4-Hydroxynonenal Induces Adaptive Response and Enhances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2

Abstract

4-Hydroxynonenal (4-HNE) is one of the major end products of lipid peroxidation. It has been widely accepted that 4-HNE can induce oxidative stress, implicating into extensive stress-related diseases. In the present study, however, 4-HNE was found to exert adaptive cytoprotective effect at low concentrations, which was primarily through induction of thioredoxin reductase 1 (TR1) via transcriptional activation of NF-E2-related factor 2 (Nrf2). Pretreatment with 4-HNE at sublethal concentrations significantly protected PC12 cells against the subsequent oxidative cell death induced by H2O2 and 6-hydroxydopamine. The cellular antioxidative glutathione system did not show any considerable changes, whereas the TR1 activity as well as the mRNA level was significantly elevated by the 4-HNE treatment. Cells treated with TR1 small interfering RNA exhibited less resistance to oxidative stress, and the adaptive response was completely abolished. The Nrf2 was transcriptionally activated by 4-HNE. Cells treated with Nrf2-small interfering RNA exerted lower constitutive levels of TR1 and exhibited less resistance to oxidative stress, and the 4-HNE-induced TR1 expression and subsequent adaptive response were again abolished in such cells. Treatment with 4-HNE at the adaptive concentration induced transient activation of extracellular signal-regulated protein kinase 1/2 and Akt/protein kinase B. Pharmacological inhibition of both these kinase pathways effectively attenuated 4-HNE-induced TR1 expression and subsequent adaptive protection. The above findings, taken together, suggest that stimulation with 4-HNE at sublethal concentrations induces adaptive response and enhances cell tolerance, primarily through induction of TR1 via transcriptional activation of Nrf2 signaling pathway, thereby protecting cells against the forthcoming oxidative stress. 4-Hydroxynonenal (4-HNE) is one of the major end products of lipid peroxidation. It has been widely accepted that 4-HNE can induce oxidative stress, implicating into extensive stress-related diseases. In the present study, however, 4-HNE was found to exert adaptive cytoprotective effect at low concentrations, which was primarily through induction of thioredoxin reductase 1 (TR1) via transcriptional activation of NF-E2-related factor 2 (Nrf2). Pretreatment with 4-HNE at sublethal concentrations significantly protected PC12 cells against the subsequent oxidative cell death induced by H2O2 and 6-hydroxydopamine. The cellular antioxidative glutathione system did not show any considerable changes, whereas the TR1 activity as well as the mRNA level was significantly elevated by the 4-HNE treatment. Cells treated with TR1 small interfering RNA exhibited less resistance to oxidative stress, and the adaptive response was completely abolished. The Nrf2 was transcriptionally activated by 4-HNE. Cells treated with Nrf2-small interfering RNA exerted lower constitutive levels of TR1 and exhibited less resistance to oxidative stress, and the 4-HNE-induced TR1 expression and subsequent adaptive response were again abolished in such cells. Treatment with 4-HNE at the adaptive concentration induced transient activation of extracellular signal-regulated protein kinase 1/2 and Akt/protein kinase B. Pharmacological inhibition of both these kinase pathways effectively attenuated 4-HNE-induced TR1 expression and subsequent adaptive protection. The above findings, taken together, suggest that stimulation with 4-HNE at sublethal concentrations induces adaptive response and enhances cell tolerance, primarily through induction of TR1 via transcriptional activation of Nrf2 signaling pathway, thereby protecting cells against the forthcoming oxidative stress. “Oxidative stress” was defined as a disturbance in the prooxidant-antioxidant balance in favor of the prooxidant (1Sies H. Oxidative Stress. Academic Press, Ltd., London1985: 1-8Crossref Google Scholar). However, even if the level of stress is less than that of antioxidant capacity, stress functions as a signal to which the body responds irrespective of its level. When the stress level exceeds defense capacity, it may induce oxidative damage, whereas the low level stress may stimulate defense network and acts as a good stress, “eustress.” 