Mitochondrial reactive oxygen species trigger hypoxia-induced transcription

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl(2)) in Hep3B cells. However, neither the mechanism of cellular O(2) sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O(2) sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O(2)) or CoCl(2) incubation, (ii) Hep3B cells depleted of mitochondrial DNA (ρ(0) cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) ρ(0) cells increase ROS generation in response to CoCl(2) and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl(2) in wild-type cells, and abolish the response to CoCl(2) in ρ° cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl(2) activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.