Thyroid Hormone Administration Induces Rat Liver Nrf2 Activation: Suppression by N -Acetylcysteine Pretreatment

Abstract

Background:

Oxidative stress associated with 3,3′,5-triiodo-l-thyronine (T₃)-induced calorigenesis upregulates the hepatic expression of mediators of cytoprotective mechanisms. The aim of this study was to evaluate the hypothesis that in vivo T₃ administration triggers a redox-mediated translocation of the cytoprotective nuclear transcription factor erythroid 2-related factor 2 (Nrf2) from the cytosol to the nucleus in rat liver. Such translocation of transcription factors is considered to be an activating step.

Materials and Methods:

The effect of T₃ administration in the presence and absence of N-acetylcysteine (NAC) on cytosol-to-nuclear translocation of Nrf2 was evaluated, with inhibition of this process by NAC taken as evidence that the process was redox mediated. Male Sprague-Dawley rats weighing 180–200 g were given a single intraperitoneal dose of 0.1 mg T₃/kg. Another group of rats were given the same dose of T₃ and were also pretreated with NAC (0.5 g/kg) at 0.5 hour before T₃ administration. Two other groups of rats received vehicle treatment and NAC, respectively. Following these treatments, rectal temperature of the animals, liver O₂ consumption, serum and hepatic levels of 8-isoprostanes, and liver protein levels of Nrf2, Akt, p38, and thioredoxin (Western blot) were determined at different times up to 48 hours.

Results:

T₃ administration induced a significant increase in the hepatic nuclear levels of Nrf2 at 1 and 2 hours after treatment and a concomitant decrease in cytosolic Nrf2. It also increased hepatic thioredoxin, a protein whose gene transcription is induced by nuclear Nrf2. Levels of nuclear Nrf2 were at a plateau from 4 to 6 hours after T₃. Rectal temperature of the animals rose from 36.6°C to 37.5°C as did liver O₂ consumption. Serum and liver 8-isoprostanes levels increased (p < 0.05) from 38.4 ± 4.0 pg/mL (n = 4) to 69.2 ± 2.0 pg/mL (n = 3) and from 0.75 ± 0.09 ng/g liver (n = 3) to 1.53 ± 0.10 ng/g liver (n = 5), respectively. In the group of rats pretreated with NAC, the increase in cytosol-to-nuclear translocation of Nrf2 was only 28% that induced by T₃. In addition, T₃ induced liver Akt and p38 activation during the period of 1–4 hours after T₃ administration. p38 activation at 2 hours after T₃ administration was abolished in NAC-pretreated animals.

Conclusions:

In vivo T₃ administration leads to a rapid and transient cytosol-to-nuclear translocation of liver Nrf2. This appears to be promoted by a redox-dependent mechanism as it is blocked by NAC. It may also be contributed by concomitant p38 activation, which in turn promoted Nrf2 phosphorylation. Nrf2 cytosol-to-nuclear translocation may represent a novel cytoprotective mechanism of T₃ to limit free radical or electrophile toxicity, as this would likely entail promoting thioredoxin production.

Introduction

T hyroid hormone (3,3′,5-triiodo-l-thyronine [T₃])–induced calorigenesis triggers increased reactive oxygen species (ROS) generation in the liver, in both hepatocytes (1,2) and Kupffer cells (3). This occurs in rodent liver and extrahepatic tissues having a calorigenic response to T₃, an effect also observed in hyperthyroid patients (4,5). T₃ also upregulates the expression of proteins by a Kupffer cell–dependent process involving the release of the cytokines tumor necrosis factor-α and interleukins 1 and 6. These interact with specific receptors in hepatocytes and induce responses affording cytoprotection from ROS (6). These include the expression of antioxidant enzymes (manganese superoxide dismutase and inducible nitric oxide synthase), antiapoptotic proteins (Bcl-2), acute phase proteins (haptoglobin and β-fibrinogen), and proliferation of hepatocytes and Kupffer cells (6). The aforementioned adaptive mechanisms induced by T₃ represent a form of hormesis (7) reestablishing redox homeostasis, promoting cell survival, and affording significant protection against ischemia-reperfusion liver injury (8 –10). In humans, these beneficial T₃ effects are consistent with what has been called liver preconditioning (11,12) and may constitute a new therapeutic option for liver surgery and liver transplantation using reduced-size grafts from living donors (8,10).

