zVAD-induced necroptosis in L929 cells depends on autocrine production of TNFα mediated by the PKC-MAPKs-AP-1 pathway

Abstract

It is intriguing that some pan-caspase inhibitors such as zVAD-fmk (zVAD) are capable of inducing necrotic cell death in a selected group of cells. As earlier reports from our laboratory have ruled out the original notion that zVAD-induced necrosis in mouse fibrosarcoma L929 cells was autophagic cell death, the main objective of this study was thus to determine the underlying mechanism of this form of cell death. In this study, we provided clear evidence that zVAD-induced necroptosis in L929 cells and such cell death is dependent on autocrine production of tumor necrosis factor-α (TNFα) at the transcriptional level. More importantly, we identified that activating protein-1 (AP-1), but not nuclear factor κ-B, is the transcription factor controlling zVAD-induced TNFα transcription. Moreover, zVAD is able to activate AP-1 through activation of two upstream mitogen-activated kinases (MAPKs), c-Jun N-terminal kinase and extracellular signal-regulated kinase. Finally, we found that protein kinase C is the important upstream signaling molecule in mediating zVAD-induced activation of MAPKs and AP-1, and subsequent autocrine production of TNFα and cell death. Data from this study reveal the molecular mechanisms underlying zVAD-induced necroptosis, an important form of programmed necrotic cell death with increasing understanding of its biological significance in health and diseases.

Figure 1

zVAD-fmk (zVAD) and BocD-fmk (BocD), but not QVD-oph (QVD), induce necrosis in L929 cells. (a) Effects of various caspase inhibitors on cell death. L929 cells were treated with zVAD (10 μM), QVD (20 μM), BocD (10 μM), IETD-fmk (20 μM), IETD-oph (20 μM), and DEVD-cho (100 μM) for 24 h. Cell death was examined under microscope ( × 200) for morphological changes (upper panel) and quantified by the PI exclusion assay (lower panel) (data were presented as mean±S.D. from three independent experiments). (b) Effects of caspase inhibitors on TRAIL-induced caspase and PARP-1 activation. L929 cells were pretreated with the caspase inhibitors as described in panel a for 1 h, followed by treatment with TRAIL (20 ng/ml) for 8 h. Cell lysate was subjected to western blotting. (c) Overexpression of CrmA is not sufficient to induce cell death in L929 cells. Cells were co-transfected with CrmA and EGFP (10 : 1), 24 h later, cells were treated with TNFα (10 ng/ml × 6 h) or remained untreated for another 24 h (Ctrl), cell death was examined under a fluorescence microscope ( × 400) and was quantified by counting the dead cells with green color (with a morphology of rounded or floated) in randomly selected 100 green cells (data were presented as mean±S.D. from three independent experiments). (d) De novo protein synthesis is required for zVAD-induced cell death. L929 cells were pretreated with CHX (10 μg/ml) or ActD (5 μg/ml) for 30 min, followed by treatment with zVAD (10 μM) for 24 h. The cell death was examined as described in panel a. (e) zVAD-induced cell death is affected by the volume of culturing medium. L929 cells were treated with different volumes of culturing medium (300, 600, and 1000 μl per well for 24-well plates) containing the same concentration of zVAD (10 μM) or TNFα (10 ng/ml) for 24 or 8 h respectively. The cell death was examined as described in panel a

