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. 2007 Jun 18;177(6):1029-36.
doi: 10.1083/jcb.200609074. Epub 2007 Jun 11.

The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production

Eric L Bell  1 , Tatyana A Klimova, James Eisenbart, Carlos T Moraes, Michael P Murphy, G R Scott Budinger, Navdeep S Chandel
Affiliations

The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production

Eric L Bell et al. J Cell Biol. .

Abstract

Mammalian cells increase transcription of genes for adaptation to hypoxia through the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein. How cells transduce hypoxic signals to stabilize the HIF-1alpha protein remains unresolved. We demonstrate that cells deficient in the complex III subunit cytochrome b, which are respiratory incompetent, increase ROS levels and stabilize the HIF-1alpha protein during hypoxia. RNA interference of the complex III subunit Rieske iron sulfur protein in the cytochrome b-null cells and treatment of wild-type cells with stigmatellin abolished reactive oxygen species (ROS) generation at the Qo site of complex III. These interventions maintained hydroxylation of HIF-1alpha protein and prevented stabilization of HIF-1alpha protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1alpha protein and prevented stabilization of HIF-1alpha protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1alpha protein and stabilized HIF-1alpha protein. These results provide genetic and pharmacologic evidence that the Qo site of complex III is required for the transduction of hypoxic signal by releasing ROS to stabilize the HIF-1alpha protein.

