The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events. Analyzing cell lines expressing an internalization-deficient receptor (TNF-R1 DeltaTRID) revealed that recruitment of RIP-1 and TRAF-2 to TNF-R1 occurred at the level of the plasma membrane. In contrast, aggregation of TRADD, FADD, and caspase-8 to establish the TNF-R1-associated DISC is critically dependent on receptor endocytosis. Furthermore, fusion of TNF receptosomes with trans-Golgi vesicles results in activation of acid sphingomyelinase and cathepsin D. Thus, TNF receptosomes establish the different TNF signaling pathways by compartmentalization of plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.

Soluble TNF-like weak inducer of apoptosis (TWEAK) trimers induce, in a variety of cell lines, translocation of cytosolic tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) to a triton X-100-insoluble compartment without changes in the total cellular TRAF2 content. TWEAK-induced TRAF2 translocation is paralleled by a strong increase in nuclear factor kappaB 2 (NFkappaB2)/p100 processing to p52, indicating that TRAF2 redistribution is sufficient for activation of the alternative NFkappaB pathway. In accordance with the crucial role of TRAF2 in proinflammatory, anti-apoptotic TNF receptor-1 (TNFR1) signaling, we observed that TWEAK-primed cells have a reduced capacity to activate the classical NFkappaB pathway or JNK (cJun N-terminal kinase) in response to TNF. Furthermore, TWEAK-primed cells are sensitized for the TNFR1-mediated induction of apoptotic and necrotic cell death. Notably, the expression of the NFkappaB-regulated, TRAF2-interacting TRAF1 protein can attenuate TWEAK-induced depletion of the triton X-100-soluble TRAF2 fraction and improve TNFR1-induced NFkappaB signaling in TWEAK-primed cells. Taken together, we demonstrate that soluble TWEAK desensitizes cells for proinflammatory TNFR1 signaling and thus identify TWEAK as a modifier of TNF signaling.

Both hypoxic and inflammatory conditions activate transcription factors such as hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB, which play a crucial role in adaptive responses to these challenges. In dendritic cells (DC), lipopolysaccharide (LPS)-induced HIF1α accumulation requires NF-κB signaling and promotes inflammatory DC function. The mechanisms that drive LPS-induced HIF1α accumulation under normoxia are unclear. Here, we demonstrate that LPS inhibits prolyl hydroxylase domain enzyme (PHD) activity and thereby blocks HIF1α degradation. Of note, LPS-induced PHD inhibition was neither due to cosubstrate depletion (oxygen or α-ketoglutarate) nor due to increased levels of reactive oxygen species, fumarate, and succinate. Instead, LPS inhibited PHD activity through NF-κB-mediated induction of the iron storage protein ferritin and subsequent decrease of intracellular available iron, a critical cofactor of PHD. Thus, hypoxia and LPS both induce HIF1α accumulation via PHD inhibition but deploy distinct molecular mechanisms (lack of cosubstrate oxygen versus deprivation of co-factor iron).