Apoptosis caused by p53-induced protein with death domain (PIDD) depends on the death adapter protein RAIDD

Abstract

The p53 tumor suppressor promotes cell cycle arrest or apoptosis in response to diverse stress stimuli. p53-mediated cell death depends in large part on transcriptional up-regulation of target genes. One of these targets, P53-induced protein with a death domain (PIDD), was shown to function as a mediator of p53-dependent apoptosis. Here we show that PIDD is a cytoplasmic protein, and that PIDD-induced apoptosis and growth suppression in embryonic fibroblasts depend on the adaptor protein receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD). We provide evidence that PIDD-induced cell death is associated with the early activation of caspase-2 and later activation of caspase-3 and -7. Our results also show that caspase-2(-/-), in contrast to RAIDD(-/-), mouse embryonic fibroblasts, are only partially resistant to PIDD. Our findings suggest that caspase-2 contributes to PIDD-mediated cell death, but that it is not the sole effector of this pathway.

Fig. 1.

PIDD protein expression. (A) Saos-2 cells were transfected with pcDNA3.1-N-myc/his (vector), pcDNA3.1-N-myc/his-mPIDD (5′Myc-PIDD), or pcDNA3.1-mPIDD-Myc (3′Myc-PIDD). After 48 h, cell extracts were resolved by SDS/PAGE and analyzed by immunoblotting by using Myc monoclonal or PIDD polyclonal antibodies. The blot was reprobed with an antibody against β-actin as a loading control. (B) DP16.1/p53ts cells were incubated at 32°C to activate p53 for the times indicated. Cell extracts were immunoprecipitated with PIDD antibodies and analyzed by immunoblotting with the same antibodies. (C) Saos-2 cells were transiently transfected with 5′Myc-PIDD or empty vector. Immunostaining was carried out 24 h later with PIDD or Myc antibodies and analyzed by confocal microscopy. (D) H460 cells were treated with 200 ng/ml adriamycin for the indicated time periods. The level of endogenous PIDD protein was determined by immunoblotting with PIDD antibodies. The blot was reprobed with an antibody against β-actin as a loading control. (E) MEFs were exposed to γ radiation (6 Gy) and at the indicated times after irradiation, cell extracts were prepared and subjected to immunoblotting with PIDD antibodies.

Fig. 2.

PIDD expression induces apoptosis. (A) DP16.1 cells were transfected with pcDNA3.1, pcDNA3.1-PIDD, or pcDNA3.1-p53, together with a plasmid expressing the CD20 receptor. Forty-eight hours later, cells were stained with an anti-CD20-FITC antibody and Annexin V-phycoerythrin. Apoptosis was then assessed by flow cytometry. CD20-positive cells were gated, and the data shown (mean ± SEM in three independent experiments) represent the percentage of CD20-positive cells that are also Annexin V-positive. (B) E1A/Ras-transformed RAIDD^+/+ and RAIDD^-/- MEFs were infected with pMIG or pMIGPIDD or treated with TNF (10 ng/ml) and cycloheximide (2 μg/ml); infected cells were fixed 48 h later, stained with Hoechst 33258, and visualized by microscopy. The infection efficiency was determined by the proportion of cells that became GFP-positive 48 h after infection and ranged between 83% and 94%. TNF-treated cells were examined 3 h after treatment. Cells with condensed chromatin or nuclear fragmentation were scored as apoptotic, and the data are summarized in C.

Fig. 3.

Activation of caspase-2, -3, and -7 in PIDD-expressing MEFs depends on RAIDD. Western blots were performed with the indicated antibodies on lysates prepared from E1A/Ras-transformed RAIDD^+/+ and RAIDD^-/- MEFs after infection with pMIG or pMIG-PIDD (A and B). The infection efficiency was determined by the proportion of cells that became GFP-positive 24 h after infection. The infection efficiency of RAIDD^+/+ MEFs was 98% with pMIG and 91% with pMIG-PIDD; the infection efficiency of RAIDD^-/- MEFs was 97% with pMIG and 92% with pMIG-PIDD. (C) Caspase activity assays were performed as described in Experimental Procedures. (Upper) The histograms show the fold increase in activity measured in RAIDD^+/+ cells 48 h after infection with pMIG-PIDD compared with pMIG infection. (Lower) The change in activity at different time points after infection is shown.

Fig. 4.

PIDD suppresses cell growth. Transformed RAIDD^-/- MEFs (A), caspase-2^-/- MEFs (B), FADD^-/- MEFs (D), caspase-8^-/- MEFs (E), and corresponding MEFs from +/+ littermate controls were coinfected with pMIG-IRES-GFP (pMIG) or pMIG-PIDD-IRES-GFP (PIDD) together with retroviruses expressing hygromycin resistance (pBabe-Hygro); 24 h later, MEFs were treated with hygromycin, and hygromycin-resistant colonies were enumerated 7-8 days after the start of drug selection. The results represent the mean of at least three independent experiments (infections) and are presented as a percentage of the colonies obtained after coinfection with pMIG and pBabe-Hygro. Error bars indicate SEM In A, the number of colonies varied between 341 and 531 colonies per plate in the pMIG-infected +/+ MEFs and between 363 and 664 colonies per plate in the pMIG-infected RAIDD^-/- MEFs. Infection efficiency was determined by the proportion of cells that became GFP-positive 48 h after infection with pMIG alone (+/+, 59%; RAIDD^-/-, 54%). In B, the number of colonies varied between 450 and 860 per plate in the pMIG-infected +/+ MEFs and between 360 and 800 per plate in the pMIG-infected caspase-2^-/- MEFs. In D, the number of colonies varied between 140 and 200 per plate in the pMIG-infected +/+ MEFs and between 180 and 260 per plate in the pMIG-infected FADD^-/- MEFs. The infection efficiency was 27% for the +/+ MEFs and 34% for the FADD^-/- MEFs. In E, the number of colonies varied between 770 and 1,080 per plate in the pMIG-infected +/+ MEFs and between 360 and 640 per plate in the pMIG-infected caspase-8^-/- MEFs. The infection efficiency was 24% for the +/+ MEFs and 52% for the caspase-8^-/- MEFs. (C) Apaf-1^-/- and Apaf-1^+/+ MEFs were cotransfected with pcDNA3.1 or pcDNA3.1-mPIDD along with a zeomycin selection plasmid. Zeomycin-resistant colonies were enumerated 7 days after the start of drug selection. The number of colonies varied between 170 and 200 per plate in the empty vector-transfected Apaf-1^+/+ MEFs and between 120 and 180 per plate in the empty vector-transfected Apaf-1^-/- MEFs. Error bars indicate SEM (n = 3).

Fig. 5.

Interaction between PIDD and FADD. (A) Saos-2 cells were transfected with 5′Myc-PIDD or empty vector. Cell extracts were prepared 48 h later and immunoprecipitated with a FADD polyclonal antibody. Immunoprecipitated proteins were separated by SDS/PAGE, and PIDD was detected by Western blotting by using a Myc monoclonal antibody. (B) H460 cells were left untreated or incubated with 200 ng/ml adriamycin for 16 h, and extracts were prepared and immunoprecipitated with purified mouse IgG1 antibody as a control or FADD monoclonal antibody before immunoblotting with PIDD polyclonal antibodies.