In addition to these cytoprotective mechanisms, induction of ROS and electrophiles secondarily upregulates antioxidant proteins and phase 2 detoxifying enzymes through activation of transcription factor nuclear transcription factor erythroid 2-related factor 2 (Nrf2) (13,14). Under physiological conditions, signaling via Nrf2 is impeded by the negative regulator Kelch-like ECH-associating protein 1 (Keap1), which acts both as a redox sensor through its cysteine-151 and as a subunit of E3 ubiquitin ligase, thus promoting continuous proteasomal degradation of Nrf2 (13 –15). Increased cellular ROS generation, in turn, promotes modification of Keap1-cysteine-151 to a Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex form, which does not have anti-Nrf2 effects (15). This conversion is associated with increasing Nrf2 levels, its translocation into the nucleus, and its interaction with antioxidant response elements to activate target gene transcription (15). Nrf2 is sensitive to a rather low oxidative stress status (16) as that induced by the calorigenic action of T₃ (4,6). The objective of this study was to test the hypothesis that in vivo T₃ administration triggers cytosol-to-nuclear translocation (a putative activation step) of Nrf2 in rat liver by a redox-dependent mechanism, such as that which would be inhibited by the antioxidant N-acetylcysteine (NAC) (9). Here, we studied not only liver Nrf2 levels in cytosol and nucleus after administration of T₃, with and without NAC pretreatment, but also hepatic levels of thioredoxin as a prototypical Nrf2-regulated protein (13) and activation of hepatic p38 and Akt (protein kinase B). Hepatic p38 and Akt are kinases that have been proposed to regulate the activity of Nrf2 by phosphorylation (15).

Materials and Methods

Animal treatments

Male Sprague-Dawley rats (Animal facility of the Institute of Biomedical Sciences, Faculty of Medicine, University of Chile) weighing 180–200 g were housed on a 12-hour light/dark cycle and were provided with rat chow and water ad libitum. Animals received a single intraperitoneal dose of 0.1 mg T₃/kg body weight or equivalent volumes of hormone vehicle (0.1 N NaOH, controls) at time zero. Studies with NAC were carried out in the described groups receiving either 0.5 g/kg of NAC or saline, 0.5 hour before T₃ administration. Therefore, there were four experimental groups: (i) controls, (ii) T₃, (iii) NAC, and (iv) NAC + T₃. Studies were performed for up to 48 hours after hormone treatment. T₃-induced calorigenesis was assessed by the rectal temperature of the animals by means of a thermocouple (Cole-Palmer Instrument Company, Chicago, IL) and the rate of O₂ consumption measured polarographically in liver perfusion experiments as previously described (3). Blood samples were obtained by cardiac puncture in rats anesthetized (1 mL/kg) with zolazepam chlorhydrate (25 mg/mL) and tiletamine chlorhydrate (25 mg/mL) ip (Zoletil 50; Virbac S/A, Carros, France) for measurement of serum 8-isoprostanes (ELISA; Cayman Chemical Co., Ann Arbor, MI), and liver samples were taken, frozen in liquid nitrogen, and kept at −80°C for measurement of 8-isoprostanes, Nrf2, p38, and Akt. Other studies were performed with the Nrf2 activator oltipraz (17). Rats were given either 150 mg/kg ip of oltipraz or corn oil vehicle (controls) per day for 4 consecutive days (LKT Laboratories, Inc., St. Paul, MN). Experimental animal protocols and animal procedures complied with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, NIH Publication 86-23, revised 1985).

