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. 1997 Sep 16;94(19):10057-62.
doi: 10.1073/pnas.94.19.10057.

Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control

Z L Chu  1 , T A McKinsey, L Liu, J J Gentry, M H Malim, D W Ballard
Affiliations

Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control

Z L Chu et al. Proc Natl Acad Sci U S A. .

Abstract

Members of the NF-kappaB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappaB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-kappaB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappaB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of IkappaB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappaB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappaB via an IkappaB targeting mechanism. Functional coupling of NF-kappaB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.

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Figures

Figure 1
Figure 1
Regulation of the c-IAP2 gene by TNF and NF-κB. Jurkat T cells stably expressing FLAG-tagged forms of wild-type IκBα (lanes 1–3) or the IκBαΔN mutant (lanes 4–6) were stimulated for 2 hr with either combinations of phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μM) (P+I, lanes 2 and 5) or TNF (20 ng/ml; lanes 3 and 6). Nuclear extracts and total RNA were prepared as described in Materials and Methods. (A) Induction of NF-κB expression. Nuclear extracts (5 μg) were incubated with a 32P-labeled κB-pd probe under standard reaction conditions (38). Resultant DNA/protein complexes were resolved on a nondenaturing 5% polyacrylamide gel and visualized by autoradiography. (B and C) Northern blot analysis of c-IAP mRNA expression. Total RNA (10 μg) was fractionated on a 1.2% agarose/formaldehyde gel, transferred to a Zeta Probe membrane, and hybridized with 32P-radiolabeled cDNA probes for either human c-IAP2 or c-IAP1 (18). After hybridization, the membrane was washed at 50°C in 2 × standard saline citrate (SSC)/0.1% SDS, and specific transcripts were detected by autoradiography.
Figure 2
Figure 2
NF-κB-dependent expression of c-IAP2 mRNA in primary T lymphocytes. Human peripheral blood T lymphocytes were purified by sheep erythrocyte rosetting (41) and cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were stimulated for 2 hr with P+I in the presence or absence of CHX (50 μg/ml), pyrrolidinedithiocarbamate (PDTC) (200 μM), or N-acetyl-Leu-Leu-norleucinal (ALLN) (100 μg/ml) as indicated. Total RNAs (≈5 μg) were fractionated by agarose gel electrophoresis, transferred to a Zeta Probe membrane, and sequentially hybridized with 32P-radiolabeled probes for human c-IAP2 (A) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (B). (C) Nuclear extracts were prepared from a portion of these cells and incubated with a photoreactive κB-pd probe (38). After exposure to UV light, DNA/protein adducts were immunoprecipitated with RelA-specific antisera and fractionated by SDS/PAGE (38, 39). Only the major adduct detected by autoradiography (≈70 kDa) is shown.
Figure 3
Figure 3
Human c-IAP2 protects cells from TNF-induced killing via an NF-κB-dependent mechanism. (A) HeLa cells were transfected with 10 μg of the indicated FLAG-tagged expression vectors. Cytoplasmic extracts were prepared 48 hr after transfection and fractionated by SDS/PAGE. Proteins were transferred to polyvinylidene difluoride membranes and subjected to immunoblotting with monoclonal anti-FLAG antibodies. (B) Effects of wild-type and mutant c-IAP2 on TNF-induced death. HeLa cells (3 × 105) were cotransfected with pZeoSVLacZ DNA (1 μg) and pCMV4-based expression plasmids (1 μg) encoding either c-IAP2WT or c-IAP2ΔC. Total DNA input was normalized with pCMV4. After 24 hr of growth, transfected cultures were split into 24-well plates and propagated for 24 hr in the presence or absence of TNF (15 