Thioredoxin, transgenic Mouse, oxidative stress, aging

Thioredoxin (Trx) has drawn much interest in biology, including aging, because of its unique ability to attenuate the level of oxidative stress/damage and alter redoxsensitive signaling, which could have diverse effects on pathophysiology. Trx is a small protein (12kDa) with two redox-active cysteine residues in the active center (CysGly-Pro-Cys) [1] and acts as the reductant for a variety of enzymes [1-7]. Trx also plays an important role in maintaining a reduced environment in cells through thioldisulfide exchange reactions [1]. This rapid and readily reversible reaction is ideal for protein function control via the redox state of structural or catalytic SH groups. In humans, Trx1 and Trx2 have been identified in different compartments of the cell, i.e., Trx1 in cytosol [8] and Trx2 in mitochondria [9]. To test the pathophysiological roles of Trx1 during aging, two transgenic mice overexpressing Trx1 were generated using: 1) a transgene containing the human thioredoxin cDNA fused to the β–actin promoter [Tg(act-TXN)^+/0 mice] [10] and 2) clones of the human TXN gene containing endogenous promoters [Tg(TXN)^+/0] [11]. The initial aging study demonstrated that Tg(act-TXN)^+/0 mice had an increased lifespan compared to their WT littermates [10,12]. However, the lifespan of WT C57BL/6 mice in their colony was shorter than WT C57BL/6 mice in aging colonies under optimal conditions, which indicates that the study was conducted under unconventional housing conditions. Therefore, an aging study with the same line of Tg(act-TXN)^+/0 mice under optimal housing conditions was conducted by our laboratory to examine the effects of increased levels of Trx1 on aging and age-related diseases. Under optimal housing conditions, male and female Tg(act-TXN)^+/0 mice showed an extension of lifespan only in the earlier part of their lifespan; however, no increase in maximum lifespan was observed [13]. Because Tg(actTXN)^+/0 mice showed that the levels of overexpression significantly decreased with age possibly due to the β-actin promoter, we subsequently generated new transgenic mice with clones of the human TXN gene containing endogenous promoters [Tg(TXN)^+/0] to ensure that the transgene would be overexpressed throughout the lifespan [11]. The aging study with Tg(TXN)^+/0 mice demonstrated that continuous overexpression of Trx1 over the lifespan slightly extended the earlier part of life, but had no significant effects on median or maximum lifespans. Tg(TXN)^+/0 mice also showed that Trx1 overexpression accelerates cancer development in old mice [11], which is similar to the pathology results in Tg(act-TXN)^+/0 mice [13]. The results from two lines of Trx1 transgenic mice showed that overexpression of Trx1 in cytosol does not have beneficial effects in aged mice, i.e., no life-extending effects with enhanced tumor development in the later part of life. These results led us to question whether increased levels of Trx2 in mitochondria could play more important roles in aging, i.e., extend both the earlier and later part of lifespan and attenuate the development of age-related diseases. The study with mCAT mice [14] clearly demonstrated the importance of antioxidant overexpression in mitochondria in aging. In their study, overexpressing catalase in mitochondria significantly increased lifespan and reduced some types of cancers, while overexpressing catalase in other compartments of the cells (nucleus or peroxisome) did not change lifespan [14]. Thus, the purpose of this study is to examine the effects of Trx2 overexpression in the mitochondria on aging. We conducted a survival study using transgenic mice generated with a clone of the human TXN2 gene containing an endogenous promoter [Tg(TXN2)^+/0]. Here, we report that increased levels of Trx in mitochondria [Tg(TXN2)^+/0 mice] showed no significant changes in lifespan compared to WT mice, although a slight extension (approximately 8-9%) of mean, median, and 10th percentile lifespans (not statistically significant) was observed. Tg(TXN2)^+/0 mice also showed no significant effects on age-related pathological changes. Therefore, our results suggest that the overexpression of Trx2 has minimal effects on aging and development of age-related diseases in male C57BL/6 mice.