Western blot analysis of Nrf2, thioredoxin, p38, and Akt

Liver samples (100–500 mg) frozen in liquid nitrogen were homogenized and suspended in a buffer solution (pH 7.9) containing 10 mM HEPES, 1 mM EDTA, 0.6% Nonidet P-40, 150 mM NaCl, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM orthovanadate). Nuclear protein extracts (100 μg) and soluble protein fractions (60 μg) were separated on 12% polyacrylamide gels using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (18) and transferred onto nitrocellulose membranes (19), which were blocked for 1 hour at room temperature with Tris buffered saline (TBS) containing 5% bovine serum albumin. The blots were washed with TBS containing 0.1% Tween 20 and hybridized with either rabbit monoclonal antibodies for phospho-Akt, phospho-p38, and Akt (Cell Signaling Technology, Inc., New York, NY), rabbit polyclonal antibodies for Nrf2, thioredoxin, and p38 (Abcam, Cambridge, MA), or mouse monoclonal antibodies for β-actin (ICN Biomedicals, Inc., Aurora, OH) and lamin A/C (BD Transduction Laboratories, San José, CA). In all determinations, anti-β-actin was used as internal control for cytosolic fractions, whereas anti-lamin A/C was employed as internal control for nuclear fractions. After extensive washing, the antigen–antibody complexes were detected using horseradish peroxidase goat anti-rabbit IgG or goat anti-mouse IgG and a SuperSignal West Pico Chemiluminescence kit detection system (Pierce, Rockford, IL).

Statistics

Values shown correspond to the means ± SEM for the number of separate experiments indicated. One-way analysis of variance and the Newman–Keuls test assessed the statistical significance (p < 0.05) of differences between mean values. Statistical analysis of the studies with combined NAC and T₃ treatments was carried out by two-way analysis of variance.

Results

There was a significant and progressive increase in the rectal temperature of rats given a single dose of 0.1 mg T₃/kg when measured at 2–24 hours. This was followed by a decline at 36 hours, with the temperature stabilizing at 48 hours (Fig. 1A). Liver O₂ consumption was increased at 2 hours compared with rats not given T₃ and progressively increased from 2 to 24 hours (Fig. 1B). Under these conditions, T₃ administration was associated with a 105% increase in the hepatic 8-isoprostanes (p < 0.05). This was suppressed by NAC pretreatment before T₃ administration (control, 0.74 ± 0.09 ng/g liver [n = 3]; T₃, 1.53 ± 0.10 [n = 5]; NAC, 0.87 ± 0.09 [n = 3]; NAC + T₃, 0.86 ± 0.10 [n = 3]). There was a 15% increase in liver O₂ consumption associated with T₃ administration (p < 0.05). This increase was reduced by 67% (p < 0.05) in NAC-pretreated rats (control, 1.95 ± 0.05 μmol/(g liver·min) [n = 5]; T₃, 2.25 ± 0.02 [n = 3]; NAC, 1.90 ± 0.04 [n = 3]; NAC + T₃, 2.00 ± 0.05 [n = 3]).

FIG. 1.

Time course study of the changes in the rectal temperature of the animals (A) and liver O₂ consumption (B) induced by 3,3′,5-triiodothyronine (T₃) administration in the rat. The rectal temperature was measured with a thermocouple in fed rats given a single dose of 0.1 mg T₃/kg ip or T₃ vehicle (controls) at time zero, and results are expressed as differences in rectal temperature at time t minus that at time zero. Liver O₂ consumption was determined polarographically in perfusion experiments. Results are expressed as means ± SEM (n = 3–5); ^asignificantly different to the respective control group (p < 0.05).

Following T₃ administration, there was a 50% decrease in hepatic cytosol Nrf2 at 1 hour and a 66% decrease at 2 hours (Fig. 2A). This was associated with a 99% increase in hepatic nuclear Nrf2 at 1 hour and a 188% increase in hepatic nuclear Nrf2 at 2 hours (Fig. 2B) (p < 0.05 for T₃ group vs. control group). Both cytosolic and nuclear Nrf2 returned to control values at 4 and 6 hours after T₃ administration (Fig. 2A, B). The change in the cytosol-to-nuclear ratio was taken here and elsewhere as being consistent with the activation of hepatic Nrf2. It occurred in a similar time frame as the development of oxidative stress. Thus, there were 105% and 55% increases (p < 0.05) in the hepatic levels of 8-isoprostanes observed at 2 and 6 hours, respectively, based on values in the control group, and there were 80%–90% increments in the serum levels of 8-isoprostanes (p < 0.05) at 1–6 hours after T₃ treatment (Fig. 3).

