The critical role of catalase in prooxidant and antioxidant function of p53

Abstract

The tumor suppressor p53 is an important regulator of intracellular reactive oxygen species (ROS) levels, although downstream mediators of p53 remain to be elucidated. Here, we show that p53 and its downstream targets, p53-inducible ribonucleotide reductase (p53R2) and p53-inducible gene 3 (PIG3), physically and functionally interact with catalase for efficient regulation of intracellular ROS, depending on stress intensity. Under physiological conditions, the antioxidant functions of p53 are mediated by p53R2, which maintains increased catalase activity and thereby protects against endogenous ROS. After genotoxic stress, high levels of p53 and PIG3 cooperate to inhibit catalase activity, leading to a shift in the oxidant/antioxidant balance toward an oxidative status, which could augment apoptotic cell death. These results highlight the essential role of catalase in p53-mediated ROS regulation and suggest that the p53/p53R2-catalase and p53/PIG3-catalase pathways are critically involved in intracellular ROS regulation under physiological conditions and during the response to DNA damage, respectively.

Figure 1

p53 and PIG3 interact with catalase. (a) U2OS and RKO cells were untreated or treated with 1 mM H₂O₂ or 20 J/m² UV. At 24 h after treatment, catalase was immunoprecipitated with anti-catalase antibody, and the immunoprecipitated proteins were detected with anti-p53 or anti-catalase antibodies. (b) U2OS cells were transfected with control or catalase siRNA. Immunoprecipitation and immunoblotting were performed 48 h post transfection, as indicated. (c) H1299 cells were infected with Ad-LacZ or Ad-p53. Immunoprecipitation and immunoblotting were performed 48 h post infection, as indicated. (d) RKO cells were untreated or treated with 20 J/m² UV and lysed. Immunoprecipitation and immunoblotting were performed, as indicated. (e) Immunoprecipitation of human p53 from a mixture of p53 and catalase. The precipitates were subjected to western blotting using catalase antibody. The level of precipitated p53 was assessed with a human p53-specific antibody. (f) Immunoprecipitation of human PIG3 from a mixture of PIG3 and catalase. The precipitates were subjected to western blotting using catalase antibody. The level of precipitated PIG3 was assessed with a PIG3-specific antibody

Figure 2

p53 and PIG3 repress catalase activity. (a and b) The indicated amount of recombinant human p53 (a) and PIG3 (b) protein were incubated with 1.0 U/ml of recombinant human catalase, and catalase activity was measured. (c and d) U2OS, RKO, HCT116, and H460 cells were transfected with control vector (white columns) or p53 expression vector (gray columns). At 48 h after transfection, catalase activity (c) and ROS levels (d) were measured. Cell lysates were analyzed by immunoblotting, as indicated (bottom). (e) p53 stimulates intracellular ROS generation in catalase-expressing H460 cells. H460 cells were infected with the indicated viral expression vectors for 48 h, and then ROS production was assayed. Cell lysates were analyzed by immunoblotting, as indicated (bottom). (f) U2OS and RKO cells were transfected with control or catalase siRNA and then infected with Ad-LacZ or Ad-p53. At 24 h after infection, ROS generation was measured. Catalase and p53 levels were analyzed by immunoblotting (bottom). Results in a–f are shown as mean±S.E.M. (n=3). **P<0.01

Figure 3

The C-terminal region of p53 and PIG3 is required for its interaction with catalase and upregulates ROS levels. (a) Plasmids encoding V5-tagged LacZ (vector), full-length p53 (wt) or deletion mutants of p53 were transfected into HEK293T cells. Catalase was immunoprecipitated with anti-catalase antibody, and the immunoprecipitated proteins were detected with anti-V5 antibody or anti-catalase antibody. (b) Lysates of HEK293T cells expressing the GFP control vector (vector), GFP-tagged full-length PIG3 (wt) or PIG3 deletion mutants were subjected to immunoprecipitation with anti-catalase, and the resulting precipitates were subjected to immunoblot analysis with anti-GFP or anti-catalase antibodies. Schematic presentation of the p53 and PIG3 deletion mutants used in the study (a and b, bottom). The catalase binding properties of these p53 and PIG3 constructs are summarized. (c and d) Control plasmid (vector), plasmids encoding full-length p53 (wt) or p53 deletion mutants (P1 and P5) were transfected into H1299 cells. After 48 h, catalase activity (c) and ROS levels (d) were measured. (e and f) Control plasmid (vector) or plasmids encoding full-length PIG3 (wt) or PIG3 deletion mutants (PG3 and PG5) were transfected into H1299 cells. After 48 h, catalase activity (e) and ROS levels (f) were measured. Results in c–f are shown as mean±S.E.M. (n=3). **P<0.01