4-Hydroxynonenal (4-HNE) 2The abbreviations used are: 4-HNE4-hydroxynonenalAREantioxidant response elementHO-1heme oxygenase-1Keap1kelch-like ECH-associated protein 1Nrf2NF-E2-related factor 26-OHDA6-hydroxydopamineROSreactive oxygen speciessiRNAsmall interfering RNATrxthioredoxinTRthioredoxin reductaseMTTdimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideRTreal timePBSphosphate-buffered saline.2The abbreviations used are: 4-HNE4-hydroxynonenalAREantioxidant response elementHO-1heme oxygenase-1Keap1kelch-like ECH-associated protein 1Nrf2NF-E2-related factor 26-OHDA6-hydroxydopamineROSreactive oxygen speciessiRNAsmall interfering RNATrxthioredoxinTRthioredoxin reductaseMTTdimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideRTreal timePBSphosphate-buffered saline. is one of the major end products of lipid peroxidation and has been found to induce oxidative stress, as it is involved in the pathogenesis of a number of degenerative diseases such as Alzheimer disease (2Sayre L.M. Zelasko D.A. Harris P.L. Perry G. Salomon R.G. Smith M.A. J. Neurochem. 1997; 68: 2092-2097Crossref PubMed Scopus (913) Google Scholar), atherosclerosis (3Uchida K. Toyokuni S. Nishikawa K. Kawakishi S. Oda H. Hiai H. Stadtman E.R. Biochemistry. 1994; 33: 12487-12494Crossref PubMed Scopus (229) Google Scholar), cataract (4Awasthi S. Srivatava S.K. Piper J.T. Singhal S.S. Chaubey M. Awasthi Y.C. Am. J. Clin. Nutr. 1996; 64: 761-766Crossref PubMed Scopus (116) Google Scholar), and cancer (5Hammer A. Ferro M. Tillian H.M. Tatzber F. Zollner H. Schauenstein E. Schaur R.J. Free Radic. Biol. Med. 1997; 23: 26-33Crossref PubMed Scopus (43) Google Scholar). However, increasing evidence has suggested that 4-HNE at low concentrations takes an important role in cell signal transduction and gene expression (6Forman H.J. Dickinson D.A. Free Radic. Biol. Med. 2004; 37: 594-596Crossref PubMed Scopus (37) Google Scholar, 7Barrera G. Pizzimenti S. Dianzani M.U. Free Radic. Biol. Med. 2004; 37: 597-606Crossref PubMed Scopus (56) Google Scholar, 8Awasthi Y.C. Yang Y. Tiwari N.K. Patrick B. Sharma A. Li J. Awasthi S. Free Radic. Biol. Med. 2004; 37: 607-619Crossref PubMed Scopus (198) Google Scholar, 9Petersen D.R. Doorn J.A. Free Radic. Biol. Med. 2004; 37: 937-945Crossref PubMed Scopus (327) Google Scholar, 10Leonarduzzi G. Robbesyn F. Poli G. Free Radic. Biol. Med. 2004; 37: 1694-1702Crossref PubMed Scopus (120) Google Scholar, 11Hayes J.D. Flanagan J.U. Jowsey I.R. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 51-88Crossref PubMed Scopus (2832) Google Scholar). 4-HNE can react in Michael addition across its carbon-carbon double bond with a wide variety of cellular components, including DNA and proteins (12Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar). Thus, it has been suggested that 4-HNE could act as a potential activator of NF-E2-related factor 2 (Nrf2) and induce the expression of phase II detoxification enzymes (13Ishii T. Itoh K. Ruiz E. Leake D.S. Unoki H. Yamamoto M. Mann G.E. Circ. Res. 2004; 94: 609-616Crossref PubMed Scopus (344) Google Scholar, 14Levonen A.L. Landar A. Ramachandran A. Ceaser E.K. Dickinson D.A. Zanoni G. Morrow J.D. Darley-Usmar V.M. Biochem. J. 2004; 378: 373-382Crossref PubMed Scopus (474) Google Scholar, 15Numazawa S. Ishikawa M. Yoshida A. Tanaka S. Yoshida T. Am. J. Physiol. Cell Physiol. 2003; 285: 334-342Crossref PubMed Scopus (267) Google Scholar). 4-hydroxynonenal antioxidant response element heme oxygenase-1 kelch-like ECH-associated protein 1 NF-E2-related factor 2 6-hydroxydopamine reactive oxygen species small interfering RNA thioredoxin thioredoxin reductase dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide real time phosphate-buffered saline. 