FIG. 2.

Time course study of the effects of T₃ administration on liver nuclear transcription factor erythroid 2-related factor 2 (Nrf2) protein expression in rats. (A) Representative blots of cytosolic Nrf2 (57 kDa) and β-actin (43 kDa) expression using 60 μg of soluble protein from a different rat of each group studied (left panel); right panel corresponds to the respective densitometric quantification expressed as Nrf2/β-actin ratios to compare lane-to-lane equivalence in total protein content. (B) Representative blots of nuclear Nrf2 and lamin A/C (65 kDa) expression using 100 μg of soluble protein from a different rat of each group studied (left panel); right panel corresponds to the respective densitometric quantification expressed as Nrf2/lamin A/C ratios. (C) Densitometric quantification of the effect of the Nrf2 activator oltipraz (150 mg/kg ip or corn oil vehicle [controls] per day for 4 consecutive days; LKT Laboratories, Inc.) (25) on the cytosolic (Nrf2/β-actin) (A) and nuclear (Nrf2/lamin A/C) (B) contents of Nrf2. Values shown are means ± SEM (n = 3–6); ^a p < 0.05, compared with the respective control values.

FIG. 3.

Time course study of the effects of T₃ administration on hepatic and serum 8-isoprostanes levels in rats. Data are means ± SEM (n = 3–15); ^a p < 0.05, compared with control values at time zero.

The methodology for discernment of cytosol and nuclear Nrf2 was evaluated by testing the effects of oltipraz administration, a known activator of Nrf2 and its controlled genes (17,20). Oltipraz administration was associated with a 28% reduction in cytosolic Nrf2 levels and 215% increase in nuclear Nrf2 content when compared with values in control rats (p < 0.05) (Fig. 2C). Similarly to T₃ administration (Fig. 3), oltipraz treatment enhanced the serum levels of 8-isoprostanes, an effect that was of a higher magnitude than T₃ (controls, 28 ± 4 pg/mL [n = 15]; oltipraz-treated rats, 203 ± 37 [n = 4]; 4.3-fold increase; p < 0.05 by Student's t-test for unpaired data).

Rats (NAC + T₃) given 0.1 mg/kg T₃ and 0.5 g/kg NAC before T₃ administration (Fig. 2A, B) had less of a decrease in cytosol Nrf2 and less of an increase in nuclear Nrf2 than rats given T₃ alone (Fig. 4A, B). At 2 hours after T₃ treatment, the decrease in cytosolic Nrf2 in NAC-pretreated rats was 9% of that in animals given T₃ alone (Fig. 4A) and the increase in nuclear Nrf2 was 28% (Fig. 4B), respectively. Further, evaluation of Nrf2, which targets thioredoxin (20), revealed 220% enhancement (p < 0.05) in its protein expression at 2 hours after T₃ administration, compared with control values (Fig. 5).

FIG. 4.

Effect of N-acetylcysteine (NAC) pretreatment on liver Nrf2 protein expression induced by T₃ administration in rats. Determinations were carried out at 2 hours after T₃ treatment. (A) Representative blots of cytosolic Nrf2 and β-actin expression using 60 μg of soluble protein from a different rat of each group studied (left panel); bar graphs correspond to the respective densitometric quantification expressed as Nrf2/β-actin ratios to compare lane-to-lane equivalence in total protein content (right panel). (B) Representative blots of nuclear Nrf2 and lamin A/C expression using 100 μg of soluble protein from a different rat of each group studied (left panel); bar graphs correspond to the respective densitometric quantification expressed as Nrf2/lamin A/C ratios (right panel). Values shown are means ± SEM (n = 3–9); significance studies (p < 0.05) are indicated by the letters identifying each experimental group.

FIG. 5.

Time course study of the effects of T₃ administration on liver thioredoxin (Trx) protein expression in rats. Representative blots of cytosolic thioredoxin (12 kDa) and β-actin (43 kDa) expression using 50 μg of soluble protein from a different rat of each group studied (upper panels). The lower panel shows the respective densitometric quantification expressed as thioredoxin/β-actin ratios to compare lane-to-lane equivalence in total protein content (lower panel). Values shown are means ± SEM (n = 3–5); ^a p < 0.05, compared with the respective control values.