Animals and animal husbandry

We generated the Tg(TXN2)^+/0 mice using the human thioredoxin 2 gene [a PAC clone (RP5-1119A7), Children’s Hospital Oakland Research Institute’s (CHORI) BACPAC Resources Center (BPRC), Oakland, CA], which contained the TXN2 gene and 13.7 kb and 6.6 kb of the 5’- and 3’-flanking sequences, respectively (Figure 1). These transgenic mice were produced by pronuclear microinjection of zygotes obtained from the mating of (C57BL/6J X SJL/J)F1 females with (C57BL/6J X SJL/ J)F1 males (Jackson Laboratory, stock no. 100012) and were backcrossed to C57BL/6 mice ten times. Male hemizygous transgenic mice [Tg(TXN2)^+/0] were crossed with C57BL/6 females to generate hemizygous transgenic and WT control mice. All mice were fed a commercial chow (Teklad Diet LM485: Madison, WI) and acidified (pH=2.6-2.7) filtered reverse osmosis water ad libitum. To measure the amount of food consumption, the amount of chow removed from the cage hopper and the spillage (the chow on the bottom of the cage) were weighed monthly. Actual food consumed was calculated by subtracting the spillage from the chow removed from the hopper. All of the mice were weighed monthly. The mice were maintained pathogen-free in microisolator units on Tek FRESH ultra laboratory bedding. Sentinel mice housed in the same room and exposed weekly to bedding collected from the cages of experimental mice were sacrificed on receipt and every six months thereafter for monitoring of viral antibodies (Mouse Level II Complete Antibody Profile CARB, Ectro, EDIM, GDVII, LCM, M. Ad-FL, M. Ad-K87, MCMV, MHV, M. pul., MPV, MVM, Polyoma, PVM, Reo, Sendai; BioReliance, Rockville, MD). All tests were negative.
We chose young (4-6 months old) and old (22-24 months old) age groups for the experiments described below because: 1) C57BL/6 mice reach their optimum reproductivity at 3-7 months of age and 2) have increased incidence of cancers after 20 months of age followed by advanced stages of cancer around 26-30 months of age. For some of the assays, only liver was used because there is a large amount of information available with respect to agerelated changes, and liver is one of the major sites for the development of spontaneous tumors in C57BL/6 mice.

PD rat model

The expressions of miR-152, MKK7 mRNA, and MKK7 mRNA in cells were detected using quantitative real-time PCR (qRT-PCR). Total RNAs were extracted from the tumor tissues and cells using Trizol reagent (Invitrogen, Waltham, MA, US). The TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, CA, US) was used to reversely transcribe RNAs into cDNAs. The qRT-PCR was conducted using the SYBR Premix Ex TaqTM II Kit (Takara, Dalian, Liaoning, China) on an ABI 7500 RealTime PCR system (Applied Biosystems, Waltham, MA, US). U6 was used as the internal control for miR-152, and GAPDH was used as the internal control for mRNAs. The relative expressions were calculated using the 2^−ΔΔCt method.

Determination of Trx2 expression

Thioredoxin 2 (Trx2) levels were measured using the mitochondrial fraction obtained from several tissues (liver, kidney, heart, brain, lung, spleen, and testes) from young (4-6 months old) and liver from old (22-24 months old) Tg(TXN2)^+/0 and WT mice as previously described [13,15]. Western blot analysis was performed using rabbit anti-Trx2 polyclonal antibody (Catalog No. LF-PA0012; LabFrontier, Seoul, South Korea). After incubation with the primary antibodies, membranes were incubated with the respective peroxidase-linked secondary antibodies (Catalog No. P0217; Dako, Carpinteria, CA). Chemiluminescence was detected using the ECL Western blot detection kit (Amersham Biosciences Corp., Piscataway, NJ).

