Multiple pathways are involved in the anoxia response of SKIP3 including HuR-regulated RNA stability, NF-κB and ATF4

Under anoxia a coordinated, cytoprotective program is induced, called the unfolded protein response (UPR). Activating transcription factor 4 (ATF4) is a mediator of the UPR and activates a gene expression program, promoting tumour growth and survival under anoxia. A key gene induced by ATF4 under normoxic conditions is SKIP3. We characterized the induction of SKIP3 during anoxic exposure to determine whether UPR alone was sufficient or there was a more complex regulatory response to anoxia. There was temporal separation of acute hypoxia-inducible factor (HIF)-1 alpha- and chronic ATF4-dependent gene expression programs. SKIP3 was regulated by chronic (48 h) rather than acute anoxia (< 24h) by a complex set of pathways and mechanisms, besides ATF4 induced by the classical UPR, there was transcriptional regulation by nuclear factor-kappa B (NF-kappa B)and RNA stabilization by HuR. Temporal activation of the NF-kappa B pathway under anoxia protected cells from negative consequences of the oxygen stress and involved the canonical signalling pathways that promote I kappa BA phosphorylation and degradation, and reduced mRNA level of the inhibitory protein I kappa BA followed by the translational repression of I kappa BA. We also show that SKIP3 acts as an inhibitor of NF-kappa B and ATF4-dependent transcription under anoxia and provides a regulatory feedback loop. Repression of the survival pathway NF-kappa B by SKIP3 sensitized cells to metabolic consequences of the anoxic stress. Thus, the response to anoxia is mediated by three pathways independently of HIF, suggesting that combined therapeutic approaches would be needed to maximize effects against this pathway.