Induction of HIF-2alpha is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line.
During low O2 (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 O2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2alpha expression and oxygen consumption in an O2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2-8% O2) caused a concentration- and time-dependent increase in HIF-2alpha 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 (rho0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 microM), myxothiazol (1 microM), antimycin A (1 microg/ml), and cyanide (1 mM), blocked HIF-2alpha 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% O2), HIF-2alpha induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2alpha induction. These data suggest that in this O2-sensitive cell line, mitochondrial O2 consumption, rather than changes in ROS, regulates HIF-2alpha during hypoxia.
Brown
Stephen T
ST
Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1 Canada. brownst@mcmaster.ca
Nurse
Colin A
CA
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D015687:Cell Hypoxia; D002460:Cell Line; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000378:metabolism; D014450:Electron Transport Complex III / Q000235:genetics / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D034622:RNA Interference; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors; D014475:Uncoupling Agents / Q000494:pharmacology; D015854:Up-Regulation; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D004272:DNA, Mitochondrial; D014450:Electron Transport Complex III; D003576:Electron Transport Complex IV; D007506:Iron-Sulfur Proteins; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D000099162:Endothelial PAS Domain-Containing Protein 1; C037989:Rieske iron-sulfur protein