. Biol. Chem. 1999; 274: 21645-21650Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). the interaction in the we that interacts with However, the of thioredoxin, also has the which the interaction with Txnip G. E. A. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). are known of Txnip, we interaction in results the inhibition of thioredoxin activity in hyperglycemia, which be by the specific induction of Txnip, the endogenous inhibitor of thioredoxin. the interaction to a functional inhibition of antioxidative thioredoxin This specific interaction results in a of the cellular redox balance that increased intracellular oxidative stress. While previous reports described an increased formation of ROS (9Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3672) Google Scholar, T. Li P. Umeda F. Yu H.Y. Kakimoto M. Imamura M. Aoki T. Etoh T. Hashimoto T. Naruse M. Sano H. Utsumi H. Nawata H. Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1273) Google Scholar), our the functional inhibition of a central cellular system in diabetes mellitus. A mechanism to oxidative stress in hyperglycemia is the increased formation of superoxide anion by the mitochondria (9Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3672) Google Scholar). anion major pathways of cellular in the increased through the activation of PKC, and the formation of M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7065) Google Scholar, T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3672) Google Scholar). Increased formation of superoxide anion has been to increased production of and that in to an of the with production of superoxide anion M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7065) Google Scholar). be by overexpression of protein-1 to a of the or overexpression of superoxide to increased of superoxide anion (9Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3672) Google Scholar). The activation of a role in the activation of the NADPH oxidase in of superoxide production (10Inoguchi T. Li P. Umeda F. Yu H.Y. Kakimoto M. Imamura M. Aoki T. Etoh T. Hashimoto T. Naruse M. Sano H. Utsumi H. Nawata H. Diabetes. 2000; 49: 1939-1945Crossref PubMed Scopus (1273) Google Scholar). anion is also known to with the highly reactive that in to of endothelial Shi C. J. 2002; PubMed Scopus Google Scholar). of also is of its or the of endothelial results in the production of molecular oxygen at the formation of Shi C. J. 2002; PubMed Scopus Google Scholar, S. L. T. J. PubMed Scopus Google Scholar). on the results of the mechanisms might also contribute to increased levels of hydrogen peroxide in hyperglycemia. Gene silencing of Txnip reduction of thioredoxin activity and reduces intracellular levels of ROS in hyperglycemia. overexpression of thioredoxin reduces hyperglycemia-induced levels of ROS, while overexpression of Txnip oxidative stress. Gene of Txnip reduced the hyperglycemia-induced in oxidative stress with controls at However, hyperglycemia levels of hydrogen peroxide in cells transfected with Txnip RNAi as measured by the and that interacts with hydrogen This might levels of reactive oxygen species that the of the thioredoxin system in the of the endogenous inhibitor Txnip. superoxide glutathione and central in the antioxidative mechanisms of the are a mechanism of the A. P. P. P. Diabetes. PubMed Google Scholar). In increased levels of hydrogen peroxide that from by thioredoxin are known to the activity S. L. S. T. Arterioscler. Thromb. Vasc. Biol. 2002; PubMed Scopus Google Scholar) and NADPH oxidase Jr., H.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). These mechanisms in to an of superoxide the induction of Txnip in hyperglycemia a molecular mechanism that results in a reduced of the antioxidative thioredoxin system to increased oxidative stress. The the activation of Txnip, while activation of p38 MAPK a in the increased expression of Txnip in hyperglycemia. inhibits activation of however, we the mechanisms of reduced activity of the in hyperglycemia. for reduced activity in hyperglycemia is the of which as a of K. S. M. Science. PubMed Scopus Google Scholar). binding to Akt, the activation of The induction of Txnip in hyperglycemia is to vascular of gene Txnip has been as of the with the induction in to glucose incubation in T. T. K. H. T. Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), cells A. M. B. 2002; PubMed Scopus Google Scholar), and cells T. S. T. H. H. Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, increased expression of Txnip was in an of diabetes mellitus T. S. T. H. H. Full Text Full Text PDF PubMed Scopus Google Scholar). The induction of Txnip by glucose to be a and be detected to h after in cells T. S. T. H. H. Full Text Full Text PDF PubMed Scopus Google Scholar). a and of Txnip by glucose in hyperglycemia. In addition to its ROS effects to oxidative stress, Txnip may also play a crucial role in cellular in hyperglycemia by (13Wang Y. De Keulenaer G.W. Lee R.T. J. Biol. Chem. 2002; 277: 26496-26500Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). gene may effects with in the of in diabetes mellitus.