4-hydroxynonenal antioxidant response element heme oxygenase-1 kelch-like ECH-associated protein 1 NF-E2-related factor 2 6-hydroxydopamine reactive oxygen species small interfering RNA thioredoxin thioredoxin reductase dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide real time phosphate-buffered saline. The transcription factor Nrf2 has been implicated as the central protein that interacts with the antioxidant response element (ARE) to activate gene transcription constitutively or in response to an oxidative stress signal (16Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (520) Google Scholar). Under homeostatic conditions, Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and facilitates the degradation of Nrf2 via the proteasome system (17Dinkova-Kostova A.T. Holtzclaw W.D. Cole R.N. Itoh K. Wakabayashi N. Katoh Y. Yamamoto M. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11908-11913Crossref PubMed Scopus (1557) Google Scholar). Upon stimulation, Nrf2 dissociates from its cytoplasmic inhibitor Keap1, translocates to the nucleus, and by heterodimerizing with a small Maf protein, transactivates the expression of ARE-dependent genes (18Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. Chem. 1999; 274: 26071-26078Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, 19Ramos-Gomez M. Kwak M.K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (968) Google Scholar, 20Itoh K. Tong K.I. Yamamoto M. Free Radic. Biol. Med. 2004; 36: 1208-1213Crossref PubMed Scopus (714) Google Scholar). Thioredoxin reductase (TR), thioredoxin (Trx), and NADPH comprise a highly conserved, ubiquitous system (21Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (747) Google Scholar) that plays an important role in the redox regulation of multiple intracellular processes, including DNA synthesis, transcriptional regulation, cell growth, and resistance to cytotoxic agents that induce oxidative stress and apoptosis (22Arner E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (1956) Google Scholar, 23Rundlof A.K. Arner E.S. Antioxid. Redox Signal. 2004; 6: 41-52Crossref PubMed Scopus (146) Google Scholar). TR can catalyze the NADPH-dependent reduction of the redox protein Trx as well as of other endogenous and exogenous compounds, including lipoic acid (24Arner E.S. Nordberg J. Holmgren A. Biochem. Biophys. Res. 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Physiol. 2005; PubMed Scopus Google Scholar). 4-HNE at low concentrations has been suggested to an important role in cell signal the not In the present to the adaptive response of 4-HNE in PC12 cells. found that stimulation of PC12 cells with sublethal concentrations of 4-HNE induced adaptive response and protected cells against the subsequent oxidative stress, which was through induction of TR1 via transcriptional activation of the Nrf2 signaling and were from NADPH was the of was from 4-HNE was from and were from tetrazolium bromide was from extracellular signal-regulated protein kinase and were from oxygenase-1 was from The of was of Cell other were of the Cell cells were in with and with and Cell were at in a of of Cell the cell the reduction was The treated cells were with in at 1 was to the by and with a the was completely The of was at a of the were the TR1 as Y. Yoshida Y. T. K. E. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were and the cell were into at and and by The cell was to the at to the TR1 the TR1 activity 1 and were used as Y. Yoshida Y. T. K. E. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of NADPH was at The TR1 activity was by of and as of and were and by to the by The and and and and The was into PC12 cells at a concentration of cells by RNA was from cells the was with a RNA time was an system and the was with the acid The gene of was used as an endogenous The are: and and or of cells were as 2004; PubMed Scopus Google Scholar). The protein concentration was by the protein were with and a were to a The were 1 at in in of were in phosphate-buffered with The were at with the with and the were with the in with 1 at The were again in and and the proteins were with 1 in with the and of at as the and by of were to 4-HNE in PC12 an the adaptive response by 4-HNE in PC12 the was used the 4-HNE-induced concentrations than 4-HNE significantly the cell in PC12 cells the above was as a at which 4-HNE in PC12 and concentrations that were to the adaptive effect of 4-HNE. Pretreatment with 4-HNE significantly protected PC12 cells against cell death induced by the subsequent of H2O2 or which both been to induce oxidative in system and implicated in D. S. N. M. A.L. 2001; PubMed Scopus Google Scholar, E. S. J. 2000; Scholar). TR1 the 4-HNE-induced to the such an adaptive response in PC12 cells. The cellular antioxidative glutathione including cellular glutathione glutathione glutathione and glutathione did not show any considerable in cells treated with 4-HNE not whereas the TR1 activity was significantly elevated by the with sublethal concentrations of 4-HNE The TR1 mRNA was significantly induced by 4-HNE at with the at the role of TR1 in the 4-HNE-induced adaptive the effect of was PC12 cells treated with exhibited a of the constitutive mRNA levels of as well as the TR1 activity of and the cells a less resistance to oxidative stress In such cells treated with 4-HNE at and at significantly the cell whereas in cells treated with RNA did not any at the concentrations the adaptive response was completely abolished in the cells. Pretreatment of such cells with low concentrations of 4-HNE or did not induce any even the a role of TR1 in the 4-HNE-induced adaptive of Nrf2 in the of TR1 and in the induction of TR1 may by transcription including Nrf2 A. M. S. N. M. M. S. J. Physiol. 2005; PubMed Scopus Google Scholar). the of Nrf2 transcription in the expression of The Nrf2 level was by the of 4-HNE a of the Nrf2 signaling in the adaptive Cells treated with lower levels of constitutive Nrf2 protein and lower TR1 mRNA that of as well as TR1 activity of the 4-HNE-induced TR1 expression was completely abolished in cells treated with In such cells 4-HNE at low concentrations or did not induce any considerable the TR1 mRNA these suggested a role of the Nrf2 transcription in regulation of TR1 in PC12 cells. to the effect of with PC12 cells to the oxidative stress induced by 4-HNE or which both again than did in cells The adaptive response was abolished in such cells as well any considerable effect by 4-HNE at low concentrations of in the transcriptional activation of Nrf2 a of phase II including which is to induced by a wide variety of and cells against oxidative J. Cook J.L. 2003; PubMed Scopus Google Scholar, D. M. G. J. A. J. Biol. 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Free Radic. Biol. Med. 2004; 37: 597-606Crossref PubMed Scopus (56) Google Scholar, 8Awasthi Y.C. Yang Y. Tiwari N.K. Patrick B. Sharma A. Li J. Awasthi S. Free Radic. Biol. Med. 2004; 37: 607-619Crossref PubMed Scopus (198) Google Scholar, 9Petersen D.R. Doorn J.A. Free Radic. Biol. Med. 2004; 37: 937-945Crossref PubMed Scopus (327) Google Scholar, 10Leonarduzzi G. Robbesyn F. Poli G. Free Radic. Biol. Med. 2004; 37: 1694-1702Crossref PubMed Scopus (120) Google Scholar, 11Hayes J.D. Flanagan J.U. Jowsey I.R. Annu. Rev. Pharmacol. Toxicol. 2005; 45: 51-88Crossref PubMed Scopus (2832) Google Scholar), the by which 4-HNE in the signal transduction and induces adaptive response not In the present that 4-HNE at low concentrations could induce adaptive protecting PC12 cells against the subsequent oxidative stress, which was through induction of TR1 via transcriptional activation of The thioredoxin system of the antioxidant enzymes Trx and The the reduction of the in Trx as well as of other endogenous and exogenous compounds, including lipoic the cytotoxic lipid and the protein NADPH (21Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (747) Google Scholar, E.S. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (1956) Google Scholar, 23Rundlof A.K. Arner E.S. Antioxid. Redox Signal. 