ABSTRACT
The Tax oncoprotein encoded by human T-cell leukemia virus induces both T-cell activation and apoptosis. The mechanism by which Tax induces apoptosis has remained unclear. Using genetically manipulated T-cell lines, we demonstrate that Tax-induced T-cell death is dependent on NF-kappaB signaling. Tax fails to induce apoptosis in T cells lacking IkappaB kinase gamma (IKKgamma), an essential component of the NF-kappaB signaling pathway. This defect was rescued when the mutant cells were reconstituted with exogenous IKKgamma. We further demonstrate that the Tax-induced T-cell death is mediated by TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), because this event can be effectively inhibited by a TRAIL-blocking antibody. Consistent with this functional aspect, Tax stimulates the expression of TRAIL mRNA. Finally, we provide genetic evidence demonstrating that the NF-kappaB signaling pathway is essential for TRAIL gene induction by both Tax and T-cell activation signals. These studies reveal a novel function of the NF-kappaB signaling pathway and suggest a key mechanism by which Tax induces T-cell death.
Subject(s)
Apoptosis/physiology , Gene Expression Regulation/physiology , Gene Products, tax/physiology , Membrane Glycoproteins/genetics , NF-kappa B/metabolism , Signal Transduction , T-Lymphocytes/cytology , Tumor Necrosis Factor-alpha/genetics , Apoptosis Regulatory Proteins , Base Sequence , DNA Primers , Humans , Jurkat Cells , NF-kappa B/physiology , RNA, Messenger/genetics , TNF-Related Apoptosis-Inducing Ligand , Transcriptional ActivationABSTRACT
Stimulation of T cells by antigens or mitogens triggers multiple signaling pathways leading to activation of genes encoding interleukin-2 and other growth-regulatory cytokines. The same stimuli also activate the gene encoding an apoptosis-inducing molecule, Fas ligand (FasL), which contributes to activation-induced cell death. It has been proposed that the signaling pathways involved in cytokine gene induction also contribute to activation-induced FasL expression; however, genetic evidence for this proposal is lacking. In the present study, the role of the NF-kappaB signaling pathway in FasL gene expression was examined using a mutant T cell line deficient in an essential NF-kappaB signaling component, IkappaB kinase gamma. These mutant cells have a blockade in signal-induced activation of NF-kappaB but remained normal in the activation of NF-AT and AP-1 transcription factors. Interestingly, the NF-kappaB signaling defect has no effect on mitogen-stimulated FasL gene expression, although it completely blocks the interleukin-2 gene induction. We further demonstrate that NF-kappaB activation is required for protecting T cells from apoptosis induction by mitogens and an agonistic anti-Fas antibody. These genetic results suggest that the NF-kappaB signaling pathway is not required for activation-induced FasL expression but rather mediates cell growth and protection from activation-induced cell death.
Subject(s)
Apoptosis/genetics , Membrane Glycoproteins/metabolism , NF-kappa B/metabolism , NF-kappa B/physiology , Annexin A5/metabolism , Blotting, Northern , Cell Line , Cell Nucleus/metabolism , Enzyme Activation , Fas Ligand Protein , Flow Cytometry , Gene Expression , Humans , I-kappa B Kinase , Immunoblotting , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Jurkat Cells , Luciferases/metabolism , Mitogens/pharmacology , Mutagenesis , NF-kappa B/genetics , Plasmids/metabolism , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Retroviridae/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , T-Lymphocytes/metabolism , Transduction, Genetic , TransfectionABSTRACT
NF-kappa B plays a pivotal role in normal T-cell activation and may also mediate human T-cell leukemia virus (HTLV)-induced T-cell transformation. Activation of NF-kappa B by both T-cell costimulatory signals and the HTLV Tax protein involves stimulation of I kappa B kinase (IKK). As a genetic approach to dissect the intermediate steps involved in NF-kappa B activation in human T cells, we performed somatic cell mutagenesis to isolate signaling-defective mutant Jurkat T-cell lines. One of the mutant cell lines was shown to have a specific blockade in the IKK signaling pathway but remained competent in the c-Jun N-terminal kinase and MAP kinase pathways. Interestingly, this mutant cell line lacks expression of IKK gamma, a non-catalytic component of the IKK complex. Expression of exogenous IKK gamma in the mutant cells restored NF-kappa B activation by both the T-cell costimulation agents and Tax. These findings provide genetic evidence for the requirement of IKK gamma in NF-kappa B signaling triggered by both T-cell costimulatory signals and HTLV-I Tax protein.