Relative Intensity 0 2 4 6 8 * Relative Intensity 0 50 100 150 ** Acrolein-induced activation of c-Jun-N-terminal kinase is mediated by alkylation of thioredoxin reductase 1 Matthew J. Randall 1 , Page C. Spiess 1 , Milena Hristova 1 , Robert J. Hondal 2 , Albert van der Vliet 1 1 Department of Pathology, 2 Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT Abstract ■ Cigarette smoke has profound health implications for several respiratory diseases including chronic obstructive pulmonary disease (COPD) and asthma. Specifically, cigarette smoke is known to increase mucus secretion and decrease epithelial barrier integrity [1,2]. The latter of which has been suggested to be a result of mitogen activated protein kinase (MAPK) activation, specifically c-Jun N-terminal kinase (JNK) [3]. ■ One component of cigarette smoke, the highly reactive aldehyde acrolein, can mimic many whole smoke-mediated epithelial responses [4,5]. This includes the depletion of the cellular antioxidant molecule glutathione [6] and activation of MAPKs including JNK [7,8]. Although acrolein can mimic the responses of cigarette smoke, the mechanism by which acrolein mediates activation of JNK and its downstream effects is not well defined. ■ Acrolein, an α,β-unsaturated aldehyde, forms adducts with cysteine (Cys), lysine and histidine amino acid residues [9,10]. Acrolein also has a high affinity for adduction to the selenocysteine (Sec) of thioredoxin reductase [11]. ■ Electrophilic adduction to thioredoxin reductase (TrxR) has been suggested to promote a gain-of-function which was identified to cause cellular apoptosis. This electrophile-adducted TrxR was coined a selenium compromised thioredoxin reductase apoptotic protein (SecTRAP) [12]. Hypothesis and Objective Based on the above literature, we hypothesized that acrolein adduction of TrxR1 would form a SecTRAP  with the potential to alter epithelial physiology. The present studies were designed to identify whether acrolein-adducted TrxR1 acquires a gain-of-function and futher identify how this modified protein, SecTRAP  effects the lung epithelium. ■ Acrolein adduction inhibits the reductase activity and enhances the NADPH consumption of mTrxR. As is known for other electrophiles, acrolein can therefore also modify TrxR to form a SecTRAP  . ■ Acrolein-mediated Trx1 redox status is independent of TrxR1 although alkylation of Trx1 is dependent on TrxR1 levels. This could indicate that although total Trx1 oxidation/alkylation is not dependent on TrxR after acrolein exposure, TrxR1 does impact the amount of reduced Trx1 available for alkylation. Potentially alkylated vs. oxidation of Trx1 is also important. ■ Acrolein adduction of TrxR1 and/or Trx1 is required for acrolein- mediated JNK1 phosphorylation which could lead to the identification of potential mechanisms by which acrolein is important for promotion of downstream epithelial pathologies resulting from cigarette smoke inhalation (PC SPIESS #131). Results (I) Methods Summary and Conclusions Cigarette smoking remains a major health concern, and many effects of cigarette smoke (CS) can be attributed to acrolein, a major electrophilic aldehyde within CS. Thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, has emerged as a highly susceptible target for electrophiles due to its unique nucleophile selenocysteine (Sec) residue. Based on previous studies indicating alkylation of TrxR1 at Sec can induce pro-apoptotic responses by a gain-of-function, we tested the importance of TrxR1 in acrolein-induced airway epithelial cell activation or injury. Human bronchial epithelial HBE1 cells exposed to acrolein (1-30 µM) resulted in dose- dependent loss of TrxR activity, which coincided with depletion of GSH and oxidation of Trx1. Acrolein- induced inactivation of TrxR1 was associated with its alkylation, as determined by biotin hydrazide labeling, and was independent of GSH status, whereas oxidation of Trx1 was potentiated by prior GSH depletion and attenuated after GSH supplementation. