The role of the mitochondria in cellular oxygen sensing

The ability of a cell to detect and respond to conditions of low O 2 is essential for normal survival and is critical during periods of acute ischemia. When responding to hypoxia, cells must initiate transcriptional and adaptive responses to help combat the imposed stress. While much is known about the cellular responses to hypoxia, the actual mechanism of cellular O2 sensing is still unknown and remains controversial. One potential candidate is the mitochondria, which has evolved for the efficient use and detection of O2. I have been able to show that reactive oxygen species originating from the mitochondria (mtROS) are responsible for a number of cellular responses to hypoxia including inhibition of prolyl hydroxylase activity and subsequent HIF-alpha stabilization and inhibition of glutathione synthesis. However, while the use of mitochondrial inhibitors and respiration deficient rho0 cells have provided valuable evidence in support of a mitochondrial role in cellular oxygen sensing, they have also produced conflicting results. To this end, I also employed a stable, genetic model of mitochondrial dysfunction to investigate the role of mitochondria in cellular oxygen sensing. Cytochrome c, an essential mitochondrial protein, is nuclear-encoded and a targeted null mutation has been made. I generated cytochrome c null embryonic cells and verified that in addition to being unable to respire, these cells are impaired in their ability to appropriately sense hypoxia as evidenced by their inability to produce mtROS or stabilize HIF-alpha in response to 1.5% O2. Stable reintroduction of cytochrome c expression restores not only mitochondrial function, but also the cell's ability to stabilize HIF-lalpha in response to 1.5% O2. By using cells engineered to stably express a HIF hydroxylation reporter, I also explored the relationship between the mitochondria and the newly identified prolyl hydroxylases, showing that during hypoxia, mtROS are responsible for inhibiting prolyl hydroxylase activity, ultimately leading to HIF stabilization. Collectively, these results provide evidence that the mitochondria play a critical role in the cellular oxygen-sensing pathway.