Impaired antioxidant defence and accumulation of oxidative stress in caspase-2-deficient mice

Abstract

Caspase-2 has been implicated in apoptosis and in non-apoptotic processes such as cell cycle regulation, tumor suppression and ageing. Using caspase-2 knockout (casp2(-/-)) mice, we show here that the putative anti-ageing role of this caspase is due in part to its involvement in the stress response pathway. The old casp2(-/-) mice show increased cellular levels of oxidized proteins, lipid peroxides and DNA damage, suggesting enhanced oxidative stress. Furthermore, murine embryonic fibroblasts from casp2(-/-) mice showed increased reactive oxygen species generation when challenged with pro-oxidants. Reduced activities of antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were observed in the old casp2(-/-) mice. Interestingly, in the old casp2(-/-) animals expression of FoxO1 and FoxO3a was significantly reduced, whereas p21 levels and the number of senescent hepatocytes were elevated. In contrast to young wild-type mice, the casp2(-/-) animals fed an on ethanol-based diet failed to show enhanced GSH-Px and SOD activities. Thus, caspase-2, most likely via FoxO transcription factors, regulates the oxidative stress response in vivo.

Figure 1

Loss of caspase-2 increases oxidative stress. Increased (a) lipid peroxidation, (b) protein oxidation levels and reduced (c) SOD activity, (d) GSH-Px activity in old Casp2^−/− mice liver. Each dot or triangle represents an individual animal. Values are mean±S.E.M. for six to seven animals per group. ^aP<0.05, ^bP<0.01 and ^cP<0.001 represent comparison between the two genotypes. (e) ROS levels in untreated and menadione treated SV40 immortalized MEFs. Results shown are representative of three independent experiments

Figure 2

Old Casp2^−/− mice display increased DNA damage. (a) Immunohistochemical localization of γ-H2AX in aged mice liver ( × 40), scale bar=50 μm. (b) Quantitation of γ-H2AX stained cells in liver sections from old Casp2^+/+ and Casp2^−/− mice. Values are average number of γ-H2AX positive cells per field of view at × 40 magnification. (c) 8-OHdG levels in liver DNA from young and old Casp2^+/+ and Casp2^−/− animals (n=5 or 4) were determined by using an EIA kit (Cayman Chemicals). Values are mean±S.E.M. ^aP<0.05 represents comparison between the two groups. (d) Casp2^−/− MEFs at early passage (P2) and late passage (P6) stained with γ-H2AX after menadione treatment and the frequency of γ-H2AX positive cells quantitated. Values are mean±S.E.M. ^aP<0.05 represents comparison between the groups

Figure 3

Old Casp2^−/− mice show increased number of senescent cells and higher p53 and p21 expression. (a) β-Gal stained cells in liver sections from old Casp2^+/+ and Casp2^−/− mice were enumerated by light microscopy. Values are average number of β-gal positive cells per field of view at × 40 magnification. Results are mean±S.E.M. (n=4), ^cP<0.001. (b) Immunoblots showing higher expression of p53 and p21 in old Casp2^−/− mice liver. Y=young, O=old. (c) Real-time PCR analysis showing increased transcript levels of p53 and p21 in old Casp2^−/− mice livers. Values represent mean±S.E.M. from three independent experiments each performed with three mice per group in triplicate. ^aP<0.05 and ^bP<0.01 represent comparison between the two groups

Figure 4

Old Casp2^−/− mice have reduced FoxO expression. (a) Real-time PCR analysis showing decreased expression of FoxO1 and FoxO3a in old Casp2^−/− mice liver. Values are mean±S.E.M. from three mice of each genotype, ^aP<0.05. (b) Expression of FoxO target genes, catalase and SOD2 was reduced, while no significant change was observed in GSH-Px levels. (c) Immunoblots showing lower expression of FoxO1 and FoxO3a in old Casp2^−/− mice liver than in old Casp2^+/+ liver. (d) Caspase-2 siRNA knockdown in U2OS cells leads to reduced expression of FoxO3a and its target gene (SOD2). (e and f) Re-expression of caspase-2-GFP in Casp2^+/+ and Casp2^−/− MEFs increases expression of FoxO1 and FoxO3a transcripts and protein compared with the GFP vector control. (g) Increase in SOD2 and GSH-Px expression and restoration of catalase expression in Casp2^−/− MEFs following caspase-2-GFP expression. Values are mean±S.E.M. from three independent experiments. ^aP<0.05 and ^bP<0.01 represents comparison between the groups

Figure 5

Old Casp2^−/− mice show reduced expression of Sestrin antioxidant enzymes in liver. (a) Sestrin 2 and 3 expression decreases in aged Casp2^−/− mice. (b) No change in peroxiredoxin 1 and 4 expression was observed. (c) Re-expression of caspase-2-GFP in MEFs restores sestrin transcript levels. Values represent mean±S.E.M. from two independent experiments each performed with three mice per group in triplicate. ^*P<0.01 represents comparison between the groups. (d) Representative images of TUNEL on liver sections of old mice ( × 20) and quantitation of frequency of TUNEL positive cells. Scale bar=100 μm

Figure 6

Oxidative stress and ROS in ethanol diet fed mice. (a) Representative H and E stained liver sections from control or ethanol diet fed mice ( × 20), scale bar=100 μm. (b) Scoring liver steatosis in the different treatment groups (n=5). (c) Serum ALT levels in Casp2^+/+ and Casp2^−/− mice fed either control diet or diet containing 5% ethanol. (d) GSH-Px activity is unchanged in the ethanol diet fed Casp2^−/− mice. (e) SOD activity is unchanged in the ethanol diet fed Casp2^−/− mice. Values are mean±S.E.M. (n=5). ^aP<0.05 and ^bP<0.01 represents comparison between the groups

Figure 7

TUNEL in ethanol diet fed mice liver. (a) Representative images of TUNEL stained liver sections from each group ( × 20), scale bar=100 μm and (b) quantitation of frequency of TUNEL positive cells, are shown. Values are mean±S.E.M. from three animals. ^aP<0.05