Figure 1

zVAD-fmk (zVAD) and BocD-fmk (BocD), but not QVD-oph (QVD), induce necrosis in L929 cells. (a) Effects of various caspase inhibitors on cell death. L929 cells were treated with zVAD (10 μM), QVD (20 μM), BocD (10 μM), IETD-fmk (20 μM), IETD-oph (20 μM), and DEVD-cho (100 μM) for 24 h. Cell death was examined under microscope ( × 200) for morphological changes (upper panel) and quantified by the PI exclusion assay (lower panel) (data were presented as mean±S.D. from three independent experiments). (b) Effects of caspase inhibitors on TRAIL-induced caspase and PARP-1 activation. L929 cells were pretreated with the caspase inhibitors as described in panel a for 1 h, followed by treatment with TRAIL (20 ng/ml) for 8 h. Cell lysate was subjected to western blotting. (c) Overexpression of CrmA is not sufficient to induce cell death in L929 cells. Cells were co-transfected with CrmA and EGFP (10 : 1), 24 h later, cells were treated with TNFα (10 ng/ml × 6 h) or remained untreated for another 24 h (Ctrl), cell death was examined under a fluorescence microscope ( × 400) and was quantified by counting the dead cells with green color (with a morphology of rounded or floated) in randomly selected 100 green cells (data were presented as mean±S.D. from three independent experiments). (d) De novo protein synthesis is required for zVAD-induced cell death. L929 cells were pretreated with CHX (10 μg/ml) or ActD (5 μg/ml) for 30 min, followed by treatment with zVAD (10 μM) for 24 h. The cell death was examined as described in panel a. (e) zVAD-induced cell death is affected by the volume of culturing medium. L929 cells were treated with different volumes of culturing medium (300, 600, and 1000 μl per well for 24-well plates) containing the same concentration of zVAD (10 μM) or TNFα (10 ng/ml) for 24 or 8 h respectively. The cell death was examined as described in panel a

Figure 2

zVAD-fmk (zVAD) promotes autocrine production of TNFα. (a) Basal level of autocrine TNFα secreted in culturing medium in untreated cells. L929 cells were washed with PBS twice and incubated in fresh medium for up to 24 h, and the secreted TNFα in the medium was determined by ELISA. (b) zVAD enhances autocrine production of TNFα in culturing medium. L929 cells were incubated in fresh medium containing zVAD (10 μM) or zVAD+CHX (10 μg/ml) for up to 24 h. The TNFα level was measured by ELISA. (c) zVAD and BocD-fmk (BocD), but not QVD-oph (QVD), promote TNFα transcription. L929 cells were treated with zVAD (10 μM), QVD (20 μM), and BocD (10 μM) for 6 h, the TNFα mRNA level was determined by RT-PCR. (d) zVAD and BocD, but not QVD, promote autocrine production of TNFα. L929 cells were treated as described in panel c in fresh medium for 8 h, and the secreted TNFα level was measured by ELISA

Figure 3

NF-κB pathway has a protective function during zVAD-fmk (zVAD)-induced necroptosis. (a) Knockdown of IKKα and IKKβ in L929 cells was performed as described in Materials and Methods section. (b) Effects of knockdown of IKKα and IKKβ on zVAD-induced cell death. After knockdown of IKKα and IKKβ, cells were treated with zVAD (10 μM) for 24 h, and cell death was measured by the PI exclusion assay (data were presented as mean±S.D. from three independent experiments). (c) Knockdown of RelA and RelB in L929 cells was performed as described in Materials and Methods section. (d) Effects of knockdown of RelA and RelB on zVAD-induced cell death. Cells with knockdown of RelA and RelB were treated and the cell death was measured as described in panel b. (e) Effects of knockdown of RelA and RelB on autocrine production of TNFα. Cells with knockdown of RelA and RelB were treated with DMSO or zVAD (10 μM) for 8 h, secreted TNFα was determined by ELISA

Figure 4

AP-1 activity is required for zVAD-fmk (zVAD)-induced TNFα production and necroptosis. (a) Knockdown of c-Jun was performed as described in Materials and Methods section. (b) Knockdown of c-Jun inhibits zVAD-induced AP-1 luciferase activity. Cells with knockdown of c-Jun were transfected with the AP-1 and Renilla luciferase vectors for 24 h and then were treated with zVAD (10 μM) for additional 10 h. The AP-1 luciferase activity was measured as described in Materials and Methods section. (c) Knockdown of c-Jun abolishes zVAD-induced TNFα transcription. Knockdown of c-Jun was performed as described in panel a, followed by treatment with zVAD (10 μM) for 6 h. TNFα mRNA level was determined by RT-PCR. (d) Knockdown of c-Jun blocks zVAD-induced cell death. Knockdown of c-Jun was performed as described in panel a, followed by treatment with zVAD (10 μM) for 24 h. Cell death was determined as described in Figure 1a