The administration of 0.1 mg/kg T₃ was associated with 180% increase (p < 0.05) in the phosphorylated Akt/nonphosphorylated Akt (Akt-OP/Akt-OH) ratios (Fig. 6A). The phosphorylated p38/nonphosphorylated p38 (p38-OP/p38-OH) ratios were increased by 498%, 221%, and 175% (p < 0.05) at 1, 2, and 4 hours after T₃ treatment, respectively (Fig. 6B). At 2 hours after T₃ administration, Akt-OP/Akt-OH ratios were enhanced by 180% over control values and by 113% in NAC-pretreated T₃-treated rats over NAC control animals, leading to a net NAC-dependent nonsignificant 28% diminution in T₃-induced Akt activation, in the absence of significant effects by NAC itself (Fig. 7A). Further, p38-OP/p38-OH ratios were increased by 222% by T₃ at 2 hours after treatment, compared with controls (p < 0.05), an effect that was suppressed by NAC pretreatment prior to T₃. Under these conditions, NAC did not modify liver p38 activation as evidenced by the lack of effects on the hepatic p38-OP/p38-OH ratios when given alone to euthyroid rats (Fig. 7B).

FIG. 6.

Time course study of the effects of T₃ administration on liver Akt and p38 phosphorylation in rats. Representative blots of liver soluble protein fractions (60 μg) for detection of nonphosphorylated Akt (Akt-OH; 60 kDa) (A) or p38 (p38-OH; 43 kDa) (B), phosphorylayed Akt (Akt-OP) (A) or p38 (p38-OP) (B), and β-actin (A and B; left panels) and the respective densitometric quantification expressed as Akt-OP/Akt-OH (A) or p38-OP/p38-OH (B) ratios (right panels). Values shown are means ± SEM (n = 3–12). Significance studies: (A) ^a p < 0.05, versus control (time zero) and T₃ at 2 hours; ^b p < 0.05, versus control (time zero) and T₃ at 1, 4, and 6 hours. (B) ^a p < 0.05, versus control (time zero) and T₃ at 2, 4, and 6 hours; ^b p < 0.05, versus control (time zero) and T₃ at 1 and 6 hours; ^c p < 0.05, versus T₃ at 1, 2, and 4 hours.

FIG. 7.

Effect of NAC pretreatment on liver Akt and p38 phosphorylation induced by T₃ administration in rats. Determinations were carried out at 2 hours after T₃ treatment. (A) Representative blots of cytosolic phosphorylated Akt (Akt-OP), nonphosphorylated Akt (Akt-OH), and β-actin expression using 60 μg of soluble protein from a different rat of each group studied (left panel); bar graphs correspond to the respective densitometric quantification expressed as Akt-OP/Akt-OH ratios (right panel). (B) Representative blots of cytosolic phosphorylated p38 (p38-OP), nonphosphorylated p38 (p38-OH), and β-actin expression using 60 μg of soluble protein from a different rat of each group studied (left panel); bar graphs correspond to the respective densitometric quantification expressed as p38-OP/p38-OH ratios (right panel). Values shown are means ± SEM (n = 3–12); significance studies (p < 0.05) are indicated by the letters identifying each experimental group in the right panels of A and B.

