PIDD-dependent activation of caspase-2-mediated mitochondrial injury in E1A-induced cellular sensitivity to macrophage nitric oxide-induced apoptosis

Abstract

Expression of the adenovirus E1A oncogene sensitizes tumor cells to innate immune rejection by apoptosis induced by macrophage-produced tumor necrosis factor (TNF)-α and nitric oxide (NO). E1A sensitizes cells to TNF-α and NO through two distinct mechanisms, by repressing NF-κB-dependent antiapoptotic responses and enhancing caspase-2 activation and mitochondrial injury, respectively. The mechanisms through which E1A enhances caspase-2 activation in response to NO were unknown. Here, we report that E1A-induced sensitization to NO-induced apoptosis is dependent on expression of PIDD (p53-inducible protein with a death domain) and enhancement of primary immunodeficiency diseases (PIDD) processing for formation of the PIDDosome, the core component of the caspase-2 activation complex. NO-induced apoptosis in E1A-expressing cells did not require expression Bak or Bax, indicating that NO-induced caspase-2-mediated mitochondrial injury does not proceed through the activities of typical, proapoptotic Bcl-2 family members that induce mitochondrial cytochrome C release. These results define a PIDD-dependent pathway, through which E1A enhances casapse-2-mediated mitochondrial injury, resulting in increased sensitivity of mammalian cells to macrophage-induced, NO-mediated apoptosis.

Fig. 1. Caspase-2 mediated mitochondrial injury in response to NO.

a Role of caspase-2 in NO-induced loss of MMP. E1A+ cells were treated with 250 μM DETA-NONOate for 6 h then stained with TMRE to estimate MMP. Loss of MMP by NO-injured (open histogram) compared to uninjured (shaded histogram) E1A-positive cells was dependent on caspase activity (E1A+ zVAD), caspase-2 activity (E1A+ zVDVAD) and expression of caspase-2 (E1AiC2). b Mitochondrial release of cytochrome c into the cytosol in E1A-positive (E1A+), E1A-negative, (E1A−) or caspase-2 siRNA-expressing E1A-positive (E1AiC2) cells following treatment with 250 μM DETA-NONOate for the times (h) indicated below the figures. c Role of caspase-2 as the initiator caspase in response to NO injury. E1A-positive cells pretreated with or without biotin-VAD (bVAD) and treated with or without 250 μM DETA-NONOate for 4 h

Fig. 2. Requirement for PIDD in NO-induced apoptosis.

a Western blot for the expression of human PIDD, actin and E1A in E1A-positive (H4-E1A), PIDD shRNA E1A+ cells (H4-E1AiPIDD) and E1A+ cells expressing scrambled control shRNA (H4-E1A scRNA). b Human fibrosarcoma cells H4, H4-E1A, H4-E1A iPDD, and H4-E1A scRNA treated with 750 μM DETA NONOate for 18 h. Cell viability was determined by MTS staining and expressed as % cell death (mean ± SEM; n = 4, ^***P < 0.001, one-way ANOVA). c Mouse NIH-3T3 cells 3T3, 3T3-E1A, 3T3-E1AiPIDD-1 and −2, and 3T3-E1A scRNA treated with 250 μM DETA-NONOate for 18 h. Cell viability was determined by MTS staining and expressed as % cell death (mean ± SEM; n = 3, ^***P < 0.001, ^**P = 0.0028, one-way ANOVA)

Fig. 3. Role of NO-induced PIDD cleavage.

a Western blot analysis of PIDD and actin in E1A-negative (H4) and E1A-positive (H4-E1A) cells in the absence or presence of DETA-NONOate. b Mouse NIN-3T3-E1A+ or E1A+ mtPIDD (S588A) cells were treated with DETA-NONOate for 18 h. Cell viability was determined by MTS staining and expressed as % cell death (mean ± SEM; n = 3, ^**P = 0.003, Student’s two-tailed t test)

Fig. 4. PIDD-mediated mitochondrial injury in response to NO.

a DilC₁ (5) staining of NIH-3T3, E1A-positive (E1A), E1A siRNA PIDD (E1A-iPIDD) and E1A PIDD-S588A (E1A-mtPIDD) cells, following treatment with DETA-NONOate for 18 h. Histograms for DETA-NONOate treated cells (unshaded histograms) are overlayed on those for untreated cells (shaded histograms). b PIDD-related changes in MMP in response to NO, expressed as a percentage of untreated control cells (mean ± SEM; n = 3, ^***P < 0.001, ^*P < 0.03, one-way ANOVA)

Fig. 5. Role of Bak/Bax in macrophage and NO-induced apoptosis.

a [³H]-thymidine-labeled BMK-E1A, BMK-E1A-Bak^−/−, BMK-E1A-Bax^−/−, or BMK-E1A-Bak/Bax^−/−/^−/− cells were incubated with ceramide (100 µM) overnight. Supernatants were collected and % specific thymidine release was assessed (mean ± SEM; n = 5, ^***P = 0.001, one-way ANOVA). b [³H]-thymidine-labeled BMK, BMK-E1A, BMK-E1A-Bak^−/−, BMK-E1A-Bax^−/−, or BMK-E1A-Bak/Bax^−/−/^−/− cells were incubated with activated bone marrow-derived macrophages at E:T of 30:1 in the absence or presence of 100 μM zVAD-fmk. After 48 h supernatants were collected and % specific thymidine release was assessed (mean ± SEM; n = 3, ^***P ≤ 0.0001, ^**P = 0.029, one-way ANOVA). c [³H]-thymidine-labeled BMK, BMK-E1A, or BMK-E1A-Bak/Bax^−/−/^−/− cells were incubated with DETA-NONOate at 650 µM alone (black bars), with 100 μM zVAD-fmk (white bars) or with cyclosporine A (100 µM) (CSA, hatched bars). After 18 h supernatants were collected and % specific thymidine release was assessed (mean ± SEM; n = 3, ^***P ≤ 0.0005, ^**P = 0.01, one-way ANOVA)