p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner

Abstract

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.

Keywords: apoptosis; calcium; endoplasmic reticulum; mitochondria-associated membranes; p53.

Fig. 1.

p53 localizes at the ER and MAMs. (A–C) Detection of p53 by immunoblotting in HCT-116 p53^+/+ fractions. (A) p53 localization in untreated condition (UNT). Accumulation of p53 at the ER and MAMs in HCT-116 p53^+/+ cells after adriamycin (ADRIA) induction (1 μM, 6 h) (B) or after H₂O₂ treatment (500 μM, 6 h) (C). (D–F) Colocalization of p53 (red) and Sec61-GFP (used as ER marker; green) in p53^+/+ MEFs under untreated conditions (UNT) (D) and after ADRIA (E) or H₂O₂ (F). (Insets) A higher magnification of the images is presented. (G) p53 activation increased its ER colocalization. Colocalization of p53 and ER in p53^+/+ MEFs quantified as the proportion of total ER marker overlapping the p53 signal (by Mander’s coefficient colocalization method). To allow for a better appreciation of colocalization of p53 with the ER, a cytoplasmic portion was selected and the contrast was increased. Bars, SEM; *P < 0.05.

Fig. 2.

Activation and accumulation of p53 at the ER/MAMs render cells more prone to death. (A and B) Percentage of apoptosis induced by (A) H₂O₂ (500 μM, 12 h) in p53^+/+ or p53^−/− MEFs or (B) ceramide (C2; 60 μM, 12 h), thapsigargin (TG; 2 μM, 12 h), tunicamycin (TUN; 6 μM, 12 h), brefeldin A (BFA; 5 mg/mL, 12 h), or menadione (MEN; 15 μM, 12 h) in p53^−/− MEFs. The data show the percentage of cell death in the whole cell population negative for annexin-V-FITC and propidium iodide (PI) staining, as analyzed by flow cytometry. (C) Detection of cytosolic cytochrome c release and supernatant HMGB1 release (a necrotic marker) by immunoblotting in p53^+/+ or p53^−/− MEFs treated with H₂O₂ (500 μM, 12 h) compared with the untreated condition. Actin was used as a loading control for the cytosolic fraction. (D) Cytosolic cytochrome c release in p53^+/+ and p53^−/− MEFs treated with C2 (60 μM, 12 h), TUN (6 μM, 12 h), or TG (2 μM, 12 h). (E) Percentage of apoptosis versus necrosis analyzed by automated imaging and cell scoring based on morphological parameters and PI staining in p53^+/+ and p53^−/− MEFs treated with H₂O₂ (500 μM, 12 h), MEN (15 μM, 12 h), TUN (6 μM, 12 h), TG (2 μM, 12 h), or C2 (60 μM, 12 h). (F) Z-VAD-FMK treatment inhibits cell death in p53^+/+ MEFs (H₂O₂, 500 μM, 6 h). (G) Quantification of cell survival induced by H₂O₂ (500 μM, 12 h) through automated nucleus count analysis. Bars, SEM. (H) Representative microscopic fields of p53^+/+ and p53^−/− MEFs under untreated conditions, pretreated with ADRIA (1 μM, 6 h) and then H₂O₂ (1 mM, 12 h). (I) Detection of apoptosis by immunoblotting in p53^+/+ and p53^−/− MEFs and p53^+/+ pretreated with ADRIA (1 μM, 6 h) under untreated conditions and with H₂O₂ (500 μM, 6 h).

Fig. 3.

Deregulation of Ca²⁺ homeostasis after p53 induction is a stress signal for mitochondrial structure and a trigger for apoptosis. (A–C) Measurements of [Ca²⁺] using recombinant aequorin upon agonist stimulation (100 μM ATP) in the ER (A), cytosol (B), and mitochondria (C). (D) ER Ca²⁺ release induced by H₂O₂ measured using a FRET-based Ca²⁺-sensitive D1ER-YC4.3 probe; the normalized FRET ratio of D1ER-YC4.3 was assumed as the intraluminal [Ca²⁺]. (Insets) A magnified portion of the first 2 min of the recording as basal. (E) Cytosolic Ca²⁺ response induced by H₂O₂ (2 mM) in MEFs loaded with the Ca²⁺-sensitive fluorescent dye Fura-2. The kinetic behavior of the [Ca²⁺]_c (Ca²⁺ concentration within cytoplasm) response is presented as the ratio of fluorescence at 340 nm/380 nm. (F) Analysis of [Ca²⁺]_m (Ca²⁺ concentration within mitochondrial matrix) during oxidative stress upon H₂O₂ stimulation (2 mM). Isosurface rendering of representative p53^+/+ (G and H) and p53^−/− (I and J) MEFs expressing mitochondrial GFP in basal conditions (UNT), after adriamycin (1 μM, 6 h), and/or H₂O₂ exposure (500 μM, 3 h). (H and J) High-resolution imaging of mitochondrial fragmentation during p53 activation and oxidative stress induction in p53^+/+ and p53^−/− MEFs.

