During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1α protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (ρ0 cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H2O2 stabilizes HIF-1α protein during normoxia and activates luciferase expression in wild-type and ρ0 cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1α stabilization during hypoxia. During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1α protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (ρ0 cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H2O2 stabilizes HIF-1α protein during normoxia and activates luciferase expression in wild-type and ρ0 cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1α stabilization during hypoxia. erythropoietin reactive oxygen species hypoxia-inducible factor-1 hypoxic response element vascular endothelial growth factor desferrioxamine diphenylene iodonium 2′,7′-dichlorofluorescein DCFH-DA, 2′,7′-dichlorofluorescein diacetate pyrrolidine dithiocarbamate 4,4′-diisothiocyanatostilbene-2,2′-disulfonate phosphatidylinositol 3-kinase Hypoxia initiates transcription of a number of gene products that help to sustain the supply of O2 to tissues and to enhance cell survival during severe O2 deprivation. Gene products that augment O2 supply at the tissue level include erythropoietin (Epo)1 which increases the proliferation of erythrocytes, tyrosine hydroxylase which is necessary for the synthesis of the neurotransmitter dopamine in the carotid bodies, and the angiogenic factor VEGF which stimulates growth of new capillaries (1Semenza G.L. Wang G.L. Mol. 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The significance of HIF-1 in transcriptional regulation was recently demonstrated by the marked decrease in mRNA expression of VEGF and glycolytic enzymes seen during hypoxia in HIF-1α- or ARNT-deficient murine embryonic stem cells (10Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M., Yu, A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2008) Google Scholar, 11Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Crossref PubMed Scopus (1318) Google Scholar, 12Maltepe E. Schmidt J.V. Baunoch D. Bradfield C.A. Simon M.C. Nature. 1997; 386: 403-407Crossref PubMed Scopus (625) Google Scholar). The mechanism by which HIF-1 activation is initiated during hypoxia remains unclear. Both HIF-1α and ARNT mRNAs are constitutively expressed, indicating that functional activity of the HIF-1α·ARNT complex is regulated by post-transcriptional events. ARNT levels are not significantly affected by [O2], whereas HIF-1α protein is rapidly degraded under normoxic conditions by the ubiquitin-proteasome system (13Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1381) Google Scholar, 14Kallio P.J. Pongratz I. Gradin K. McGuire J. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5667-5672Crossref PubMed Scopus (333) Google Scholar). Hypoxia enhances HIF-1α protein levels by inhibiting its degradation, thereby allowing it to accumulate, to dimerize with ARNT, and to bind to the hypoxia-responsive element (HRE) in the promoter or enhancer regions of various genes. Thus, the functional HIF-1α·ARNT complex is primarily regulated by the abundance of the HIF-1α subunit. Although much has been learned about the role of HIF-1 in controlling the expression of hypoxia-responsive genes, the underlying mechanism by which cells detect the decrease in [O2] and initiate the stabilization of HIF-1α is not known. Presently, four diverse O2-sensing mechanisms have been proposed to mediate the transcriptional response to hypoxia (15Semenza G.L. Cell. 1999; 98: 281-284Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Two of these models postulate the involvement of an iron-containing unit in the form of either a heme group or an iron/sulfur cluster, which undergoes a change in activity during hypoxia that triggers the transcriptional response. These models are supported by the observation that cobaltous ions, or alternatively the iron chelator desferrioxamine (DFO), stabilize HIF-1α under normoxic conditions (16Wang G.L. Semenza G.L. Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar). However, no specific proteins with this role have been identified in mammalian systems. Two other models involve the generation of reactive oxygen species (ROS) by a flavoprotein-containing NAD(P)H oxidase or by mitochondria. The NAD(P)H oxidase theory postulates that a decrease in ROS production triggers the transcriptional response to hypoxia (17Fandrey J. Frede S. Jelkman W. Biochem. J. 1994; 303: 507-510Crossref PubMed Scopus (223) Google Scholar, 18Ehleben W. Bolling B. Merten E. Porwol T. Strohmaier A.R. Acker H. Respir. Physiol. 1998; 114: 25-36Crossref PubMed Scopus (46) Google Scholar). In support of that model, exogenous H2O2 was found to inhibit subsequent hypoxic stabilization of HIF-1α (19Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar). However, diphenylene iodonium (DPI), a specific flavoprotein inhibitor that blocks ROS generation by NAD(P)H oxidase, abolishes the hypoxic induction of HIF-1-dependent genes (20Gleadle J.M. Ebert B.L. Ratcliffe P.J. Eur. J. Biochem. 1995; 234: 92-99Crossref PubMed Scopus (87) Google Scholar), whereas the model would predict that DPI should activate the response during normoxia. We previously proposed that hypoxia partially inhibits mitochondrial electron transport, producing redox changes in the electron carriers that increase the generation of ROS. These oxidants then enter the cytosol and function as second messengers in the signaling pathway leading to stabilization of HIF-1α (21Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11715-11720Crossref PubMed Scopus (1559) Google Scholar). In support of this model, hypoxia failed to increase ROS production or the expression of EPO, VEGF, and glycolytic enzymes in ρ0 cells, which lack mitochondrial DNA and electron transport activity. Also, the response to hypoxia was abolished by the DPI, which abrogates mitochondrial ROS generation by inhibiting electron transport at the flavin site in mitochondrial Complex I (22Majander A. Finel M. Wikstroem M. J. Biol. Chem. 1994; 269: 21037-21042Abstract Full Text PDF PubMed Google Scholar). ROS are required for the DNA of HIF-1 and the subsequent mRNA expression of VEGF, and glycolytic enzymes during hypoxia. However, protein levels of HIF-1α not that not reveal mitochondrial ROS required to stabilization of HIF-1α during hypoxia. ROS found to necessary for the transcriptional response to hypoxia, it was not ROS by sufficient to initiate HIF-1α the the mitochondrial ROS are required for HIF-1α stabilization during hypoxia, or ROS are sufficient to HIF-1α and mitochondrial as the O2 responsible for the redox changes underlying the increase in ROS generation during hypoxia. cells and the cells in and to as previously P.T. J. Physiol. 