Eoin P. Cummins

Hypoxia is a feature of the microenvironment of a growing tumor. The transcription factor NFκB is activated in hypoxia, an event that has significant implications for tumor progression. Here, we demonstrate that hypoxia activates NFκB through a pathway involving activation of IκB kinase-β (IKKβ) leading to phosphorylation-dependent degradation of IκBα and liberation of NFκB. Furthermore, through increasing the pool and/or activation potential of IKKβ, hypoxia amplifies cellular sensitivity to stimulation with TNFα. Within its activation loop, IKKβ contains an evolutionarily conserved LxxLAP consensus motif for hydroxylation by prolyl hydroxylases (PHDs). Mimicking hypoxia by treatment of cells with siRNA against PHD-1 or PHD-2 or the pan-prolyl hydroxylase inhibitor DMOG results in NFκB activation. Conversely, overexpression of PHD-1 decreases cytokine-stimulated NFκB reporter activity, further suggesting a repressive role for PHD-1 in controlling the activity of NFκB. Hypoxia increases both the expression and activity of IKKβ, and site-directed mutagenesis of the proline residue (P191A) of the putative IKKβ hydroxylation site results in a loss of hypoxic inducibility. Thus, we hypothesize that hypoxia releases repression of NFκB activity through decreased PHD-dependent hydroxylation of IKKβ, an event that may contribute to tumor development and progression through amplification of tumorigenic signaling pathways.

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1α (but not HIF-2α) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxia-inducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1α mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1α in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1α mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1α stabilization and activity. Based on these data and a mathematical model of HIF-1α suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1α activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1α-dependent transcription.