Type I interferons induce apoptosis by balancing cFLIP and caspase-8 independent of death ligands

Abstract

Interferons induce a pleiotropy of responses through binding the same cell surface receptor. Here we investigated the molecular mechanism driving interferon-induced apoptosis. Using a nonbiased small interfering RNA (siRNA) screen, we show that silencing genes whose products are directly engaged in the initiation of interferon signaling completely abrogate the interferon antiproliferative response. Apoptosis-related genes such as the caspase-8, cFLIP, and DR5 genes specifically interfere with interferon-induced apoptosis, which we found to be independent of the activity of death ligands. The one gene for which silencing resulted in the strongest proapoptotic effect upon interferon signaling is the cFLIP gene, where silencing shortened the time of initiation of apoptosis from days to hours and increased dramatically the population of apoptotic cells. Thus, cFLIP serves as a regulator for interferon-induced apoptosis. A shift over time in the balance between cFLIP and caspase-8 results in downstream caspase activation and apoptosis. While gamma interferon (IFN-γ) also causes caspase-8 upregulation, we suggest that it follows a different path to apoptosis.

Fig 1

siRNA screen of apoptosis-related genes. (A) The gene count was plotted as a function of the average Z score of the YNS-treated group. cFLIP and PLK1 had a Z score of <−3, indicating enhanced antiproliferative activity, while IFNARs, CUL3, and DAP3 had a Z score of >3, indicating a rescue effect. (B) WISH cells were transfected with OnTargetPlus siRNA oligonucleotides as single K/Ds or with cFLIP oligonucleotides as double K/Ds for 48 h following treatment with 1 nM IFN-YNS for 72 h. Cell density was normalized to values for nontransfected (Non trx), nontreated cells. Data are represented as means ± standard errors of the means. (C) Western blot analysis validating downregulation upon siRNA transfection.

Fig 2

Cell death inhibitors and apoptosis markers. (A) WISH cells were treated with Z-VAD at 100 μM, Z-IETD at 50 μM, Nec-1 at 63 μM, 3-MA at 1 mM, or their combination with or without (not treated [NT]) 1 nM IFN-YNS for 72 h. Cell density was normalized to values for nontreated cells. Data are represented as means ± standard errors of the means. (B) WISH cells were transfected with OnTargetPlus siRNA following treatment with 1 nM IFN-YNS and/or 100 μM Z-VAD. Cell density was normalized to values for nontransfected, nontreated cells. (C) WISH cells were transfected with siRNA targeting IFNAR1, cFLIP, or caspase-8 or with nontargeting siRNA (control) prior to treatment with 1 nM IFN-YNS for 48 h. At the end of the process, the cells were stained with annexin V (Ax) and PI to determine apoptosis. (D) Similar to panel C, but apoptosis was analyzed by TUNEL staining.

Fig 3

Cell cycle arrest in antiproliferative activity of IFN. (A) WISH cells were transfected with cFLIP or caspase-8 siRNA followed by treatment with 1 nM YNS with or without Z-VAD for the indicated hours. Trypan blue staining was used to count live cells. (B) Cell cycle analysis was performed on WISH cells treated with 100 μM Z-VAD and/or 1 nM YNS for 48 h or left untreated (not treated [NT]).

Fig 4

cFLIP knockdown enhances sensitivity to interferon. (A and B) Continuous IFN-YNS treatment (A) and pulse treatment (B) were performed on WISH cells transfected with siRNA targeting cFLIP or IFNAR1 following treatment with 1 nM YNS for the indicated hours. (A) The cells were treated with YNS for the indicated times. (B) All the cells were treated at time zero, and after the indicated times, the medium was replaced with fresh medium without YNS but containing 200 nM extracellular domain of IFNAR2, to exclude residual IFN, for the remaining 72 h. Data are represented as means ± standard errors of the means. (C) WISH cells were transfected with up to 10 nM siRNA targeting cFLIP and/or caspase-8 for 48 h prior to treatment with 1 nM YNS for 72 h. Caspase-8 silencing was measured by Western blotting using 0, 0.5, 1, 2.5, 5, and 10 nM siRNA (inset). The protein level was calculated compared to the level in control cells. Data are represented as means ± standard errors of the means.

Fig 5

Caspase-3/7, -8, and -9 activity. (A) WISH cells were transfected with siRNA targeting cFLIP or caspase-8 following 24 h of treatment with 1 nM YNS. Cotreatment with the pancaspase inhibitor Z-VAD at 100 μM was used to inhibit the caspase activity. Data are represented as means ± standard errors of the means. (B) Caspase kinetics was measured in WISH cells transfected with siRNA targeting cFLIP or mock transfected following treatment with 1 nM YNS for the indicated hours. Data are represented as means ± standard errors of the means. (C) Protein cleavage was tested in nontransfected WISH cells and cells transfected with siRNA targeting cFLIP before treatment with 1 nM YNS. Fas was used as a positive control for caspase-3 and -8 cleavage. Cotreatment with 100 μM Z-VAD was used to inhibit the caspase activity. (D) WISH cells were transfected with siRNA targeting the indicated genes following treatment with 1 nM YNS for 48 and 72 h. Cells were stained with annexin V and PI to determine apoptosis. Data are represented as means. Multiple repeats and experiments have shown an average error of ±10% for the results.

