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).

Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes.

Most antineoplastic chemotherapies eliminate cancer cells through activation of the mitochondria-controlled intrinsic apoptotic pathway. Therein, BAX, BAK, and/or BOK function as the essential pore-forming executioners of mitochondrial outer membrane permeabilization (MOMP). The activation threshold of BAX and BAK also correlates inversely with the required strength of an apoptotic stimulus to induce MOMP and thereby effectively determines a cell’s readiness to undergo apoptosis. Consequently, the ‘gatekeepers’ BAX and BAK emerged as therapeutic targets, but functional or genetic loss renders BAX/BAK-targeting strategies prone to fail. Here, we show that the small molecule Raptinal overcomes this limitation by triggering cytochrome c release in a BAX/BAK/BOK-independent manner. Raptinal exerts a dual cytotoxic effect on cancer cells by rapid activation of the intrinsic apoptotic pathway and simultaneous shutdown of mitochondrial function. Together with its efficacy to eliminate cancer cells in vivo, Raptinal could be useful in difficult-to-treat cancer entities harboring defects in the intrinsic apoptosis pathway.