Discussion

Previous studies have shown that induction of genes involved in cytoprotection occurs after the administration of T₃ to rats at doses that have a calorigenic effect (6,8). T₃ induced a biphasic response in rectal temperature probably related to upregulation of the expression of respiratory genes through genomic mechanisms (4,6). The late decline in rectal temperature at 36 hours after T₃ treatment was probably mediated, at least in part, by a negative feedback of T₃ on TSH (21). In the present study, we have found that administration of T₃ at calorigenic doses, involving higher rates of liver O₂ consumption, was associated with apparent translocation of the cytoprotective transcription factor Nrf2 from the cytosol to the nucleus, consistent with activation of Nrf2. The enhancement in the hepatic nuclear abundance of Nrf2 with diminution in that of cytosolic Nrf2 was an early event occurring at 1–2 hours after T₃ treatment, similar to most rapid nongenomic actions of thyroid hormones (22 –25). This is the time period during which there is a development of oxidative stress as evidenced by the enhanced hepatic and serum levels of 8-isoprostanes. Interestingly, one of the target genes for Nrf2, that is, the thioredoxin gene, also appears to be activated, as thioredoxin levels are increased at 2 hours after T₃ administration. Thioredoxin is a major component of the cellular antioxidant system (14,15). The major finding of the present study was that NAC pretreatment markedly decreased the T₃-induced activation (cytosol-to-nuclear translocation) of liver Nrf2. It also suppressed the T₃ induction of increases in liver 8-isoprostane levels and O₂ consumption. In the present study, we did not measure serum NAC levels. In prior studies under similar experimental conditions, however, the administration of 0.5 g/kg of NAC to rats led to a rapid increase of the serum NAC levels at 0.5 hour and a fall to undetectable concentrations after 48 hours (9). We postulate that the increase in the oxidative stress status of the liver associated with T₃-induced calorigenesis is an important factor for Nrf2 upregulation, which may expand the pool of free Nrf2. This can be achieved by Keap1 inactivation associated with T₃-induced ROS promoting Keap1-cysteine-151 oxidation, which inhibits its anti-Nrf2 effects (14,15,26).

In addition to the ROS-induced Keap1-mediated ubiquitination/degradation mechanism controlling Nrf2 activity, subcellular localization and posttranslational modification of Nrf2 are also involved (15,26). In the latter case, it has been proposed that Nrf2 phosphorylation plays a role in Nrf2 activation either (i) by increasing free Nrf2 levels, as phosphorylated Nrf2 is no longer a substrate for the Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex (15,27), and/or (ii) by enhancement of the binding affinity of Nrf2 to antioxidant response elements (15). In this respect, data presented suggest that Nrf2 phosphorylation by p38, rather than Akt, may play a role in Nrf2 upregulation by T₃ administration, considering (i) that mitogen-activated protein kinase (MAPK) p38 is concomitantly activated as shown by the significant increase in the hepatic p38-OP/p39-OH ratio over control values, and (ii) that both Nrf2 and p38 activation by T₃ are suppressed by NAC treatment prior to T₃. Activation of p38 consists of the initial stimulation of an MAPK kinase kinase by ROS, with the sequential phosphorylation of an MAPK kinase and p38 (28). Although p38 phosphorylation coincides with Nrf2 activation by T₃, recent studies revealed that phosphorylation of Nrf2 by MAPKs including p38 has a limited contribution in modulating Nrf2 activity and suggested that indirect mechanisms at the translational level may be involved through control of Nrf2 protein synthesis by MAPKs (29). This contention is supported by the interaction of thyroid hormones with a plasma membrane receptor on integrin αVβ3 to activate MAPK signaling, the affinity of the receptor for T₃ being lower than that for thyroxine (22).

Although T₃-induced redox upregulation of liver Nrf2 activity and content is a rapid event occurring within 1–2 hours after T₃ administration, it returned toward basal levels from 4 hours onward. Surprisingly, downregulation of hepatic Nrf2 after T₃-induced activation is observed under conditions in which a pro-oxidant state is present, as evidenced by the calorigenic response lasting for 24 hours and involving higher serum and hepatic 8-isoprostanes levels, an indicator of free radical-dependent lipid peroxidation. Liver Nrf2 downregulation under sustained oxidative stress conditions induced by T₃ may be ascribed to the enhancement in the expression of Nrf2 inhibitor Keap1. This proposal is based on data concerning the role of Nrf2 in cancer promotion, showing that enhanced Nrf2 activity and Nrf2-dependent gene expression in tumor cells is related to dysfunctional Keap1-Nrf2 interaction and/or Keap1 downregulation by promoter hypermethylation (30 –32). Conversely, T₃-induced Nrf2 downregulation after activation may be explained in terms of Keap1 upregulation due to hypomethylation of the Keap1 gene, a feature that is required for active gene transcription (33). This contention is supported by the finding that T₃ administration results in the demethylation of a specific DNA cytosine in the 3' region of the S14 gene, which is associated with increased expression of tissue-specific genes (34). Recently, an autoregulatory loop between Keap1 and the ubiquitination-related proteins Cul3 and Rbx1 controlling Nrf2 cellular abundance has been established, with Nrf2 activation upregulating the expression of Keap1, Cul3, and Rbx1, which in turn increase Nrf2 degradation (35,36). The influence of T₃ on liver Keap1, Cul3, and Rbx1 expression is under investigation in our laboratory.

