ICANCER RESEARCH 58. 882-886. March I. 19981 Advances in Brief CD95 (Fas)-induced Caspase-mediated Proteolysis of NF-KB1 Rajani Ravi, Asheesh Bed!, Ephraim J. Fuchs, and Atul Bedi2 John* Hopkins Onct>Iof>\Center. Division of Experimental Therapeutics und Pharmacology, Baltimore, Maryland 21287 Abstract Activation of the nuclear factor (NF)-KB transcription factor is instru mental for the immune response and the survival of peripheral activated T cells. We demonstrate that ligation of CD95 (Fas/APOl), a potent apoptotic stimulus in lymphocytes, results in repression of NF-KB activity in Jurkat T cells by inducing the proteolytic cleavage of NF-KB p65 (Rei A) and p50. Inhibition of caspase-3-related proteases by a specific acety- lated aldehyde (Ac-DEVD-CHO) prevented CD95-induced cleavage of p65 (RelA) or p50 and restored the inducibility of NF-KB in cells treated with an antibody against CD95. The addition of recombinant caspase-3 also resulted in proteolytic cleavage of RelA p65 and p50 in vitro. INI-« treatment, unlike CD95 ligation, did not result in the death of Jurkat cells but did so in the presence of Ih-HuM. a transdominant inhibitor of NF-KB. These results suggest that intact, functional NF-KB maintains the survival of activated T cells, and that CD95-induced cleavage of NF-KB subunits sensitizes T cells to apoptosis and, hence, facilitates the decay of an immune response. Introduction The immune response entails a cascade of cellular and molecular events commencing with T-cell proliferation and the generation of effector T cells, and culminating with the death of the majority of activated T cells and persistence of a small cadre of T cells respon sible for immunological memory (1). The activation and decay phases of the immune response involve certain key receptor-ligand interac tions. Activation of T cells requires at least two signals (2). Signal one is generated by ligation of the clonotypic T-cell receptor by a complex of the antigenic peptide in the groove of a major histocompatibility complex molecule on the surface of the antigen-presenting cell. A second, or costimulatory, signal involves ligation of the CD28 recep tor on the T cell by members of the B7 family of molecules expressed by the antigen-presenting cell. A prominent function of CD28 ligation in a primary immune response is the maintenance of T-cell survival until decay of the immune response (3). Apoptosis of ATCs3 occurs upon ligation of the CD95 (Fas/APO-1) cell surface receptor, a member of the TNF receptor superfamily, whose ligand, CD95L (FasL), is expressed on activated but not resting T cells (4). CD95- CD95L interactions are required for T-cell homeostasis, because mice deficient in either molecule accumulate excessive numbers of CD4 CDS", B220+ T cells that are otherwise marked for apoptosis (5). As the signals responsible for T-cell activation and apoptosis be- Received 11/17/97; accepted 1/19/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was funded in pan by Grant l R29CA7I660-OIA1 from the National Cancer Institute (to A. B.). A. B. is a recipient of a Passano Physician Scientist award, a Jose Carreras American Society of Hematology Scholar award, and grants from the American Cancer Society and the Wendy Will Case Cancer Fund. " To whom requests for reprints should be addressed, at 3-127 Johns Hopkins Oncol ogy Center. 6(X) North Wolfe Street. Baltimore. Maryland 21287. Phone: (410) 955-8797; Fax: (410)955-1969. 1The abbreviations used are: ATC, activated T cell; TNF. tumor necrosis factor; NF. nuclear factor; PHA. phytohemagglutinin; Ac-DEVD-CHO. acetyl-Asp-Glu-Val-Asp- aldehyde; 1KB«,inhibitor «Ber:EMSA, eleclrophoretic mobility shift assay; PMSF. phenylmethylsulfonyl fluoride; TRAF. TNF receptor-associated factor. come better defined, the intracellular mediators of these signals are increasingly sought after as targets for therapeutic intervention. One important mediator of the immune response is NF-KB, a family of heterodimeric transcription factors that has a well-established role in the development and activation of T cells (6-9). The survival of peripheral ATCs is also dependent upon NF-KB activity because excessive ATC apoptosis is observed in transgenic mice expressing constitutively high levels of iKBa, an inhibitor of NF-KB (9, 10). NF-KB comprises two subunits (p65 or RelA and p50), which are held in an inactive complex with IKBa (p35: Refs. 6-10). Extracellular signals, such as CD28 ligation, release active NF-KB from the com plex by inducing the phosphorylation and ubiquitin-mediated proteo- somal degradation of IKBa (11-15). Although the events leading to NF-KB activation upon T-cell activation have been well defined, the signals leading to its decay at the tail-end of an immune response have yet to be elucidated. One possibility is that signals through CD95 are somehow involved with its repression. The CD95L promoter contains an NF-KB binding site, and expression of CD95L is induced upon T-cell activation (4). CD95 ligation initiates the sequential activation of a cascade of cysteine proteases, or caspases, which are evolution- arily conserved mediators of cell death in response to diverse stimuli (16-21). Putative recognition sequences for caspase-3-related pro teases are present in the amino-acid sequences of NF-KB p65 (RelA) and NF-KB p50 (22-24). These observations prompted us to investi gate the possibility that CD95-transduced signals may be responsible for the autoregulatory repression of NF-KB in ATCs by caspase- mediated cleavage of RelA p65 or p50. Materials and Methods Cell Lines. A transdominant negative NH2- and COOH-terminal phospho rylation mutant of IKBa (iKBaM) has been shown to repress transcriptional activation mediated by all five NF-KB/Rel subunits (25. 26). The generation of Jurkat T cells transduced with the IKBaM retrovirus (pLIxBaMSN) has been described previously (26). IKBaM was constructed by combining the NH2- and COOH-terminal phosphorylation mutants of IKBa into a single cDNA (26). Jurkat cells expressing I/<BaM (IxBaM Jurkat) and control cells transduced with empty vector (Jurkat-LXSN) were a generous gift from Drs. Douglas Green (La Jolla Institute for Allergy and Immunology. La Jolla, CA) and Inder M. Verma (Salk Institute. La Jolla. CA; Ref. 26). IKBaM expression in pools of infected cells was confirmed by immunoblot analysis of IxBa: as described previously, murine IKBaM migrated faster than endogenous 1KB«on SDS- PAGE (26). Control Jurkat cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum and 100 units/ml penicillin/strepto mycin. IKBaM Jurkat cells were grown in the same medium supplemented with G418 (500 fig/ml). Assessment of Apoptosis in Response to TNF-a or Anti-CD95 Anti body. Jurkat cells transduced with IxBaM (IxEaM Jurkat) or empty LXSN vector (control Jurkat; 5 X 104/ml) were treated with permutations of recom binant human TNF-a (100 ng/ml; Genzyme Diagnostics. Cambridge. MA), anti-human CD95 antibody (1.0 /j.g/ml; United Biomedicai, Inc., Lake Placid. NY), and PHA (10 nM), as described. Cells were assessed for apoptosis 6 and 24 h later by Hoechst staining of nuclei and survival by trypan blue dye exclusion. The cell viability was measured by scoring at least 200 cells in each group, and the average percentage of viability was calculated from three different experiments. 882 Research. on September 11, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from