1993; Google and in with and The and cells by wild-type cells in and PubMed Scopus Google Scholar). conditions as previously (21Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11715-11720Crossref PubMed Scopus (1559) Google Scholar). 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The cells failed to HIF-1α protein during hypoxia and HIF-1α protein in cells expression was not in cells, these cells demonstrated expression in response to or The increase in expression in cells was abolished in cells to catalase the role of mitochondria in the hypoxic wild-type cells to hypoxia, or in the of DPI or DPI, and abolished the accumulation of HIF-1α during hypoxia not during or ROS generation at Complex A. B. Biochem. J. PubMed Scopus Google Scholar, A. Biochem. PubMed Scopus Google Scholar), which should the response to hypoxia and to stabilization of failed to stabilize HIF-1α during normoxia and not the induction of HIF-1α during hypoxia, or We previously that the ROS response to during normoxia was the response to hypoxia in cells (21Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. 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Hypoxia and both an increase in that of the increases to the stabilization of The abolished the stabilization of HIF-1α and of in response to hypoxia or the involvement of ROS. cells catalase an expression response to hypoxia or the for H2O2 in a second cell of H2O2 HIF-1α stabilization and expression of under normoxic conditions in both cell these findings support the that ROS are both required and sufficient to activate the signaling system in the stabilization of findings are with increases in ROS generation in Johnson J. Physiol. 1999; PubMed Scopus Google and cells B. B. B. A. J. A. H. W. W. 1999; PubMed Scopus Google Scholar). exogenous of H2O2 not detect stabilization of HIF-1 under normoxia. In H2O2 abolished gene transcription during subsequent hypoxia O2 for (19Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar). H2O2 that a of is rapidly degraded by cells in that of required to sustain cells to H2O2 for by accumulation of whereas cells H2O2 for the expression of These that levels of an are sufficient to stabilize The iron chelator HIF-1α stabilization and HIF-1-dependent gene it to at a site in the signaling of HIF-1α by not to involve as during normoxia. The response to was not abolished by either or both of which the of The flavin inhibitor DPI and the Complex I inhibitor failed to HIF-1α stabilization during and ρ0 cells to to However, and both abolished the HIF-1α stabilization during These findings that does not mitochondrial electron transport or ROS and to at a site the the ubiquitin-proteasome is the of ROS that stabilization of HIF-1α during Complex during normoxia A. B. Biochem. J. PubMed Scopus Google Scholar, A. Biochem. PubMed Scopus Google reveal that hypoxia increases the generation of ROS at Complex to an sufficient to stabilize HIF-1α In support of ρ0 cells failed to stabilize to and failed to increase of during hypoxia. However, to to and to indicating that the to cells to exogenous H2O2 by expression of these that an electron transport is required for hypoxic stabilization of HIF-1α and that ROS during hypoxia are sufficient to this The Complex I inhibitor ROS generation by the supply of Complex This abolished the increase in during hypoxia and the increase in and both inhibit electron transport and the abolished the increases in and HIF-1α protein during hypoxia. inhibiting electron transport at the of Complex III, to detect HIF-1α stabilization that during normoxia. a increase in with hypoxia (21Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. 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PubMed Scopus Google Scholar). that by abolished the during hypoxia or with during normoxia. the of mitochondrial should the that are required for HIF-1α stabilization in hypoxia have no the response to or to these not mitochondria. the that HIF-1α accumulation was abolished by during hypoxia in cells and during in cells during normoxia. However, not the response to or to These support the that an to the cytosol is required for the stabilization of of HIF-1α protein by or does not a functional mitochondrial electron transport as both to stabilize HIF-1α protein and expression of in ρ0 cells. However, the of and the expression was in ρ0 cells of HIF-1α by was not by DPI, that stimulates ROS generation by a The observation that does not ROS or mitochondria to as a signaling hypoxia, and stabilization of and growth have been to stabilize HIF-1α protein and HIF-1-dependent gene expression during normoxia E. Levy M. B. EMBO J. 1998; 17: PubMed Scopus Google Scholar). 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, to at with that oxidase in required under hypoxia to a decrease N.S. Schumacker P.T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), these cells HIF-1α protein accumulation in the the the of hypoxia required to a change in and the required to stabilize it is that oxidase as the O2 in hypoxia. it that Complex to [O2], allowing it to its generation of ROS with the O2 observation that ROS generation at [O2] in cells with (21Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11715-11720Crossref PubMed Scopus (1559) Google is with this electron transport by would Complex III, its redox to the redox of In the generation by Complex under hypoxia was in mitochondria. to Complex III, ROS generation during hypoxia the in cells. ROS generation to decrease during hypoxia to to Complex the of this response for Complex We that Complex as the during hypoxia by ROS generation with In a it is to that mitochondria are in cells and a mechanism of O2 in other observation of an by a bacterium that to this by that ROS generation by Complex is not to E. of at O2 indicating that not an under hypoxia. The to increase ROS generation by E. to its lack of a allowing electron to the of a Biochem. Sci. 12: Full Text PDF Scopus Google Scholar). This the that electron a to as the of in cells that a to the that mitochondria function as a and ROS as signaling in other systems. We M. for the in this