Author(s): Enrico De Smaele; Francesca Zazzeroni; Salvatore Papa; Dung U. Nguyen; Rongguan Jin; Joy Jones; Rong Cong; Guido Franzoso (corresponding author)
In addition to coordinating immune and inflammatory responses, NF-[kappa]B/Rel transcription factors control cell survival . Normally, NF-[kappa]B dimers are sequestered in the cytoplasm by binding to inhibitory I[kappa]B proteins, and can be activated rapidly by signals that induce the sequential phosphorylation and proteolysis of I[kappa]Bs . Activation of NF-[kappa]B antagonizes apoptosis or programmed cell death by numerous triggers, including the ligand engagement of 'death receptors' such as tumour-necrosis factor (TNF) receptor . The anti-apoptotic activity of NF-[kappa]B is also crucial to oncogenesis and to chemo- and radio-resistance in cancer . Cytoprotection by NF-[kappa]B involves the activation of pro-survival genes ; however, its basis remains poorly understood. Here we report that NF-[kappa]B complexes downregulate the c-Jun amino-terminal kinase (JNK) cascade , thus establishing a link between the NF-[kappa]B and the JNK pathways. This link involves the transcriptional upregulation of gadd45 [beta]/myd118 (ref. 4), which downregulates JNK signalling induced by the TNF receptor (TNF-R). This NF-[kappa]B-dependent inhibition of the JNK pathway is central to the control of cell death. Our findings define a protective mechanism that is mediated by NF-[kappa]B complexes and establish a role for the persistent activation of JNK in the apoptotic response to TNF-[alpha].
To understand mechanisms controlling TNF-R-induced programmed cell death (PCD)- and NF-[kappa]B-dependent survival, we used the method of 'death trap' screening  in NF-[kappa]B null cells. Complementary DNA expression libraries derived from TNF-[alpha]-treated wild-type cells were transfected into NF-[kappa]B/RelA-/- fibroblasts . Apoptosis was induced with TNF-[alpha], and plasmids were recovered from surviving cells. After four cycles of selection, about 35% of the library cDNAs protected RelA null cells from killing by TNF-[alpha]. Known inhibitors of TNF-R-triggered apoptosis, including RelA, cFLIP (cellular FLICE inhibitory protein) and dominant-negative forms of FADD  (Fas-associated death domain protein), were highly enriched during selection.
A cDNA enriched by selection with TNF-[alpha] was found to encode full-length Gadd45[beta], a member of the Gadd45 family of inducible factors  associated with cell-cycle control and DNA repair . gadd45 [beta] was strongly and rapidly induced by TNF-[alpha] in wild-type mouse embryo fibroblasts (MEFs), but not in RelA-/- cells (Fig. 1a). This mirrored the expression of ikb[alpha] , a target of NF-[kappa]B . gadd45 [beta] was also upregulated by TNF-[alpha] in parental and Neo 3DO T cells, but not in 3DO clones expressing I[kappa]B[alpha]M, a variant of I[kappa]B[alpha] that blocks activation of NF-[kappa]B  (Fig. 1b; see also Supplementary Information Fig. 1). gadd45 [beta] was also induced by NF-[kappa]B after treatment with daunorubicin or phorbol 12-myristate-13-acetate (PMA) plus ionomycin (Fig. 1d and c, respectively).
In both MEFs and 3DO cells, expression of the other family members gadd45[alpha] /gadd45 and gadd45[gamma] /oig37/cr6/grp17 (ref. 11) was independent of NF-[kappa]B (Fig. 1b-d; and data not shown). Thus, unlike these latter genes, gadd45 [beta] is a TNF-[alpha]-inducible gene and a physiological target of NF-[kappa]B. Mutational analyses confirmed the presence of three functional [kappa]B elements...