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we used siRNA silencing of TrxR1 or supplemented cells with selenite, which enhanced TrxR1 levels by about 3-fold. Induction of TrxR1 by selenite markedly enhanced the ability of acrolein to promote phosphorylation of the MAP kinases c-Jun N-terminal kinase (JNK) and extracellular regulated kinase (ERK), which was associated with increased alkylation of TrxR1. Conversely, acrolein- induced JNK phosphorylation and alkylation of TrxR1 were attenuated after siRNA silencing of TrxR1. Although these manipulations of TrxR1 by siRNA or selenite supplementation affected initial Trx1 redox status, they had no impact on total Trx1 oxidation/alkylation by acrolein. These findings indicate that the activation of JNK and ERK by acrolein are directly related to alkylation of TrxR1 and are independent of oxidation/alkylation of Trx1. In conclusion, we suggest that alkylation of TrxR1 may actively contribute to acrolein-induced epithelial alterations associated with JNK or ERK activation, such as alterations in epithelial integrity or epithelial cytokine production. Background Results (III) p-JNK1 (normalized to b-actin) 0 3 10 30 0 5 10 15 20 ** * [Acrolein] (  M) p-JNK2 (normalized to b-actin) 0 3 10 30 0 2 4 6 8 10 * No Supplement Na 2 SeO 3 [Acrolein] (  M) p-JNK2 (normalized to  -actin) 0 3 10 30 0 2 4 6 8 NT-siRNA TXNRD1 siRNA p-JNK1 (normalized to  -actin) 0 3 10 30 0 2 4 6 # # # p-JNK1 TrxR1 β-actin p-JNK2 JNK 1 JNK2 0 3 10 30 0 3 10 30 Na 2 SeO 3 Acr (µM) p-ERK 1/2 ERK 1/2 P-P38 P38 p-JNK 1 TrxR1 β-actin p-JNK 2 JNK 1 JNK 2 0 3 10 30 0 3 10 30 NT-siRNA TXNRD1 siRNA Acr (µM) p-ERK 1/2 ERK 1/2 p-P38 P38 Figure 5. Acrolein-mediated MAPK phosphorylation is significantly increased by increased cellular TrxR1. HBE1 cells (± Na 2 SeO 3 supplementation) treated with indicated concentrations of acrolein for 30 min were analyzed for p-JNK1/2, p-ERK1/2, and p-P38 by Western blot. Analysis of these blots was done by densitometry. Conclusion: Acrolein-mediated JNK phosphorylation is increased significantly as a result of increased SecTRAP which indicates that TrxR1 regulates acrolein-mediated JNK activity. Figure 6. Acrolein-mediated MAPK phosphorylation is significantly attenuated in TrxR1 silenced HBE1 cells. Non-targeting (NT- siRNA) and TXNRD1 siRNA transfected HBE1 cells were treated with 30 µM acrolein for 30 min and p-JNK, p-ERK, and p-P38 were analyzed by Western Blot. Analysis of these blots was done by densitometry. Conclusion: Acrolein-mediated JNK-1 phosphorylation is significantly attenuated by a decrease in SecTRAP, which indicates that TrxR1 is required for acrolein-mediated JNK activation 0 30 0 30 [Acr](µM) Na 2 SeO 3 Trx1 0 30 0 30 [Acr](µM) NT-siRNA TXNRD1 siRNA Trx1  Abs. (340nm) x 60 sec -1 0.00 0.01 0.02 0.03 * [Acrolein] ( M) Partially Oxidized Trx1 (% of total Trx1) 0 10 20 30 0 20 40 60 80 100 [Acrolein] ( M) Fully & Partially Oxidized Trx1 (% of total Trx1) 0 10 20 30 0 20 40 60 80 100 ** * Trx1 — + — + — + — + Acrolein — — + + — — + + mTrxR-GCUG mTrxRΔ3  Abs. (340nm) x 30 sec -1 0.00 0.02 0.04 0.06 0.08 0.10 Figure 1. Acrolein inhibits the reductase activity and enhances the NADPH consumption of mTrxR. Thioredoxin reductase functions as a homodimer whereby the N-terminal FAD domain utilizes reducing equivalents to reduce the C-terminal selenothiol couple. The Sec further transfers these reducing equivalents to reduce thioredoxin (Trx). Semisynthetic mTrxR-GCUG and mTrxR∆3 were incubated with 30 μM acrolein for 30 min. Reductase activity was measured by insulin assay in the presence/absence of thioredoxin. NADPH oxidase activity was measured in the presence/absence of lipoic acid. Conclusion: Together these data suggest that while acrolein inhibits the reductase activity of mTrxR it also enhances the NADPH consumption which is in agreement with the idea that electrophilic adduction of TrxR can promote the formation of a SecTRAP . Figure 2. The role of cellular glutathione levels on acrolein-mediated effects of the thioredoxin redox system. HBE1 cells pretreated with GSH synthesis inhibitor, buthionine sulfoximine (BSO) (24hr) or glutathione mimetic, glutathione ethyl ester (GEE) (4hr) were treated with acrolein at indicated concentrations for 30 min after which TrxR activity, GSH levels, and Trx1 oxidation were measured. Conclusion: Acrolein inhibited TrxR1 activity independently of cellular GSH status. Alternatively, the total Trx1 oxidation/alkylation