2004; 6: 41-52Crossref PubMed Scopus (146) Google Scholar, E.S. Nordberg J. Holmgren A. Biochem. Biophys. Res. Commun. 1996; 225: 268-274Crossref PubMed Scopus (157) Google Scholar, M. 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Res. 1996; Google Scholar). did not the role of Trx in the 4-HNE-induced adaptive response was The inhibition of TR in of the multiple functions of the thioredoxin system in DNA and as well as in the defense against oxidative inhibition of TR cells to oxidative stress, which TR a potential agents that induced oxidative stress D. Res. 2004; 64: PubMed Scopus Google Scholar). inhibition of TR activity the cancer of such as J. J. Holmgren A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In with the above it has been found in that inhibition of TR1 by abolished the adaptive response and PC12 cells to oxidative stress, a role of TR1 in the adaptive response as well as in the cell defense against oxidative stress. It has to in PC12 of at concentrations or significantly the cell not which is not TR1 and Trx been to an important role in cell and apoptosis (21Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (747) Google Scholar). The by which TR1 is not been A. M. S. N. M. M. S. J. Physiol. 2005; PubMed Scopus Google Scholar) that Nrf2 an important role in the expression of TR1 by in cells. H. J. Nutr. 2003; PubMed Scopus Google Scholar, K. J. Chem. 2005; PubMed Scopus Google Scholar) that a gene is transcriptionally by through an which has to the that the regulation of to phase II responds to transcriptional activation through the In the present the of Nrf2 transcription in the adaptive response and in the induction of Nrf2 was in the by the of which to the thereby the expression of including and The of PC12 cells with the constitutive TR1 mRNA level as well as the TR1 activity and abolished the 4-HNE-induced TR1 mRNA a role of Nrf2 in the regulation of TR1 in PC12 cells. It that the induction of in the present to a less role in the 4-HNE-induced adaptive it is widely accepted that is to of cells against oxidative stress D. M. G. J. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, P. B. Stewart D. M. J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, D. H. J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). In PC12 has been found as the major of the cytoprotective effect induced by factor M. Ruiz A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and Li Biochem. Biophys. Res. Commun. 2005; PubMed Scopus Google Scholar). such is whereas could as a potential cellular antioxidative TR1 a cells against oxidative in cells highly with may a The in that the activation of Nrf2 by 4-HNE at by the extracellular signal-regulated protein kinase and signaling protein kinase such as protein protein kinase and been to a role in Nrf2 activation D. M. G. J. A. J. Biol. 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Cell Physiol. 2003; 285: 334-342Crossref PubMed Scopus (267) Google Scholar). Under conditions, the cellular concentration of 4-HNE from to (12Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar, K. Res. 2003; PubMed Scopus Google Scholar). However, of oxidative stress, 4-HNE can concentrations of to (12Esterbauer H. Schaur R.J. Zollner H. Free Radic. Biol. Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, K. Res. 2003; PubMed Scopus Google Scholar). Thus, the present and other it to at of oxidative stress, the of 4-HNE and other lipid peroxidation products as a that an signal and cells of the oxidative thereby the response enzymes or proteins to with the forthcoming oxidative In the present that 4-HNE at sublethal concentrations induces adaptive in PC12 cells. Treatment of PC12 cells with 4-HNE to a of which to the thereby the cell against the forthcoming oxidative stress. an to the 4-HNE-induced adaptive at sublethal concentrations and to TR1 as a potential to the oxidative that induce adaptive in with TR1 as the the cell against oxidative stress or cells to oxidative