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. 2007 Nov 1;43(9):1299-312.
doi: 10.1016/j.freeradbiomed.2007.07.025. Epub 2007 Aug 6.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

Ye-Shih Ho  1 , Ye Xiong, Dorothy S Ho, Jinping Gao, Balvin H L Chua, Harish Pai, John J Mieyal
Affiliations

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

Ye-Shih Ho et al. Free Radic Biol Med. .

Abstract

To understand the physiological function of glutaredoxin, a thiotransferase catalyzing the reduction of mixed disulfides of protein and glutathione, we generated a line of knockout mice deficient in the cytosolic glutaredoxin 1 (Grx1). To our surprise, mice deficient in Grx1 were not more susceptible to acute oxidative insults in models of heart and lung injury induced by ischemia/reperfusion and hyperoxia, respectively, suggesting that either changes in S-glutathionylation status of cytosolic proteins are not the major cause of such tissue injury or developmental adaptation in the Glrx1-knockout animals alters the response to oxidative insult. In contrast, mouse embryonic fibroblasts (MEFs) isolated from Grx1-deficient mice displayed an increased vulnerability to diquat and paraquat, but they were not more susceptible to cell death induced by hydrogen peroxide (H(2)O(2)) and diamide. A deficiency in Grx1 also sensitized MEFs to protein S-glutathionylation in response to H(2)O(2) treatment and retarded deglutathionylation of the S-glutathionylated proteins, especially for a single prominent protein band. Additional experiments showed that MEFs lacking Grx1 were more tolerant to apoptosis induced by tumor necrosis factor alphaplus actinomycin D. These findings suggest that various oxidants may damage the cells via distinct mechanisms in which the action of Grx1 may or may not be protective and Grx1 may exert its function on specific target proteins.

