NF-κB Signal Triggering and Termination by Tumor Necrosis Factor Receptor 2

Abstract

Tumor necrosis factor receptor 2 (TNFR2) activates transcription factor B (NF-B) and c-Jun N-terminal kinase (JNK).The mechanisms mediating these activations are dependent on the recruitment of TNF receptor-associated factor 2 (TRAF2) to the intracellular region of the receptor. TNFR2 also induces TRAF2 degradation. We show that in addition to the well characterized TRAF2 binding motif 402-SKEE-405, the human receptor contains another sequence located at the C-terminal end (amino acids 425-439), which also recruits TRAF2 and activates NF-B. In addition to that, human TNFR2 contains a conserved region (amino acids 338 -379) which is responsible for TRAF2 degradation and therefore of terminating NF-B signaling. TRAF2 degradation and the lack of NF-B activation when both TNFR1 and TNFR2 are co-expressed results in an enhanced ability of TNFR1 to induce cell death, showing that the cross-talk between both receptors is of a great biological relevance. Induction of TRAF2 degradation appears to be independent of TRAF2 binding to the receptor. Amino acids 343-TGSSDSS-349 are essential for inducing TRAF2 degradation because deletion mutants of this region or point mutations at serine residues 345 and 346 or 348 and 349 obliterate the ability of TNFR2 to induce TRAF2 degradation. Tumor necrosis factor receptor 2 (TNFR2)5 is one of the two receptors known to bind TNF, this cytokine can be found as a transmembrane (mTNF) or a soluble (sTNF) form. Whereas both mTNF and sTNF activate TNFR1, TNFR2 is mainly activated by mTNF (1, 2), which upon binding to the receptor, causes its trimerization and activation. The fact that mTNF is the optimal activator of TNFR2 has implied serious limitations in the study of this receptor. Because the activation of TNFR2 depends on its aggregation, receptor activation can be forced by its overexpression or by the use of specific antibodies against the receptor (3, 4).TNFR2 lacks any intrinsic catalytic activity within its cytoplasmic tail, thus any signal emerging from the receptor depends on the recruitment of adaptor proteins. TNFR2 can bind directly TRAF2 and through this interaction signals for NF-B and JNK activation, as the expression of a dominant negative form of the adaptor protein (TRAF2dn) can suppress both the activation of NF-B and JNK (5).Seven different TRAF proteins have been identified so far (6). All of them share a highly conserved TRAF domain at the protein C terminus and, with the exception of TRAF1, a N-terminal-RING finger domain followed by five to seven zinc-finger motifs (7,8). TRAF proteins were initially considered as adaptor proteins between TNFRs and the kinases implicated in the activation of JNK or IB kinase IKK (9). It was then described that TRAF proteins, because of their RING finger domain, might act as E3 ubiquitin ligases, which catalyze K63-linked ubiquitination (10). In the case of TRAF2 this requires the interaction with cellular inhibitor of apoptosis 1 (cIAP1) and 2 (cIAP2) (11). More recently, it has been suggested that TRAF2 is by itself unable to ac...

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

“…28 As expected, NH 4 Cl further increased the protection conferred by starvation (Fig. 3C) and, though reducing the staining of cells with intact lysosomes, markedly diminished the frequency of pale cells independently of concomitant starvation, thus confirming that inhibiting lysosomal degradation significantly affects TNF and CHX toxicity (Fig.…”

“…Should this be the case, preventing intralysosomal degradation of autophagocytosed material would be expected to enhance starvation-associated protection. To test this possibility, starvation was performed in the presence of NH 4 Cl in order to Figure 1. Iron affects TNF and CHX toxicity in HTC cells.…”

“…To confirm that the effect of NH 4 Cl actually relies on impairment of lysosomal degradative activity, and not merely on their alkalinization, an E-64-d and leupeptin mixture was added simultaneously with starvation to selectively block acidic proteolysis. In keeping with NH 4 Cl, the cathepsin inhibitors potentiated starvation-induced protection from TNF and CHX (Fig. 3C), thus supporting the view that blocking lysosomal degradation of autophagocytosed materials increases protection from death achieved by starvation-induced autophagy.…”

“…The relative amount of SQSTM1 was expressed vs that of time 0 for each condition (histograms, right panel). (C) Cells starved as above for 2 h were exposed to TNF and CHX for 6 h in the absence or presence of 20 mM NH 4 Cl, added during the last 60 min of starvation, or of 100 mM E-64-d and 10 mM leupeptin for the whole period of starvation. The percentage of apoptotic-like cells (phosphatidylserine-positive and PI-negative) was then quantified by flow cytometry (5,000 cells were analyzed for each sample).…”

“…The TNF-binding capability of TNFRSF1B has been demonstrated since more than 15 y ago, 3 but the full biological role of this receptor remains elusive. It is believed to propagate prosurvival 4 as well as cytotoxic 5 signals, the latter in synergism with TNFRSF1A, which clearly transduces both forms of signals. TNF toxicity was initially accounted for by induction of caspasedependent apoptotic death, 6 but later alternative non-necrotic, caspase-independent apoptotic-like death mechanisms have been reported with increasing frequency.…”