Fig 6

Death ligand elimination assay. (A and B) WISH cells were transfected with cFLIP siRNA or mock and treated with YNS at 1 nM, TNF-α at 25 ng/ml, 1% Fas ligand, TRAIL at 25 ng/ml, Z-VAD at 100 μM, and anti-TNF-α/Fas TRAIL fused protein at 1 μg/ml for 72 h prior to determination of cell density (A) and for 24 h for the caspase-3/7 activity assay (B). Data are represented as means ± standard errors of the means. (C and D) WISH cells were transfected with siRNA targeting cFLIP, RIPK1, cIAP1, or cIAP2 or not transfected (control) following treatment with 1 nM YNS with or without Z-VAD, 1 μg/ml anti-death ligand mix, or 63 μM necrostatin-1 (Nec-1) for 48 and 72 h. Cells were stained with annexin V and PI to determine apoptosis. Data are represented as means. Multiple repeats and experiments have shown an average error of ±10% for the results. (E and F) WISH cells were transfected with siRNA targeting the indicated genes following treatment with 1 nM YNS for an additional 72 h to determine cell density (E) or 24 h for caspase-3/7 activity (F). Caspase activity is relative to untreated cells.

Fig 7

FADD-DN overexpression rescues cells from IFN-induced apoptosis. (A) Extrinsic death signal blocking was validated in FADD-DN-expressing cells versus wild-type cells (w.t.). WISH cells were treated using cross-linked anti-Fas monoclonal antibody, following crystal violet staining. (B) FLAG-tagged FADD-DN expression was monitored following treatment with TNF-α (25 ng/ml) or 1 nM YNS for 24 h in wild-type and FADD-DN WISH cells using anti-FLAG antibody. (C and D) Wild-type and FADD-DN cells were transfected with siRNA targeting the indicated genes following treatment with 1 nM YNS for 72 h, after which cell density was monitored (C), or were treated for 24 h with YNS before caspase-3/7 activity was monitored (D). Data are represented as means ± standard errors of the means.

Fig 8

In situ quantification of caspase-8 and cFLIP interactions with FADD following YNS treatment. (A) cFLIP and caspase-8 localization and levels were monitored with immunofluorescence using specific antibodies. (B and C) cFLIP-FADD (B) and caspase-8–FADD (C) interactions visualized by an in situ proximity ligation assay. WISH cells were transfected or not with cFLIP or caspase-8 siRNA following treatment with 1 nM YNS, as indicated, or were left untreated (not treated [NT]). (D) Quantification of PLA interactions per cell (shown in panels B and C). (E) Same as panels B and C. WISH cells were transfected or not with DR4 or DR5 siRNA following treatment with 1 nM YNS, as indicated.

Fig 9

Interferon-induced caspase-8 upregulation in interferon-sensitive and -resistant cell lines. (A) MDA-MB-321, T47D, and HS578T cells were treated with 1 nM YNS for 3 days following crystal violet staining for cell density evaluation. (B) Caspase-3/7 activity was measured in T47D and HS578T IFN-resistant cell lines following treatment with 1 nM YNS for 48 h. (C) WISH, MDA-MB-321, T47D, BT549T, and HS578T cells were treated with 1 nM YNS for the indicated hours following protein extraction for Western blotting. Error bars represent the standard errors of 2 to 4 independent replicates. The dashed line marks a 3-fold increase in the caspase-8 level. (D) Representative Western blotting for WISH cells treated with 1 nM YNS and/or 1,000 U/ml IFN-γ for the indicated times. (E and F) WISH cells were transfected with siRNA targeting the indicated genes following treatment with 1 nM YNS and/or 1,000 U/ml IFN-γ for 72 h (E) or 24 h (F), after which after cell density (E) or caspase-3/7 activity (F) was monitored. For panels A to C, E, and F, data are represented as means ± standard errors of the means.

Fig 10

Schematic model of IFN-induced apoptosis in WISH cells. IFN binding to its receptors activates the canonical JAK-STAT pathway, leading to cell cycle arrest and expression of caspase-8, which progresses within days. Meanwhile, the cFLIP cytoplasmic concentration increases rapidly, binding FADD within hours after IFN induction to inhibit caspase-8 cleavage. This results in slow and gradual caspase-8 activation, followed by caspase-3 cleavage and apoptosis. Caspase-8–cFLIP competition is FADD dependent and requires DR5 as a platform independent of death ligands.