Trx1 levels

Cytosolic fractions obtained from tissues homogenized as previously described [13] were used to determine Trx1 levels in liver obtained from young (4-6 months old) Tg(TXN2)^+/0 and WT mice by Western blot analysis using goat anti-human Trx1 polyclonal antibodies (Catalog No. 705; American Diagnostica, Inc., Greenwich, CT). These antibodies recognize total Trx1 (both oxidized and reduced forms). After incubation with the primary antibody, membranes were incubated with the peroxidase-linked secondary antibody (Catalog No. P0449; Dako, Carpinteria, CA). Chemiluminescence was detected with an ECL Western blot detection kit (Amersham Biosciences Corp., Piscataway, NJ).

Glutaredoxin and total glutathione levels

Glutaredoxin (Grx) levels were measured using total homogenate fractions obtained from the liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice as previously described [13]. Western blot analysis was performed using goat anti-human glutaredoxin polyclonal antibody (Catalog No. 710; American Diagnostica, Inc., Greenwich, CT). After incubation with the primary antibodies, membranes were incubated with the respective peroxidase-linked secondary antibodies (Catalog No. P0449; Dako, Carpinteria, CA). Chemiluminescence was detected using the ECL Western blot detection kit (Amersham Biosciences Corp., Piscataway, NJ). The levels of total glutathione were determined using the Bioxytech GSH-420 kit (Catalog No. 21023; Oxis International, Inc., Foster City, CA) in several tissues (liver, kidney, heart, brain, lung, spleen, and testes) from young (4-6 months old) Tg(TXN2)^+/0 and WT mice.

Determination of major antioxidant enzyme activities: Cu/ZnSOD, MnSOD, glutathione peroxidase, and catalase

The activities of major antioxidant enzymes (Cu/ZnSOD, MnSOD, glutathione peroxidase (GPx), and catalase) were measured in tissue homogenates obtained from the liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice. The supernatants were used for the antioxidant defense enzymatic activity assay. GPx activity in tissue homogenates was measured by the assay described by Sun et al. [16]. Catalase activity was determined by measuring the decomposition of hydrogen peroxide at 520 nm using the Catalase-520TM assay kit (OxisResearchTM, Portland, OR). MnSOD and Cu/ZnSOD levels were measured by activity gels as previously described [17,18]. Images of the gels were analyzed by ImageQuant software.

Reactive oxygen species (ROS) production from mitochondria

H₂O₂ release from isolated mitochondria obtained from skeletal muscle was measured using the fluorogenic probe, Amplex Red (Molecular Probes, Eugene, OR) as previously described [19].

Assays for lipid peroxidation(F2-isoprostane levels)

The levels of F2-isoprostanes were determined using gas chromatography/mass spectrometry as described by Morrow and Roberts [20]. The plasma samples obtained from young (4-6 months old) Tg(TXN2)^+/0 and WT mice were added to HPLC (pH 3.0) water and mixed by vortex. After centrifugation (2,500 x g for 3 minutes at 4°C), the F2- isoprostanes were extracted from the clear supernatants with a C18 Sep-Pak column and a silica Sep-Pak column. The F2-isoprostanes were then converted to pentafluorobenzyl esters and subjected to thin layer chromatography. The F2-isoprostanes were further converted to trimethylsilyl ether derivatives, and the F2-isoprostane levels were quantified by gas chromatography/mass spectrometry. An internal standard, 8-isoPGF2a-d4 (Cayman Chemical, Ann Arbor, MI), was added to the samples at the beginning of extraction to correct the yield of the extraction process. The amounts of F2-isoprostanes were expressed as picograms of 8-Iso-prostaglandin F2 per milliliter of plasma sample.

Assays for DNA oxidation (8-oxodG levels)

The levels of oxidative damage to DNA were measured by the amount of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA as described by Hamilton et al. [21]. DNA was isolated from liver obtained from young (4-6 months old) Tg(TXN2)^+/0 and WT mice by NaI extraction using the DNA Extractor WB Kit (Wako Chemicals USA, Inc., Richmond, VA). The data are expressed as the ratio of nmoles of oxo8dG to 105 nmoles of 2dG.

