Abstract
Tumor necrosis factor (TNF) receptor associated factor-2 (TRAF2) knockout (KO) cells were generated to investigate the role of TRAF2 in signaling by TNFR1 and the CD95-type death receptors (DRs) TRAILR1/2 and CD95. To prevent negative selection effects arising from the increased cell death sensitivity of TRAF2-deficient cells, cell lines were used for the generation of the TRAF2 KO variants that were protected from DR-induced apoptosis downstream of caspase-8 activation. As already described in the literature, TRAF2 KO cells displayed enhanced constitutive alternative NFκB signaling and reduced TNFR1-induced activation of the classical NFκB pathway. There was furthermore a significant but only partial reduction in CD95-type DR-induced upregulation of the proinflammatory NFκB-regulated cytokine interleukin-8 (IL8), which could be reversed by reexpression of TRAF2. In contrast, expression of the TRAF2-related TRAF1 protein failed to functionally restore TRAF2 deficiency. TRAF2 deficiency resulted furthermore in enhanced procaspase-8 processing by DRs, but this surprisingly came along with a reduction in net caspase-8 activity. In sum, our data argue for (i) a non-obligate promoting function of TRAF2 in proinflammatory DR signaling and (ii) a yet unrecognized stabilizing effect of TRAF2 on caspase-8 activity.
Highlights
The death domain (DD)-containing receptors (DRs) of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) can be subdivided into three groups according to how DD-containing signaling proteins and caspase-8 are used to induce apoptosis [1]
While Fas-associated death domain (FADD) is essentially required for CD95-type DR group (CD95)-type death receptor (DR)-induced necroptosis, TNFR1-induced necroptosis is even enhanced in the absence of FADD [4, 5]
We failed in apoptosis-competent cell lines to obtain TRAF2 knockout (KO) cells with an otherwise well working CRISPR/Cas9 protocol pointing to negative selection of TRAF2 KO cells
Summary
The death domain (DD)-containing receptors (DRs) of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) can be subdivided into three groups according to how DD-containing signaling proteins and caspase-8 are used to induce apoptosis [1]. The receptors of the CD95-type DR group (CD95) (Apo, Fas), TNF-related apoptosis inducing ligand (TRAIL) receptor-1 (TRAILR1, DR4), and TRAILR2 (DR5) recruit procaspase-8 directly in the receptor signaling complex by means of the DD-containing adapter protein Fas-associated death domain (FADD). The receptors of the third group of DRs (p75NGFR, EDAR, DR6) induce cell death under poorly defined circumstances by heterogeneous mechanisms without obvious involvement of FADD and caspase-8. The CD95- and TNFR1-type DRs. TRAF2 in DR Signaling and DR6 have the ability to trigger necroptotic cell death via the receptor-interacting protein serine/threonine kinase-1 (RIPK1)–RIPK3 mixed lineage kinase domain-like (MLKL) pathway [2, 3]. The importance of FADD for DR6-induced necroptosis has not been investigated, yet
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