Figure 5

zVAD-fmk(zVAD)-induced AP-1 activation is mediated by JNK and ERK. (a) zVAD activates JNK, ERK, and c-Jun. L929 cells were treated with zVAD (10 μM) for up to 6 h. Cell lysate was subjected to western blotting. (b) zVAD activates JNK, ERK, and c-Jun in the presence of TNFα-neutralizing antibody. Cells were pretreated with TNFα-neutralizing antibody (2.0 μg/ml) for 1 h, followed by treatment with zVAD as described in panel a, cell lysate was subjected to western blotting. (c) MAPKs inhibitors suppress zVAD-induced c-Jun phosphorylation. L929 cells were treated with zVAD (10 μM) with or without the presence of SP600125 (SP, 20 μM) or PD98059 (PD, 20 μM) for 6 h, and cell lysate was subjected to immunoblotting. (d) MAPKs inhibitors suppress zVAD-stimulated AP-1 luciferase activity. L929 cells were transfected with luciferase vectors for 24 h, and then were treated as described in panel c for 10 h, the AP-1 activity was measured by the luciferase assay. (e) MAPKs inhibitors block zVAD-induced autocrine production of TNFα. L929 cells were incubated in fresh medium and were treated as described in panel c for 8 h, the TNFα protein level in the culturing medium was determined by ELISA. (f) MAPKs inhibitors protect zVAD-induced cell death. L929 cells were treated as described in panel c for 24 h, and cell death was measured as described in Figure 3b

Figure 6

PKC has a critical function in zVAD-fmk (zVAD)-induced JNK, ERK, andAP-1 activation, and consequent TNFα production and necroptosis. (a) zVAD activates PKC. L929 cells were treated with zVAD (10 μM) for up to 6 h, with or without the presence of PKCi (1 μM). TPA (80 nM × 1 h) was used as a positive control. Activation of PKC was determined by the phosphorylation of PKC substrates using immunoblotting. (b) Effects of TPA and PKCi on zVAD-induced activation of JNK and ERK. L929 cells were treated with zVAD (10 μM), TPA (80 nM), or PKCi (1 μM) as indicated in the figure for 6 h, and cell lysate was subjected to immunoblotting. (c) Effects of TPA and PKCi on zVAD-induced TNFα transcription. Cells were treated as described in panel b for 6 h, TNFα mRNA level was determined by RT-PCR. (d) PKC activation contributes to zVAD-induced autocrine production of TNFα. L929 cells were treated as described in panel b for 8 h, concentration of the TNFα protein level in the medium was determined by ELISA. (e) PKC activation enhances zVAD-induced cell death but suppresses TNFα-induced cell death. L929 cells were treated with zVAD (10 μM × 24 h) or TNFα (1 ng/ml × 8 h) with or without the presence of PKCi (1 μM) or TPA (80 nM). Cell death was determined as described in Figure 3b

Figure 7

TPA combining with caspase 8 inhibition induces necroptosis in L929 cells. (a) zVAD-fmk (zVAD) and BocD-fmk (BocD), but not QVD-oph (QVD), activate the PKC-MAPKs-AP-1 signaling pathway. L929 cells were treated with zVAD (10 μM), QVD (20 μM), or BocD (10 μM) for 6 h, and cell lysate was subjected to immunoblotting. (b) QVD, when combining with TPA, induces cell death. L929 cells were treated with zVAD (10 μM), QVD (20 μM), or DEVD-cho (DEVD, 100 μM) with or without the presence of TPA (80 nM) for 24 h, and cell death was quantified as described in Figure 3b. (c) TPA induces cell death in caspase 8 knockdown cells. L929 cells with knockdown of caspase 8, were treated with TPA (80 nM) or zVAD (10 μM) for 24 h, cell death was determined as described in Figure 3b. (d) Illustration of the signaling pathways for zVAD-induced necroptosis. Both promotion of autocrine production of TNFα by the PKC–MAPKs–AP-1 signaling pathway and suppression of caspase activation to stabilize RIP1 are required for necroptosis induced by zVAD