by acrolein was significantly effected by cellular GSH levels. This could indicate that the alkylation of TrxR1 is not regulated by the overall cell redox status and TrxR1 alkylation may therefore be important for cellular responses to acrolein. Figure 3. Modulation of cellular TrxR1- acrolein adduct levels. Protein lysates from Na 2 SeO 3 supplemented (significantly increased TrxR1 protein) or TXNRD1 siRNA transfected (significantly decreased TrxR1 protein) HBE1 cells treated with 30 µM acrolein were labeled with a biotin hydrazide tag. Labeled proteins were further isolated with neutravidin resin and analyzed by Western blot for TrxR1. Conclusion: Through Na 2 SeO 3 supplemenation and TXNRD1 siRNA transfection or HBE1 cells, followed by acrolein treatment, we are able to alter cellular levels of TrxR1-acrolein adducts. This method of altering cellular SecTRAP levels allows us to further attempt to identify a specific role for this alkylated protein. Fully Reduced Partially Oxidized Fully Oxidized Fully Reduced Partially Oxidized Fully Oxidized Na 2 SeO 3 No supplement 0 3 10 30 [Acrolein] (µM) Figure 4. Role of TrxR1 on acrolein-mediated Trx1 oxidation/alkylation. HBE1 cells supplemented with Na 2 SeO 3 or transfected with TXNRD1 siRNA were treated with the indicated concentrations of acrolein after which S-carboxymethylation was performed and the oxidation/alkylation state of Trx1 was observed by redox Western blot. Densitometry was done to analyze the relative levels of partially oxidized (solid lines) and fully oxidized (dotted lines) Trx1. Trx1 alkylation was analyzed using the biotin hydrazide labeling and affinity chromatography method. Conclusion: The acrolein-mediated total Trx1 oxidation was not significantly altered due to cellular TrxR1 levels although Trx1 alkylation was significantly dependent upon cellular TrxR1 protein level. Therefore, in regards to acrolein exposure, the overall redox status of Trx1 may not be relevant for downstream effects of SecTRAP, but may rely specifically on Trx1 alkylation. Relative Intensity 0 2 4 6 8 p = 0.065 Relative Intensity 0 10 20 30 40 50 * 0 30 0 30 [Acr] (µM) Na 2 SeO 3 TrxR1 β-actin 0 30 0 30 [Acr] (µM) NT-siRNA TXNRD1 siRNA TrxR1 β-actin 0 10 20 30 0 50 100 [Acrolein] ( M) TrxR activity (% of Control) no pretreatment BSO GEE 0 10 20 30 0 50 100 [Acrolein] ( M) % of Control * ** ** GSH TrxR Activity 0 10 20 30 0 50 100 150 [Acrolein] ( M) [GSH] (% of Control) ** ** ** ** no pretreatment BSO GEE 0 10 20 30 0 10 20 30 40 [Acrolein] ( M) Partially Oxidized Trx1 (% of total Trx1) * ** ** no pretreatment BSO GEE Lipoic Acid — + — + — + — + Acrolein — — + + — — + + mTrxR-GCUG mTrxRΔ3 Acknowledgement: This work was supported by grants from NIH ES021476, HL68865, and FAMRI O H O H SH + S N S H 2 N N H O H N O S HN NH N O H N O S HN NH S N H O H N O S HN NH N H + NaBH 4 Acrolein Protein thiol Biotin hydrazide ■ Using a purified semisynthetic mitochondrial TrxR (mTrxR) as well as a truncated variant (mTrxR∆3) lacking the C-terminal –UCG amino acids we analyzed the reductase activity and NADPH consumption of the enzyme, following acrolein adduction. ■ HBE1 cells exposed to a bolus of acrolein (30 μM) were lysed after 30 min. TrxR activity, cellular GSH, and Trx1 oxidation were analyzed. Further, proteins were labeled with biotin hydrazide and subsequently isolated using high affinity neutravidin agarose resin to identify acrolein adducted proteins. ■ Protein modifications (oxidation/phosphorylation/alkylation) were visualized by Western blotting. MAPK Activation Cytokine Release -- Barrier Disruption -- Cell Death Thioredoxin Reductase Acrolein Adduction Inhibition of Function Trx1 Oxidation ROS Production Gain-of- Function NADPH Flexible Tail NADP + TrxR1 C-terminus TrxR1 N-terminus TrxR1 C-terminus TrxR1 N-terminus Results (II) TXNRD1 siRNA NT-siRNA 0 3 10 30 [Acrolein] (µM) Fully Reduced Partially Oxidized Fully Oxidized Fully Reduced Partially Oxidized Fully Oxidized Suggested mechanism for JNK activation via SecTRAP ASK1 JNK Alkylated TrxR1 ROS generation P Trx1 Alkylation P ??? Reductase Activity ? + NADPH Oxidase Activity 1. Shao, M. X.; Nakanaga, T.; Nadel, J. A. 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