Survival study

Mice in the survival groups were allowed to live out their lives, and the lifespan for individual mice was determined by recording the age of spontaneous death. A survival study consisting of 19 Tg(TXN2)^+/0 and 22 WT male mice was conducted. The survival curves were compared statistically using the log-rank test [22]. The mean, median, and 10th percentile (when 90% of the mice had died) survival were calculated for each group. The mean survivals for each experimental group were compared to the respective WT group by performing a Student’s t-test upon log-transformed survival times. The median and 10th percentile survivals for each group were compared to the WT group using a score test adapted from Wang et al. [23].

Cross–sectional pathological assessment

The cross-sectional pathological analyses were conducted with 23 Tg(TXN2)^+/0 mice and 19 WT male mice. After the gross pathological examinations, the following organs and tissues were excised and preserved in 10% buffered formalin: brain, pituitary gland, heart, lung, trachea, thymus, aorta, esophagus, stomach, small intestine, colon, liver, pancreas, spleen, kidneys, urinary bladder, reproductive system (prostate, testes, epididymis, and seminal vesicles), thyroid gland, adrenal glands, parathyroid glands, psoas muscle, knee joint, sternum, and vertebrae. Any other tissues with gross lesions were also excised. The fixed tissues were processed conventionally, embedded in paraffin, sectioned at 5 m, and stained with hematoxylineosin. The diagnosis of each histopathological change was made with histological classifications in aging mice as previously described [24,25]. A list of pathological lesions was constructed for each mouse that included both neoplastic and non-neoplastic diseases. Based on these histopathological data, the tumor burden, disease burden, and severity of each lesion in each mouse were assessed as previously described [25-27].

Determination of c-Jun and c-Fos levels

Total cell lysates from liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice were prepared, and detection of c-Jun (Catalog No. 9165; Cell Signaling Technology, Inc., Danvers, MA) and c-Fos (Catalog No. 4384; Cell Signaling Technology, Inc., Danvers, MA) were performed using Western blots. The amount of c-Jun and cFos was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.

Determination of mTOR signaling pathway activity and HIF-1α levels

Levels of p70S6K1 and 4E-BP1 (phosphorylated and nonphosphorylated forms) were measured in the total cell lysates from liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice by Western blot analysis using mouse p70S6K1, phospho-p70S6K1, 4E-BP1, and phospho-4EBP1 antibodies (Cell Signaling Technology, Inc., Danvers, MA).
Total cell lysates from liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice were prepared, and detection of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was performed using Western blots. The amount of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard. Total cell lysates from liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice were prepared, and detection of HIF-1α (Catalog No. Ab82832; Abcam, Cambridge, MA) was performed using Western blots. The amount of HIF-1α was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.

Measurement of the NFκB pathway

The levels of NFκB (p65 and p50) were measured in the total cell lysates from liver of young (4-6 months old) Tg(TXN2)^+/0 and WT mice by Western blot analysis using mouse NFκB p65 (Catalog No. 3034; Cell Signaling Technology, Inc., Danvers, MA) and p50 (Catalog No. 3035; Cell Signaling Technology, Inc., Danvers, MA) antibodies.

Statistical analysis

Unless otherwise specified, all experiments were done at least in triplicate. Data were expressed as means ± SEM and were analyzed by the non-parametric test ANOVA. All pair-wise contrasts were computed using Tukey error protection at 95% CI, unless otherwise indicated. Differences were considered statistically significant at p<0.05.

Overexpression of Trx2 in tissues from Tg(TXN2)^+/0 mice

The levels of Trx2 in tissues (liver, kidney, heart, brain, lung, spleen, and testes) from young (4-6 months old) Tg(TXN2)^+/0 and WT mice were measured using Western blot analysis. The Trx2 protein levels were significantly higher (approximately 1.6- to 5-fold) in all of the seven tissues examined in young Tg(TXN2)^+/0 mice compared to their WT littermates (Figure 2a; p< 0.05). The levels of Trx2 overexpression were maintained in the liver of old (22-24 months old) Tg(TXN2)^+/0 mice (Figure 2b; p< 0.05).