In summary, data presented show that T₃ administration to rats leads to a rapid and transient increase in liver Nrf2 activation, as evidenced by the lower cytosolic and higher nuclear Nrf2 protein levels observed. Liver Nrf2 activation induced by T₃ appears to be a redox-dependent process, which may be contributed by Nrf2 phosphorylation related to p38 activation. This would represent an alternate cytoprotective mechanism of T₃ action against free-radical or electrophile toxicity, in addition to that afforded by NF-κB, STAT3, and/or AP-1 upregulation (6).

Footnotes

Acknowledgment

This work was supported by a grant (no. 1090020) from FONDECYT (Chile).

Disclosure Statement

The authors declare that no competing financial interests exist.

References

Fernández

, Videla

. 1993. Influence of hyperthyroidism on superoxide radical and hydrogen peroxide production by rat liver submitochondrial particles. Free Radic Res Commun, 18:329–335.

Venditti

, De Rosa

, Di Meo

. 2003. Effect of thyroid state on H₂O₂ production by rat liver mitochondria. Mol Cell Endocrinol, 205:185–192.

Tapia

, Pepper

, Smok

, Videla

. 1997. Kupffer cell function in thyroid hormone-induced liver oxidative stress. Free Radic Res, 26:267–279.

Fernández

, Tapia

, Varela

, Romanque

, Cartier-Ugarte

, Videla

. 2006. Thyroid hormone-induced oxidative stress in rodents and humans: a comparative view and relation to redox regulation of gene expression. Comp Biochem Physiol Part C, 142:231–239.

Venditti

, Di Meo

. 2006. Thyroid hormone-induced oxidative stress. Cell Mol Life Sci, 63:414–434.

Videla

. 2010. Hormetic responses of thyroid hormone calorigenesis in the liver: association with oxidative stress. IUBMB Life, 62:460–466.

Calabrese

. 2008. Converging concepts: adaptive response, preconditioning, the Yerkes-Dobson law manifestations of hormesis. Ageing Res Dev, 7:8–20.

Fernández

, Castillo

, Tapia

, Romanque

, Uribe-Echevarría

, Uribe

, Cartier-Ugarte

, Santander

, Vial

, Videla

. 2007. Thyroid hormone preconditioning: protection against ischemia-reperfusion liver injury in the rat. Hepatology, 45:170–177.

Fernández

, Tapia

, Varela

, Gaete

, Vera

, Mora

, Vial

, Videla

. 2008. Causal role of oxidative stress in liver preconditioning by thyroid hormone in rats. Free Radic Biol Med, 44:1724–1731.

10.

Tapia

, Santibáñez

, Farías

, Fuenzalida

, Varela

, Videla

, Fernández

. 2010. Kupffer-cell activity is essential for thyroid hormone rat liver preconditioning. Mol Cell Endocrinol, 323:292–297.

11.

Casillas-Ramírez

, Mosbah

, Ramalho

, Roselló-Catafau

, Peralta

. 2006. Past and future approaches to ischemia-reperfusion lesion associated with liver transplantation. Life Sci, 79:1881–1894.

12.

de Rougemont

, Lehmann

, Clavien

. 2009. Preconditioning, organ preservation, and postconditioning to prevent ischemia-reperfusion injury to the liver. Liver Transpl, 15:1172–1182.

13.

Itoh

, Tong

, Yamamoto

. 2004. Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free Radic Biol Med, 36:1208–1213.

14.

Nguyen

, Yang

, Pickett

. 2004. The pathways and molecular mechanisms regulating Nrf2 activation in response to chemical stress. Free Radic Biol Med, 37:433–441.

15.

