The interaction of superoxide with nitric oxide destabilizes hypoxia-inducible factor-1alpha.

In renal carcinoma cells (RCC4) hypoxia inducible factor-1 (HIF-1) is constitutively expressed due to a von Hippel Lindau protein deficiency, but can be degraded by calpain, independently of the 26S proteasome, when exposed to hypoxia/nitric oxide (NO). In this study we examined molecular mechanisms to explain calpain activation. The inability of hypoxia/NO to degrade HIF-1alpha in respiratory-deficient RCC4-rho0 cells pointed to the requirement for mitochondria-derived reactive oxygen species. A prerequisite for O(2)(-) in combination with NO to destabilize HIF-1alpha was corroborated in RCC4-rho0 cells, when the redox cycler 2,3-dimethoxy-1,4-naphthoquinone was used as a source of superoxide. Degradation of HIF-1alpha required intracellular calcium transients and calpain activation. Using uric acid to interfere with signal transmission elicited by NO/O(2)(-) blocked HIF-1alpha degradation and attenuated a calcium increase. We conclude that an oxidative signal as a result of NO/O(2)(-) coformation triggers a calcium increase that activates calpain to degrade HIF-1alpha, independently of the proteasome.