Levels of Trx1, glutaredoxin, total glutathione, and major antioxidant enzymes in tissues from Tg(TXN2)^+/0 mice

To examine whether the increased levels of Trx2 altered the levels of Trx1, glutaredoxin, and glutathione, which have biological functions similar to Trx2, we measured the levels of Trx1, glutaredoxin, and total glutathione in liver and other tissues obtained from young (4-6 months old) Tg(TXN2)^+/0 and WT mice. The data in Figure 3 show that Trx1 (Figure 3a) and glutaredoxin (Figure 3b) levels in the liver were similar between Tg(TXN2)^+/0 and WT mice at 4-6 months of age (p> 0.05). The levels of total glutathione in the tissues (liver, kidney, heart, brain, lung, spleen, and testes) were also similar between young (4-6 months old) Tg(TXN2)^+/0 and WT mice (Figure 3c; p> 0.05).
The activities of major antioxidant enzymes (Cu/ZnSOD, MnSOD, glutathione peroxidase (GPx), and catalase) were similar between young (4-6 months old) Tg(TXN2)^+/0 and WT mice (data not shown).

Hydrogen peroxide production from mitochondria

H₂O₂ release from isolated mitochondria obtained from skeletal muscle was measured using the fluorogenic probe, Amplex Red (Molecular Probes, Eugene, OR) as previously described [19]. The H₂O₂ release from isolated mitochondria from skeletal muscle was significantly less in both young (4-6 months old) and old (22-24 months old) Tg(TXN2)^+/0 mice compared to their WT littermates (Figures 4a and 4b; p< 0.05).

Lipid peroxidation (F2-isoprostane levels) and DNA oxidation (8-oxodG levels)

We tested whether Trx2 overexpression protects against oxidative damage by measuring levels of lipid peroxidation (F2 isoprostanes) in serum and DNA oxidation (8-oxodG) in liver from young (4-6 months old) Tg(TXN2)^+/0 and WT mice. Levels of F2-isoprostanes were significantly lower in young (4-6 months old) Tg(TXN2)^+/0 mice compared to WT mice (Figure 5a; p< 0.05), however, Tg(TXN2)^+/0 mice had only 13-14% lower levels of F2-isoprostanes compared to WT mice, in spite of the significant ROS production from mitochondria (Figures 4a and 4b). Although Trx2 overexpression reduced the ROS production from mitochondria and oxidative damage to lipids, levels of DNA oxidation measured by 8-oxodG were similar between young (4-6 months old) Tg(TXN2)^+/0 and WT mice (Figure 5b; p> 0.05).

Survival curves, body and organ weights

The survival curves of Tg(TXN2)^+/0 and WT mice are presented in Figure 6. The survival study was conducted with 19 Tg(TXN2)^+/0 and 22 WT male mice. The survival curves were not significantly different between Tg(TXN2)^+/0 and WT mice (Log-rank: p> 0.05). The mean, median, and 10th percentile survival for WT mice were 852, 833, and 1,094 days, respectively (Figure 6). The mean, median, and 10th percentile survival for Tg(TXN2)^+/0 mice were 922, 907, and 1,183 days, respectively (Figure 6). Tg(TXN2)^+/0 mice had slightly longer mean (8.2%), median (8.9%), and 10th percentile (8.1%) lifespans compared to WT mice, however, these differences did not reach statistical significance (p> 0.05).

The body and organ weights were similar between Tg(TXN2)^+/0 and WT mice (Table 1). Food consumption was also similar between Tg(TXN2)^+/0 and WT mice (data not shown).