Fig. 4.

p53 controls mitochondrial Ca²⁺ homeostasis and, in turn, apoptotic sensitivity from ER/MAM compartments. (A) Agonist-dependent [Ca²⁺]_m response in p53^+/+ MEFs after pharmacological block of the transcriptional arm of p53. (B) Schematic representation of p53-ΔNLS and ER-p53 chimeras. (C) Immunofluorescence images of p53^−/− MEF cells expressing the p53-ΔNLS or ER-p53 constructs stained with anti-p53 antibody (green) and Hoechst (nuclear marker). (D) Mitochondrial Ca²⁺ response in p53^−/− MEFs after the reintroduction of an ER-targeted chimera, ER-p53 or p53-ΔNLS. (E) Representative microscopic fields, from three independent experiments, of p53^−/− MEFs expressing p53-ΔNLS and ER-p53 before and after H₂O₂ treatment (1 mM, 12 h). (F) Evaluation of cell-death induction by H₂O₂ (500 μM, 12 h) through automated nucleus count analysis in p53^−/− MEFs, p53^−/− MEFs expressing ER-p53, and p53^−/− MEFs expressing p53-ΔNLS. Bars, SEM. (G) Analysis of apoptotic markers by immunoblot in p53^−/− MEFs and p53^−/− MEFs expressing p53-ΔNLS and ER-p53 under untreated conditions and after H₂O₂ treatment (500 μM, 6 h).

Fig. 5.

p53 mutants cannot modulate the mitochondrial Ca²⁺ response and thus apoptosis. (A) Mitochondrial Ca²⁺ response after agonist stimulation in HCT-116 p53^−/− cells and HCT-116 p53^−/− cells after reintroduction of the p53-ΔNLS and ER-p53 chimeras or naturally occurring p53 mutants R175H and R273H. (B) Mitochondrial [Ca²⁺] after ATP stimulation measured in MDA-MB 468 cells, harboring p53 273H mutation, under control conditions and after adriamycin treatment (1 μM, 6 h). (C and D) Evaluation of apoptosis induction in HCT-116 p53^−/− cells expressing p53-ΔNLS and ER-p53 chimeras or naturally occurring p53 mutants R175H and R273H after treatment with H₂O₂ using (C) immunoblot detection of cleaved PARP and cleaved caspase 3 (500 μM, 6 h) and (D) automated cell count analysis (500 μM, 12 h). Bars, SEM. (E) Representative images of HCT-116 p53^−/− cells expressing different p53 constructs under untreated conditions and after H₂O₂ treatment (1 mM, 12 h).

Fig. 6.

p53 interacts with SERCA and stimulates Ca²⁺ accumulation in the ER, changing the SERCA oxidative state. (A) In vitro binding of endogenous SERCA to MBP-p53. H1299 cell lysates were incubated with bacterially expressed MBP-p53 protein or MBP as a control. Ponceau staining shows the amount of MBP proteins used in the experiments. (B) Full-length (FL) and HA-tagged p53 deletion mutants transiently expressed in H1299 cells were immunoprecipitated by anti-HA antibody and analyzed by Western blot (WB). IP, immunoprecipitation. (C) H1299 cells were transiently transfected with different p53 constructs (FL, full-length p53 WT; ΔNLS, p53-ΔNLS; R175H, p53 R175H; R273H, p53 R273H) and then harvested for immunoprecipitation and immunoblotting as indicated. WCL, whole cell lysate. (D and E) Rate analysis of Ca²⁺ uptake measured in the ER vesicles isolated from the (D) liver of p53^−/− and p53^+/+ mice and p53^+/+ mice treated with adriamycin (1 μM, 6 h) or (E) ER compartments of p53^−/− and p53^+/+ MEFs at different times after ADRIA (1 μM, 30 min, 3 h, 6 h) treatments. (F) Immunoblot with an antibody reactive to dimedone-conjugated cysteine residues of the protein sample extracts from HCT-116 p53^+/+ and MDA-MB 468 cells after ADRIA induction that were immunopurified using a monoclonal SERCA2 antibody. Cells with an active p53 reveal lower cysteinyl sulfenic acid-modified SERCA. (G) Analysis of ER Ca²⁺ uptake in HCT-116 p53^+/+ and MDA-MB 468 cells after ADRIA induction. Bars, SEM.