Zhang

. 2006. Mechanistic studies of the Nrf2-Keap1 signaling pathway. Drug Metab Rev, 38:769–789.

16.

Gloire

, Legrand-Poels

, Piette

. 2006. Nf-κB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol, 72:1493–1505.

17.

Fisher

, Jackson

, Lickteig

, Augustine

, Cherrington

. 2008. Drug metabolizing enzyme induction pathways in experimental non-alcoholic steatohepatitis. Arch Toxicol, 82:959–964.

18.

Laemmli

. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227:680–685.

19.

Towbin

, Staehelin

, Gordon

. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA, 76:4350–4354.

20.

Buetler

, Gallagher

, Wang

, Stahl

, Hayes

, Eaton

. 1995. Induction of phase I and phase II drug-metabolizing enzymes mRNA, protein, and activity by BHA, ethoxyquin, and oltipraz. Toxicol Appl Pharmacol, 135:45–57.

21.

Scanlon

. Toft AD 1996 Regulation of thyrotropin secretion. Braverman

, Utiger

. Werner and Ingbar's The Thyroid. Lippincott-Raven Publishers: Philadelphia, 220–240.

22.

Davis

, Leonard

, Davis

. 2008. Mechanisms of nongenomic actions of thyroid hormones. Front Neuroendocrinol, 29:211–218.

23.

Arnold

, Goglia

, Kadenbach

. 1998. 3,5-Diiodothyronine binds to subunit Va of cytochrome c oxidase and abolishes the allosteric inhibition of respiration by ATP. Eur J Biochem, 252:325–330.

24.

Mezosi

, Szabo

, Nagy

, Borbely

, Varga

, Paragh

, Varga

. 2005. Nongenomic effect of thyroid hormone on free-radical production in human polymorphonuclear leukocytes. J Endocrinol, 185:121–129.

25.

Hiroi

, Kim

, Ying

, Furuya

, Huang

, Simoncini

, Noma

, Ueki

, Nguyen

, Scanlan

, Moskowitz

, Cheng

, Liao

. 2006. Rapid nongenomic actions of thyroid hormone. Proc Nat Acad Sci USA, 103:14104–14109.

26.

, Kong

. 2009. Molecular mechanisms of Nrf2-mediated antioxidant response. Mol Carcinogenesis, 48:91–104.

27.

Huang

, Nguyen

, Pickett

. 2002. Phosphorylation of Nrf2 at Ser40 by protein kinase C regulates antioxidant response element-mediated transcription. J Biol Chem, 277:42769–42774.

28.

Kim

, Choi

. 2010. Pathological roles of MAPK signaling pathways in human diseases. Biochim Biophys Acta Mol Basis Dis, 1802:396–405.

29.

Sun

, Huang

, Zhang

. 2009. Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response. PLoS ONE, 4:e6588.

30.

Singh

, Misra

, Thimmulappa

, Lee

, Ames

, Hoque

, Herman

, Baylin

, Sidranski

, Gabrielson

, Borck

, Biswal

. 2006. Dysfunctional Keap-1-Nfr2 interaction in non-small-cell lung cancer. PLoS Med, 3:e420.

31.

Wang

, An

, Ji

, Jiao

, Sun

, Zhou

. 2008. Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues. Biochem Biophys Res Commun, 373:151–154.

32.

Lau

, Villeneuve

, Sun

, Wong

, Zhang

. 2008. Dual roles of Nrf2 in cancer. Pharmacol Res, 58:262–270.

33.

Klose

, Bird

. 2006. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci, 31:89–97.

34.

Jump

, Wong

NCW

, Oppenheimer

. 1987. Chromatin structure and methylation state of a thyroid hormone-responsive gene in rat liver. J Biol Chem, 262:778–784.

35.

Lee

, Jain

, Papusha

, Jaiswal

. 2007. An auto-regulatory loop between stress sensors INef2 and Nrf2 controls their cellular abundance. J Biol Chem, 282:36412–36420.

36.

Kaspar

, Jaiswal

. 2010. An autoregulatory loop between Nrf2 and Cul3-Rbx1 controls their cellular abundance. J Biol Chem, 285:21349–21358.