articles NATURE CELL BIOLOGY VOL 3 APRIL 2001 http://cellbio.nature.com 409 Regulation of death receptor expression and TRAIL/Apo2L- induced apoptosis by NF- κB Rajani Ravi*, Gauri. C. Bedi† , Laura W. Engstrom*, Qinwen Zeng*, Bijoyesh Mookerjee*, Céline Gélinas‡, Ephraim J. Fuchs* and Atul Bedi*§ *Johns Hopkins Oncology Center, The Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, Maryland 21231, USA †Department of Surgery, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, Maryland 21287, USA ‡Center for Advanced Biotechnology and Medicine, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 679 Hoes Lane, Piscataway, New Jersey 08854-5638, USA §e-mail: rbedi@jhmi.edu TRAIL (tumour-necrosis factor-related apoptosis ligand or Apo2L) triggers apoptosis through engagement of the death receptors TRAIL-R1 (also known as DR4) and TRAIL-R2 (DR5). Here we show that the c-Rel subunit of the tran- scription factor NF-κB induces expression of TRAIL-R1 and TRAIL-R2; conversely, a transdominant mutant of the inhibitory protein IκBα or a transactivation-deficient mutant of c-Rel reduces expression of either death receptor. Whereas NF-κB promotes death receptor expression, cytokine-mediated activation of the RelA subunit of NF-κB also increases expression of the apoptosis inhibitor, Bcl-x L , and protects cells from TRAIL. Inhibition of NF-κB by blocking activation of the IκB kinase complex reduces Bcl-x L expression and sensitizes tumour cells to TRAIL-induced apopto- sis. The ability to induce death receptors or Bcl-x L may explain the dual roles of NF-κB as a mediator or inhibitor of cell death during immune and stress responses. A poptosis has an essential role in embryogenesis, adult tissue homeostasis and the cellular response to stressful stimuli, such as DNA damage, hypoxia or aberrations in cell-cycle progression 1 . Increased apoptosis is involved in the pathogenesis of diverse ischaemic, degenerative and immune disorders 2 . Conversely, genetic aberrations that render cells incapable of exe- cuting their suicide program promote tumorigenesis and underlie the observed resistance of human cancers to genotoxic anticancer agents 3 . Unravelling mechanisms to unleash the apoptotic program in tumour cells might aid the design of effective therapeutic inter- ventions against resistant human cancers. The molecular machinery of cell death comprises an evolution- arily conserved family of cysteine aspartate proteases (caspases) 4 . Caspases can be activated by the engagement of death receptors belonging to the tumour-necrosis factor (TNF) receptor gene superfamily 5 , such as TNFR1, CD95 (Fas), TRAIL-R1 (DR4) 6 and TRAIL-R2 (DR5, TRICK2, KILLER) 7–13 , by their respective cognate ‘death ligands’, TNF-α, CD95L (Apo1L) and TRAIL (also known as Apo2L) 14,15 . TRAIL induces apoptosis in several tumour cell lines, including those that resist chemotherapeutic agents or ionizing radiation because of inactivating mutations of the p53 tumour suppressor gene 16–20 . TRAIL-R1 and TRAIL-R2 are type I transmembrane proteins con- taining cytoplasmic sequences, termed ‘death domains’, that recruit adaptor proteins and activate caspases 16 . Two other TRAIL receptors, TRAIL-R3 (TRID/DcR1) and TRAIL-R4 (TRUNDD/DcR2), have extracellular domains similar to TRAIL-R1 and TRAIL-R2, but lack a functional cytoplasmic death domain 7,8,21–24 . TRAIL-R3 and TRAIL- R4 may serve as ‘decoys’ that compete with TRAIL-R1/TRAIL-R2 for binding to TRAIL, and overexpression of either protein confers pro- tection against TRAIL-induced death 7,8 . The NF-κB family of dimeric transcription factors is important in modulating cell survival during stress and immune responses 25 . NF-κB protects cells from apoptosis 26–31 by promoting expression of survival factors, such as members of the inhibitor of apoptosis (IAP) family (c-IAP1, c-IAP2, XIAP) 32 and the Bcl-2 homologues, Bfl-1/A1 (refs 33, 34) and Bcl-x L (ref. 35). In contrast, much evi- dence highlights an apparently paradoxical pro-apoptotic role for NF-κB 36–39 . These observations raise the possibility that κB sites in pro- or anti-apoptotic genes may exhibit different preferences for particular subunits comprising the NF-κB dimer, and that NF-κB may have signal-specific effects on cell survival. Here we show that the RelA and c-Rel subunits of NF-κB are critical determinants of the expression of death receptors and sur- vival genes that modulate TRAIL-induced apoptosis. The signal- specific activation of dimers that induce expression of either death receptors or survival genes might explain how NF-κB adopts either of its dual personalities as a mediator or inhibitor of cell death dur- ing immune and cellular stress responses. The identification of NF- κB as a key determinant of cellular susceptibility to TRAIL may have important implications for anticancer therapy. Results Subunit-specific effects of NF-κB on death receptor expression and on sensitivity to TRAIL. NF-κB exists in almost all cell types in an inactive cytoplasmic complex with an inhibitory protein, IκB. Signal-dependent phosphorylation and ubiquitin-mediated degra- dation of IκB by IκB kinases (IKKs) releases the active complex, which functions in transcriptional regulation of target genes after nuclear translocation 25 . Trimerization of TNFR1 by TNF-α leads to degradation of IκB and activation of NF-κB. Mouse embryonic fibroblasts (MEFs) stably transduced with a retrovirus carrying a combined amino- (residues 32 and 36) and carboxy-terminal PEST sequence phosphorylation mutant of IκBα (IκBαM) 28 show reduced basal and TNF-α-inducible κB DNA-binding activity and lower expression of TRAIL-R2 messenger RNA compared with wild-type MEFs carrying a control vector (Fig. 1a, b). The subunits of NF-κB are known to exhibit different prefer- ences for variations of the 10-base-pair (bp) consensus sequence © 2001 Macmillan Magazines Ltd