Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I
© Hagen et al; licensee BioMed Central Ltd. 2011
Received: 28 December 2010
Accepted: 12 April 2011
Published: 12 April 2011
The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein.
We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux.
This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.
Compound C has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α (HIF-1α) stabilization by interacting with the mitochondrial electron transport chain (ETC) and suppressing mitochondria generated reactive oxygen species . This finding coincides with the hypothesis that increased reactive oxygen species (ROS) released from mitochondrial complex III during hypoxia stabilize HIF-1α [2–4]. However our recent findings showed that the mitochondrial electron transport chain controls the stability of HIF-1α during hypoxia independently of reactive oxygen species production . We therefore studied the mechanism through which Compound C interacts with the ETC in detail.
Effect of Compound C on hypoxia-induced HIF-1α protein stabilization
Effect of Compound C on mitochondrial respiration
To study the effects of Compound C on the mitochondrial ETC, we measured the oxygen consumption in 0.5 mg of mouse liver mitochondria with a Clarke-type oxygen electrode. Mitochondria were subjected to one freeze-thaw cycle before the measurement and NADH (2.5 mM) was used as the respiratory substrate for complex I. Treatment of mitochondria with 80 μM Compound C inhibited respiration by 74.5%. Oxygen consumption resumed when the respiratory substrate for complex II, succinate (5 mM) was added, as illustrated in Figure 1B. These results suggest that Compound C is an inhibitor of complex I and does not inhibit downstream complexes. To determine if Compound C interacts with complex II, III or IV, Compound C was added to mitochondria respiring on succinate. Compound C did not inhibit succinate dependent oxygen consumption (Figure 1C) and therefore does not interact with complex II, III or IV. To directly test if Compound C is an inhibitor of complex IV, 0.2 mM 2,2,4-trimethyl-1,3-pentanediol (TMPD)/1 mM ascorbate was added. TMPD is an artificial electron donor that transfers electrons from ascorbate to complex IV via cytochrome c. Compound C had no effect on the oxygen consumption when TMPD/ascorbate was used as the respiratory substrate (Figure 1D). These results imply that Compound C inhibits mitochondrial respiration through its interaction with complex I.
Effect of Compound C on ROS production from isolated mouse liver mitochondria
To measure the cellular H2O2 and superoxide production in mammalian cells, fluorescence of the 2',7'-dichlorofluorescein (DCFDA) and dihydroethidium (DHE) dye, respectively, were monitored by flow cytometry. No significant difference in both the H2O2 and superoxide concentration was observed in cells treated with Compound C (at concentrations up to 100 μM) compared with untreated cells (data not shown). Taken together, the results suggest that Compound C mediated inhibition of hypoxia dependent HIF-1α stabilization is not due to inhibition of complex III derived ROS as previously suggested  but likely due to an effect on mitochondrial oxygen consumption.
The aim of this study was to determine the mechanism through which Compound C inhibits the stabilization of the HIF-1 α protein in hypoxia. We provide evidence that Compound C acts as a complex I inhibitor that affects the stabilization of the HIF-1 α protein independently of ROS production. Our results suggest that Compound C, like other mitochondrial ETC inhibitors, regulates HIF-1 α stability by controlling the cellular oxygen concentration leading to increased cellular oxygen availability and reactivation of prolyl hydroxylases in hypoxia [5, 7].
The study was supported by grant, R-183-001-216-112, from the Academic Research Fund.
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