1-1-diethyl-2-hydroxy-2-nitrosohydrazine and cobaltous-chloride

The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation.

Topics: Animals; Benzoates; Cell Hypoxia; Cell Line; Cobalt; Coculture Techniques; Deferoxamine; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Glutathione; Guanylate Cyclase; Hydrazines; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Inflammation; Kidney Tubules, Proximal; Macrophage Activation; Macrophages; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Oxides; Nitroso Compounds; Nuclear Proteins; Oxadiazoles; Oxazines; S-Nitrosoglutathione; Spermine; Swine; Transcription Factors