Induction of HIF-2α is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line

During low O-2 (hypoxia), hypoxia-inducible factor (HIF)-alpha is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O-2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2 alpha expression and oxygen consumption in an O-2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2-8% O-2) caused a concentration-and time-dependent increase in HIF-2 alpha induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA (rho 0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 mu M), myxothiazol (1 mu M), antimycin A (1 mu g/ml), and cyanide (1 mM), blocked HIF-2 alpha induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O2 concentration such that at moderate-to-severe hypoxia (6% O-2), HIF-2 alpha induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O-2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2 alpha induction. These data suggest that in this O-2-sensitive cell line, mitochondrial O-2 consumption, rather than changes in ROS, regulates HIF-2 alpha during hypoxia.