Cross-sectional pathology

The cross-sectional pathological analyses of 23 Tg(TXN)^+/0 and 19 WT mice (22-24 months old) showed that the major disease in these mice was neoplastic disease. The tumors observed were lymphoma, hemangioma/ hemangiosarcoma in the liver and spleen, pulmonary adenocarcinoma, hepatocellular carcinoma, and adenoma in the thyroid gland, and the most prevalent tumor was lymphoma, which is consistent with the pathology data from C57BL/6 mice and mice overexpressing Trx1 [11,13]. First, we compared the number of different types of tumors (tumor burden) for each mouse in both Tg(TXN2)^+/0 and WT groups because aging mice may have several different types of tumors. As the data in Figure 7a show, the tumor burden for the Tg(TXN2)^+/0 mice is similar to WT mice (p > 0.05). The incidence of lymphoma was similar between Tg(TXN2)^+/0 and WT mice (data not shown). Tg(TXN2)^+/0 mice had a slightly higher severity of lymphoma than WT mice, which was not statistically significant (Figure 7b; p> 0.05).
Next, we compared the severity of major non-neoplastic disease between the two groups. Severity of glomerulonephritis and inflammation, which were the most common non-neoplastic lesions observed in these mice, were compared between Tg(TXN2)^+/0 and WT mice. No significant changes were found regarding the severity of glomerulonephritis or inflammation in Tg(TXN2)^+/0 and WT mice (data not shown). The disease burden, defined as the total number of histopathological changes in a body, which can also serve as a good index of age-related accumulation of tissue and cell injury [24,26,27], were similar between Tg(TXN2)^+/0 and WT mice (data not shown).

Measurement of the c-Fos and c-Jun levels

Since c-Fos and c-Jun are redox-sensitive transcription factors, and their levels are correlated to cancer development, the levels of c-Fos and c-Jun were measured using Western blot. The data in Figure 8 show that Tg(TXN2)^+/0 mice had significantly higher levels of c-Fos and c-Jun compared to their WT littermates (Figures 8a and 8b, respectively; p< 0.05).

Determination of mTOR signaling pathway activity and HIF-1α levels

Since substantial evidence showed that mTOR activity is one of the key pathways for cancer development and lifespan, levels of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) were measured in the total cell lysates from liver of Tg(TXN2)^+/0 and WT mice by Western blot analysis using mouse p70S6K1, phosphop70S6K1, 4E-BP1, and phospho-4E-BP1 antibodies. The data in Figure 9a show that Tg(TXN2)^+/0 mice had slightly higher levels of phospho-p70S6K1 compared to WT littermates, although the difference was not statistically significant (Figure 9a; p> 0.05). Levels of phospho-4E-BP1 were similar between Tg(TXN2)^+/0 and WT mice (Figure 9b; p> 0.05). In addition, the levels of HIF-1α, which also plays important roles in cancer development, were similar between Tg(TXN2)^+/0 and WT littermates (data not shown).

Measurement of the NFκB pathway

Since NFκB is one of the redox-sensitive transcription factors, which plays important roles in oxidative stress, inflammation, apoptosis, and cancer, the levels of NFκB (p65 and p50) were measured using Western blot. The data in Figure 10 show that Tg(TXN2)^+/0 mice had similar levels of NFκB p65 and NFκB p50 compared to their WT littermates (Figures 10a and 10b, respectively; p> 0.05).

Acknowledgments

We acknowledge the Pathology Core in the San Antonio Nathan Shock Center (P30- AG013319) for pathological analyses. This research was supported by the VA Merit Review grant from the Department of Veteran Affairs (Y.I.), NIH grant AG13319 (Y.I.), The American Federation for Aging Research (AFAR) grant (Y.I.), and a grant from the Glenn Foundation (Y.I.).

Conflict of interest

Yuji Ikeno is a member of the Editorial Board of Aging Pathobiology and Therapeutics. All authors declare no conflict of interest and were not involved in the journal’s review or desicions related to this manuscript.

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