Hypoxia-inducible factor induction by tumour necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor κB activation

Tumour necrosis factor alpha (TNF-alpha) binds to its receptor (TNFR1) and activates both death- and inflammation/survival-related signalling pathways. The inflammation and survival-related signalling cascade results in the activation of the transcription factor, nuclear factor kappaB (NF-kappaB) and requires recruitment of receptor-interacting protein (RIP) to TNFR I. The indispensable role of RIP in TNF-induced NF-kappaB activation has been demonstrated in RIP-/- mice and in cell lines derived from such mice. In the present study, we show that the TNF-alpha-induced accumulation of hypoxia-inducible factor 1alpha (HIF-1alpha) protein in normoxic cells is RIP-dependent. Exposing fibroblasts derived from RIP-/- mice to either cobalt or PMA resulted in an equivalent HIF-1alpha induction to that seen in RIP+/+ fibroblasts. In contrast, RIP-/- cells were unable to induce HIF-1alpha in response to TNF-alpha. Further, transient transfection of NIH 3T3 cells with an NF-kappaB super-repressor plasmid (an inhibitor of NF-kappaB activation) also prevented HIF-1alpha induction by TNF-alpha. Surprisingly, although HIF-1alpha mRNA levels remained unchanged after induction by TNF, induction of HIF-1alpha protein by the cytokine was completely blocked by pretreatment with the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside. Finally, TNF failed to induce both HIF1alpha, made resistant to von Hippel-Lindau (VHL), and wild-type HIF-1alpha transfected into VHL-/- cells. These results indicate that HIF-1alpha induction by TNF-alpha in normoxic cells is mediated by protein stabilization but is nonetheless uniquely dependent on NF-kappaB-driven transcription. Thus the results describe a novel mechanism of HIF-1alpha up-regulation and they identify HIF-1alpha as a unique component of the NF-kappaB-mediated inflammatory/ survival response.