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Figures

Fig. 1
Fig. 1
Targeted disruption of the mouse Glrx1 gene. (A) Genomic structure and partial restriction map of the mouse Glrx1 locus (top), the targeting vector (middle), and the targeted locus (bottom) are shown. The opened and black boxes represent the protein coding regions and noncoding regions in the exons. The number of the exon is indicated under each exon. The shaded box on top of the restriction map of the Glrx1 locus represents the 3' external sequence used for probing the DNA blot filters. B, BamHI; E, EcoRI; H, HindIII; N, NotI; P, PstI; neo, neomycin resistance cassette; TK, herpes thymidine kinase gene under the control of a mouse promoter of the phosphoglycerate kinase-1 (PGK-1) gene [33]. Arrows show the directions of transcription of the neo and TK genes. The sizes of the PstI restriction fragments from the wild-type and targeted loci hybridized with the probe are shown on the top and bottom of the figure, respectively. (B) DNA blot analysis of wild-type, heterozygous Glrx1 knockout, and homozygous Glrx1 knockout mice. DNA isolated from mouse tails was digested with PstI and probed with the 3' external probe shown in (A). +/+, +/−, and −/− represent wild-type, heterozygous Glrx1 knockout, and homozygous Glrx1 knockout mice, respectively. The 4.3 kb hybridization band is derived from the wild-type allele, and 2.9 kb hybridization band the mutated allele.
Fig. 2
Fig. 2
Expression analysis of Glrx1 knockout mice. (A) RNA blot analysis of Glrx1 mRNA expression in tissues of wild-type and Glrx1 knockout mice. The RNA blot membrane was first hybridized with a mouse Glrx1 cDNA, and then re-hybridized with a mouse cDNA for glutaredoxin 2 (Glrx2), and then a rat cDNA for glyceraldehydes 3-phosphate dehydrogenase (Gapd) (B) Protein blot analysis of Glrx1 in tissues of wild-type and Glrx1 knockout mice. The protein blot membrane was initially reacted with rabbit anti-human Grx1 antibodies and then rereacted with rabbit anti-human copper-zinc superoxide dismutase (CuZnSOD) antibodies. In (A) and (B), +/+, +/−, and −/− represent wild-type, heterozygous Glrx1 knockout, and homozygous Glrx1 knockout mice, respectively.
Fig. 3
Fig. 3
Lack of effect of Grx1 deficiency on myocardial infarction in mice. Animals were subjected to 60 min of LAD coronary artery ligation followed by 4 h of reperfusion. The infarct size was standardized against LV and area at risk (AR). Each value represent mean ± SD of 5 to 6 hearts.
Fig. 4
Fig. 4
Lung edema (wet/dry ratio) in Glrx1+/+ and Glrx1−/− mice following exposure to >99% oxygen for 72 hours. *, p<0.05 vs. Glrx1+/+ mice exposed to air. **, p<0.01 vs. Glrx1−/− mice exposed to air. N ≥ 4 for each group of mice. Each value represent mean ± SD.
Fig. 5
Fig. 5
The susceptibility of MEFs to oxidant-induced cell damage. MEFs isolated from Glrx1+/+ and Glrx1−/− mice were treated with different concentrations of H2O2 in D-PBS for 1 hr (A), or with different concentrations of diamide (B), diquat (C), and paraquat (D) in culture medium for 24 hrs. Cell viability was determined by the MTT assay. Results are shown as the percent absorbance of oxidant-treated MEFs compared to that of the same MEFs cultured in D-PBS or medium without oxidants. The data from Glrx1+/+ and Glrx1−/− MEFs with the same treatment were compared. Data are representatives from at least three independent experiments. Each bar represents mean ± SD, n = 4 measurements. *, p<0.001, Glrx1+/+ MEFs vs. Glrx1−/− MEFs with the same treatment.
Fig. 6
Fig. 6
Morphology of MEFs treated with diquat and paraquat. Glrx1+/+ and Glrx1−/− MEFs were cultured in medium in the absence (as control) or presence of 0.05 mM diquat or 0.4 mM paraquat at 37 °C for 24 hrs. The genotypes of the MEFs and treatments are shown on top and left of the figure, respectively. A more extensive detachment of cell monolayer and cell death (shown as rounded cells) were found in Glrx1−/− MEFs treated with diquat or paraquat compared to Glrx1+/+ MEFs.
Fig. 7
Fig. 7
Grx1-deficient MEFs are more tolerant to apoptosis induced by tumor necrosis factor α (TNFα) plus actinomycin D. Glrx1+/+ and Glrx1−/− MEFs were cultured medium in the absence (as control) or presence of 10 ng/ml tumor necrosis factor α (TNFα) plus 50 ng/ml actinomycin D (AD) at 37 °C for 24 hrs. Cell viability was determined by the MTT assay as described in Fig. 5. Data are representatives from two independent experiments. Each bar represents mean ± SD, n = 4 measurements. *, p<0.001, Glrx1+/+ MEFs vs. Glrx1−/− MEFs.
Fig. 8
Fig. 8
Formation and decay of protein-SSG mixed disulfides in Glrx1+/+ and Glrx1−/− MEFs treated with 5 mM H2O2. (A) S-glutathionylation of proteins in Glrx1+/+ and Glrx1−/− MEFs in response to different concentrations of H2O2. The cells were treated with H2O2 in D-PBS for 1 hr and total proteins were separated on a 12% SDS-gel without the use of reducing agent (β-mercaptoethanol) for blot analysis using an anti-GSH antibody. The 44-kDa and 34-kDa glutathionylated proteins are indicated by arrows on right of the figure. (B) Reversal of protein S-glutathionylation in Glrx1+/+ and Glrx1−/− MEFs. The cells were treated with 5 mM H2O2 in DPBS for 1 hr and then cultured in regular medium for different periods of time before harvesting for blot analysis as described in (A). The proteins were separated on a 10% non-reducing SDS-gel to allow better separation of the high-molecular-weight proteins. The 44-kDa glutathionylated protein is indicated by an arrow on right of the figure.
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