ng/ml). Cells were stained for β-galactosidase expression and scored. Cell viability is expressed as the percentage of blue cells in TNF-treated versus unstimulated cultures. The data shown represent the mean viability (± SEM) of cells determined from triplicate transfections in two separate experiments. (C) Requirement of NF-κB for c-IAP2-mediated protection. HeLa cells were cotransfected with pZeoSVLacZ (1 μg) and the indicated combinations of expression vectors for IκBαΔN (0.1 μg), c-IAP2 (1 μg), and CrmA (1 μg). The percentage of cells (± SEM) surviving exposure to TNF (24 hr) under each experimental condition was determined as described for B. Similar results were obtained in three separate experiments.
Figure 4
Figure 4
Overexpressed c-IAP2 activates NF-κB. (A) Stimulation of NF-κB-directed transcription. Jurkat cells stably expressing either IκBαWT (filled bars) or IκBαΔN (empty bars) were cotransfected with HIV-κB-CAT (5 μg) and 10 μg of either empty pCMV4 vector or the wild-type c-IAP2 expression plasmid. After 24 hr of growth, the indicated cultures were exposed to TNF (20 ng/ml) for 16 hr. Whole cell extracts were prepared at 40-hr posttransfection and assayed for CAT activity. Results from triplicate transfections are reported as the fold induction in CAT activity (mean ± SEM) relative to that measured in unstimulated Jurkat/IκBαWT cells transfected with HIV-κB-CAT alone (normalized to 1). (B) Induction of NF-κB DNA binding activity. HeLa cells (2 × 106) were cotransfected with pHook-1 (5 μg) and 15 μg of either blank pCMV4 plasmid (lanes 1 and 2) or the c-IAP2WT expression vector (lane 3). Transfected cells were selected by magnetic bead capture (36) after 24 hr of growth. Selected transfectants were propagated for 24 hr and then treated with CHX (50 μg/ml; 4 hr) in the presence (lane 2) or absence (lanes 1 and 3) of TNF (20 ng/ml). Nuclear extracts were prepared and analyzed in gel retardation assays with a radiolabeled κB probe (see Fig. 1A). (C) Stimulation of IκBα degradation. HeLa cells were cotransfected with an expression vector for wild-type IκBα (1 μg) and 10 μg of either pCMV4 (Upper) or c-IAP2WT (Lower). Cytoplasmic extracts were prepared from transfected cells after treatment with CHX (50 μg/ml) for the indicated times. Ectopic forms of IκBα were isolated by immunoprecipitation with anti-FLAG antibodies, resolved by SDS/PAGE, and immunoblotted with IκBα-specific antisera.
Figure 5
Figure 5
Dominant-negative effects of RING-deleted c-IAP2 on TNF-mediated NF-κB activation. (A) c-IAP2ΔC inhibits TNF-induced degradation of IκBα. HeLa cells were cotransfected with a plasmid encoding FLAG-tagged IκBα (1 μg) and 10 μg of either pCMV4 (lanes 1 and 2) or the indicated c-IAP2 expression vectors (lanes 3–6). After 48 hr of growth, transfected cells were treated for 1 hr with CHX (50 μg/ml) in the presence or absence of TNF (20 ng/ml). Ectopic IκBα was purified from cytoplasmic extracts by immunoprecipitation with anti-FLAG antibodies, subjected to SDS/PAGE, and detected by immunoblotting with IκBα-specific antibodies. Molecular mass markers (in kDa) are indicated. (B) Effects of c-IAP2ΔC on NF-κB-directed transcription in TNF-treated cells. Jurkat T lymphocytes were cotransfected with HIV-κB-CAT (5 μg), a CrmA expression vector (5 μg), and graded doses of the indicated c-IAP2 expression vectors. Total DNA input in each transfection was normalized with pCMV4. After 24 hr of growth, transfectants were stimulated for 16 hr with TNF (20 ng/ml). Whole cell extracts were prepared and assayed for CAT activity. For each titration point (n = 3), results are expressed as the mean percentage (± SEM) of CAT activity relative to that induced by TNF in cells lacking ectopic IAP protein (fold-induction over basal level = 7.6 ± 1.5; normalized to 100%).
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