Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the α-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1α is unstable in cells maintained at 21% O2, but is progressively stabilized as the O2 concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 µM) stabilize HIF-1α at all O2 concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (ρ0-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1α stabilized by exposure of the cells to 3% O2. This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1α by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance.
Regulation of mitochondrial respiration and apoptosis through cell signaling: Cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation