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Figures

Figure 1.
Figure 1.
Cells that contain a cytochrome b–deficient bc1 complex are respiratory incompetent but still generate ROS and stabilize HIF-1α in hypoxia. (A) Mitochondrial complex III generates ROS through the ubiquinone (Q) cycle. The Q cycle generates ROS at the Qo and Qi site. The Rieske Fe-S protein is required for ROS production at the Qo site. The loss of complex III subunit cytochrome b abolishes the Qi site. (B) Oxygen consumption of 143B cells, 143Bρ0 cells, WT cybrids, and ΔCyt b cybrids with and without 1 μM of the mitochondrial electron transport inhibitor stigmatellin. (C) HIF-1α protein levels in WT and ΔCyt b cybrids subjected to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from four independent experiments. (D) Intracellular H2O2 levels were measured by Amplex red in 143B cells, 143Bρ0 cells, WT cybrids, and ΔCyt b cybrids exposed to 1.5 or 21% O2 for 4 h. n = 4 (mean ± SEM); *, P < 0.05 (all groups were compared with the normoxia sample of WT cells).
Figure 2.
Figure 2.
Mitochondrial-targeted antioxidant MitoQ prevents hypoxic stabilization of HIF-1α protein. (A) Intracellular H2O2 levels were measured in ΔCyt b cybrids using Amplex red in the presence of 1 μM MitoQ or 1 μM of the control compound TPMP for 4 h. n = 4 (mean ± SEM); *, P < 0.05 (TPMP hypoxia samples compared with MitoQ hypoxia samples). (B and C) HIF-1α protein levels of whole cell lysates from ΔCyt b cybrids (B) and WT cybrids (C) preincubated with 1 μM MitoQ or 1 μM of the control compound TPMP for 4 h and then subjected to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from four independent experiments.
Figure 3.
Figure 3.
Cytosolic antioxidant EUK-143 prevents hypoxic stabilization of HIF-1α protein. HIF-1α protein levels of whole cell lysates from WT cybrids (A) and ΔCyt b cybrids (B) preincubated with 10 μM EUK-134 for 2 h and then subjected to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from three independent experiments.
Figure 4.
Figure 4.
RNAi of TFAM diminishes hypoxic stabilization of HIF-1α protein. Quantitative real-time PCR of cDNA (A) or total DNA (B) generated from ΔCyt b cybrids stably expressing shRNA for D. melanogaster HIF (dHIF) or TFAM. n = 3 (mean ± SEM); *, P < 0.05 (dHIF shRNA compared with TFMA shRNA). (C) HIF-1α protein levels from whole cell lysates of ΔCyt b cybrids stably expressing shRNA for either D. melanogaster HIF (dHIF) or TFAM, exposed to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from three independent experiments.
Figure 5.
Figure 5.
Electron transfer at the Qo site of the bc1 complex is necessary for hypoxic generation of ROS and stabilization of HIF-1α protein. (A) Immunoblot analysis for Rieske Fe-S protein of whole cell lysates from ΔCyt b cybrids stably expressing shRNA for D. melanogaster HIF (dHIF), TFAM, or Rieske Fe-S protein (FE-S) at 21% O2. (B) HIF-1α protein levels from whole cell lysates of ΔCyt b cybrids stably expressing shRNA against either D. melanogaster HIF (dHIF) or Rieske Fe-S protein (FE-S) exposed to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from four independent experiments. (C) Intracellular H2O2 levels measured with Amplex red in ΔCyt b cybrids stably expressing shRNA for D. melanogaster HIF, TFAM, or Rieske Fe-S protein exposed to either 21 or 1.5% O2 for 4 h. n = 4 (mean ± SEM); *, P < 0.05 (hypoxic dHIF shRNA compared with hypoxic TFMA shRNA and hypoxic Rieske Fe-S shRNA).
Figure 6.
Figure 6.
Hypoxic HIF-1α protein stability is attenuated by pharmacological inhibitors of the Qo but not Qi site in WT cells. (A) HIF-1α protein levels of whole cell lysates from WT cybrids incubated with 1 μM stigmatellin and subjected to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from three independent experiments. (B) HIF-1α protein levels of whole cell lysates from WT cybrids incubated with 1 μM antimycin A and subjected to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from three independent experiments.
Figure 7.
Figure 7.
Hypoxic increase in cytosolic ROS generated from the Qo site of complex III inhibits hydroxylation of HIF-1α protein. Immunoblot analysis of whole cell lysates for hydroxylated HIF-1α protein from WT cybrids (A) and ΔCyt b cybrids (B) treated with 20 μM MG132 to stabilize HIF-1α protein preincubated with 1 μM MitoQ or 1 μM control compound TPMP for 4 h and then subjected to 21% O2 (N), 1.5% O2 (H), or 1 mM DMOG (D) for 4 h. Representative blot from three independent experiments.
Figure 8.
Figure 8.
ROS are sufficient to prevent hydroxylation of HIF-1α protein. (A) Intracellular ROS measured by DCFH in WT cybrids subjected to 21% O2 (N), 1.5% O2 (H), 21% O2 plus glucose oxidase (10 μg/ml), or 21% O2 plus glucose oxidase and catalase for 2 h. n = 4 (mean ± SEM); *, P < 0.05 (normoxia compared with hypoxia or normoxia + glucose oxidase compared with normoxia + glucose oxidase + catalase). (B) Immunoblot analysis of whole cell lysates for HIF-1α protein from WT cybrids subjected to 21% O2 (N), 1.5% O2 (H), 21% O2 plus glucose oxidase, or 21% O2 plus glucose oxidase and catalase for 2 h. Representative blot from three independent experiments. (C) Immunoblot analysis for hydroxylated HIF-1α protein from whole cell lysates of WT cybrids treated with 20 μM MG132 subjected to 21% O2 (N), 1.5% O2 (H), 21% O2 plus glucose oxidase, or 21% O2 plus glucose oxidase and catalase for 2 h. Representative blot from three independent experiments.
Figure 9.
Figure 9.
Reduced cytochrome c is not sufficient to stabilize the HIF-1α protein. (A) HIF-1α protein levels of 143B and A549 cells subjected to 21% O2 (N), 1.5% O2 (H), and 1 mM DMOG (D) for 2 h. Representative blot from three independent experiments. (B) HIF-1α protein levels of 143Bρ0 and A549ρ0 whole cell lysates <21% O2 (N) or 1.5% O2 (H) for 2 h after a 15-min pulse of 100 μM of the cytochrome c–reducing agent TMPD and 400 μM ascorbate (T) or 1 mM DMOG (D). Representative blot from three independent experiments. (C) Cytochrome c and p66Shc protein levels in 143Bρ0 and A549ρ0 whole cell lysates. Representative blot from three independent experiments.
Figure 10.
Figure 10.
Schematic model of mitochondrial-generated ROS stabilization of HIF-1α in hypoxic conditions. Hypoxia increases generation of ROS from the Qo site of the bc1 complex. These ROS are released into the intermembrane space and enter the cytosol to decrease PHD activity, thus preventing hydroxylation of the HIF-1α protein. We speculate that ROS decrease the PHD activity from a combination of a posttranslational modification of the PHDs, such as phosphorylation or decreasing the availability of Fe (II), which is required for hydroxylation to occur.
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