Figure 4

p53R2 interacts with catalase and is required for antioxidant function of p53. (a and b) U2OS, RKO, and HCT116 cells were transfected with control (white columns) or p53 (gray columns) siRNA, and ROS levels (a) and catalase activity (b) were measured 48 h later. Cell lysates were analyzed by immunoblotting, as indicated (bottom). (c) Catalase activity was measured in reactions containing equal amounts of catalase (0.3 U/ml) and various amounts of p53R2 protein. (d) U2OS cells were untreated or treated with UV (20 J/m²). At 24 h after irradiation, catalase was immunoprecipitated with anti-catalase antibody, and the immunoprecipitated proteins were detected with anti-p53R2 or anti-catalase antibodies. (e) Plasmids encoding GFP (vector), GFP-tagged full-length p53R2 (wt) or p53R2 deletion mutants were transfected into HEK293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection, as indicated. Schematic presentation of p53R2 deletion mutants used in the study (bottom). The catalase binding properties of these p53R2 constructs are summarized. (f) U2OS cells were transfected with control or p53 siRNA. At 48 h after transfection, immunoprecipitation and immunoblotting were performed, as indicated. (g and h) U2OS cells were transfected with control or p53 siRNA and then infected with Ad-LacZ or Ad-p53R2. The cells were then analyzed for catalase activity (g) and ROS levels (h). Immunoblots of p53, p53R2, and catalase from transfected cells are shown (bottom). Results in a–c, g and h are shown as mean±S.E.M. (n=3). **P<0.01, *P<0.05

Figure 5

The direct binding of p53 and p53R2 to catalase was visualized by BiFC imaging before and after UV irradiation. (a and b) U2OS cells were co-transfected with either the combination of p53-VN/catalase-VC (A) or p53R2-VN/catalase-VC (b) and untreated (CTL) or treated with 50 J/m² UV. BiFC fluorescence was obtained at 2 h after UV radiation. (c) Fold inductions of the BiFC-positive cells after UV irradiation are indicated to quantify the UV effects on protein interactions. The fold inductions of BiFC-positive cells after the UV treatment were indicated as means±s.e.m. of CTL. ** P<0.01. (d) After either VN-p53 and VC-catalase or VN-p53R2 and VC-catalase were expressed in U2OS cells, time-lapse images of single cells were captured with 5-min interval for up to 2 h after UV irradiation. (e) To quantify the changing p53/catalase (upper panel) and p53R2/catalase (lower panel) BiFC signals, fluorescence intensities of 14 individual cells were measured using an automated intensity recognition feature of the imaging analysis system. (f) Time-course changes of BiFC signal intensities in individual cells were integrated by statistical analysis as a function of mean values

Figure 6

Catalase overexpression inhibits p53- and PIG3-induced apoptotic cell death (a and b) Saos-2 and U2OS cells (left panel) and HCT116 p53^–/– and HCT116 cells (right panel) were irradiated with 10 J/m² UV for the indicated times, and catalase activity (a) and ROS levels (b) were measured. (c) Control (white columns) and catalase-overexpressing (gray columns) H1299 cells were infected with Ad-p53 for the indicated times. Apoptotic cells (sub-G1) were then identified according to DNA content and were counted. (d) Control and catalase-overexpressing H1299 cells were infected with Ad-p53 for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. (e) PIG3-overexpressing HCT116 p53^−/− cells were infected with either Ad-LacZ (white columns) or Ad-catalase (gray columns) for 24 h, and cells were then treated with 0, 20, or 40 J/m² UV irradiation. At 48 h post irradiation, apoptotic cells (Sub-G1) were analyzed. (f) PIG3-overexpressing HCT116 p53^–/– cells were infected with either Ad-LacZ or Ad-catalase for 24 h, and the cells were then untreated or treated with 10 J/m² UV irradiation for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Results in a–c and e are shown as mean±S.E.M. (n=3). *P<0.05; **P<0.01

Figure 7

Model of p53-mediated ROS regulation. In the basal state, the p53–p53R2 pathway increases catalase activity, leading to a decrease in ROS levels. After DNA damage, however, p53 cooperates with PIG3 to lower catalase activity, causing an increase in ROS accumulation