Mitochondrial Autophagy Is an HIF-1-dependent Adaptive Metabolic Response to Hypoxia

Abstract

Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death. Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death. The survival of metazoan organisms is dependent upon their ability to efficiently generate energy through the process of mitochondrial oxidative phosphorylation in which reducing equivalents, derived from the oxidation of acetyl CoA in the tricarboxylic acid cycle, are transferred from NADH and FADH2 to the electron transport chain and ultimately to O2, a process which produces an electrochemical gradient that is used to synthesize ATP (1Lehninger A.L. Biochemistry. Worth Publishers, Inc., New York1975Google Scholar). Although oxidative phosphorylation is more efficient than glycolysis in generating ATP, it carries the inherent risk of generating reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; HIF-1, hypoxia-inducible factor-1; MEF, mouse embryo fibroblast; WT, wild type; KO, knockout; HET, heterozygous-null; SNC, scrambled negative control short hairpin RNA; siRNA, small interfering RNA; MnTMPyP, Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride; NAO, nonyl acridine orange; EV, empty vector; GFP, green fluorescent protein; 7-AAD, 7-aminoactinomycin D. as a result of electrons prematurely reacting with O2 at respiratory complex I or complex III. Transient, low level ROS production is utilized for signal transduction in metazoan cells, but prolonged elevations of ROS result in the oxidation of protein, lipid, and nucleic acid leading to cell dysfunction or death. O2 delivery and utilization must, therefore, be precisely regulated to maintain energy and redox homeostasis. Hypoxia-inducible factor 1 (HIF-1) plays a key role in the regulation of oxygen homeostasis (2Melillo G. Cell Cycle. 2004; 3: 154-155Crossref PubMed Google Scholar, 3Brahimi-Horn M.C. Pouyssegur J. FEBS Lett. 2007; 581: 3582-3591Crossref PubMed Scopus (296) Google Scholar). HIF-1 is a heterodimer composed of a constitutively expressed HIF-1β subunit and an O2-regulated HIF-1α subunit (4Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5105) Google Scholar). Under aerobic conditions, HIF-1α is hydroxylated on proline residue 402 and/or 564 by prolyl hydroxylase 2 a dioxygenase that utilizes O2 and α-ketoglutarate as co-substrates with ascorbate as co-factor in a reaction that generates succinate and CO2 as side products (5Dann III, C.E. Bruick R.K. Biochem. Biophys. Res. 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First, HIF-1 coordinates a switch in the composition of cytochrome c oxidase (mitochondrial electron-transport chain complex IV) from COX4-1 to COX4-2 subunit utilization, which increases the efficiency of cytochrome c oxidase under hypoxic conditions (19Fukuda R. Zhang H. Kim J.W. Shimoda L. Dang C.V. Semenza G.L. Cell. 2007; 129: 111-122Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar). Second, HIF-1 activates transcription of the PDK1 gene encoding a kinase that phosphorylates and inactivates pyruvate dehydrogenase, thereby shunting pyruvate away from the mitochondria by preventing its conversion to acetyl CoA (20Kim J.W. Tchernyshyov I. Semenza G.L. Dang C.V. Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar, 21Papandreou I. Cairns R.A. Fontana L. Lim A.L. Denko N.C. Cell Metab. 2006; 3: 187-197Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). 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G. A. F. P. E. G. K. J.A. G. J. 2007; PubMed Scopus Google Scholar, A. Y. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). of for and Beclin-1 that protein at levels in and cells of O2 of the with increased of BNIP3 with in cells under hypoxic as with conditions as on the increased levels of BNIP3 protein The increased of with BNIP3 by of with Beclin-1 in hypoxic cells In the of with Beclin-1 by O2 in cells, in which BNIP3 induced by of increased mitochondrial levels and autophagy in under hypoxic of cells to hypoxia in increased BNIP3 and Beclin-1 with in WT, but in KO, as by the that hypoxia-induced BNIP3 with Beclin-1 for to and thereby increases the levels of which The of autophagy of the in an that hypoxia-induced mitochondrial autophagy cell survival under hypoxic cell increased in as with In BNIP3 hypoxia-induced cell Beclin-1 or BNIP3 or increased hypoxia-induced cell in In to of prevent hypoxia-induced cell in of cells by that which more in than in to hypoxia-induced cell the role of ROS in hypoxia-induced cell with the which in the of ROS is to the fluorescent to the of to O2 for in a increase in ROS in to in which ROS levels in response to hypoxia expression of BNIP3 in ROS levels Although ROS levels in with prolonged hypoxia in increased ROS levels in in ROS levels under conditions of are with the in of adaptive to hypoxia that in cells, as subunit (19Fukuda R. Zhang H. Kim J.W. Shimoda L. Dang C.V. Semenza G.L. Cell. 2007; 129: 111-122Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar) and PDK1 expression (20Kim J.W. Tchernyshyov I. Semenza G.L. Dang C.V. Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar). of for BNIP3 Beclin-1 or with increased ROS as increased expression increased ROS levels to cell subjected to hypoxia in the or of the with on but ROS levels and cell in hypoxic that HIF-1 plays an role by an the of energy and redox homeostasis the of O2 (19Fukuda R. Zhang H. Kim J.W. Shimoda L. Dang C.V. Semenza G.L. Cell. 2007; 129: 111-122Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, J.W. Tchernyshyov I. Semenza G.L. Dang C.V. Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar, H. Gao P. Fukuda R. Kumar G. Krishnamachary B. Zeller K.I. Dang C.V. Semenza G.L. 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Commun. 2005; 338: 627-638Crossref PubMed Scopus (179) Google Scholar, 7Schofield C.J. Ratcliffe P.J. Biochem. Biophys. Res. Commun. 2005; 338: 617-626Crossref PubMed Scopus (284) Google Scholar, 10Guzy R.D. Hoyos B. Robin E. Chen H. Liu L. Mansfield K.D. Simon M.C. Hammerling U. Schumacker P.T. Cell Metab. 2005; 1: 401-408Abstract Full Text Full Text PDF PubMed Scopus (1170) Google Scholar). molecular mechanisms by which cells their energy to hypoxic subunit (19Fukuda R. Zhang H. Kim J.W. Shimoda L. Dang C.V. Semenza G.L. Cell. 2007; 129: 111-122Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar), of by of PDK1 (20Kim J.W. Tchernyshyov I. Semenza G.L. Dang C.V. Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar), and of mitochondrial biogenesis by of (25Zhang H. Gao P. Fukuda R. Kumar G. Krishnamachary B. Zeller K.I. Dang C.V. Semenza G.L. Cancer Cell. 2007; 11: Full Text Full Text PDF PubMed Scopus Google Scholar). In of to mitochondrial in response to hypoxia with increased ROS levels and increased cell death. The study that mitochondrial autophagy is a of the metabolic that is required to prevent increased ROS levels and cell in hypoxic that to O2 as O2 as The of the O2 from to O2 and ATP production by and or with of BNIP3 and Beclin-1 and or and In to we that O2 is in hypoxic of substrate but is under hypoxic conditions the utilization of O2 for respiration is as in KO, or to levels of ROS and cell death. mitochondrial autophagy through BNIP3 subunit PDK1 and a adaptive response that cells to prolonged hypoxia with M.C. G. A. F. P. E. G. K. J.A. G. J. 2007; PubMed Scopus Google Scholar, A. Y. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, M.C. A. E. J.M. J.A. G. 2007; 3: PubMed Scopus Google Scholar), that hypoxia autophagy of mitochondria but and that expression of BNIP3 plays an role in the of hypoxia-induced mitochondrial autophagy by the of Beclin-1 with Although Beclin-1 required for hypoxia-induced autophagy in for regulation of Beclin-1 by HIF-1, in to a that of HIF-1 in with Beclin-1 levels J. S. V. 2007; 3: PubMed Scopus Google Scholar). The that of of a key of the autophagy or the of hypoxic that is with HIF-1α or BNIP3 of for the role of autophagy in survival under conditions of prolonged be that of autophagy in at BNIP3 expressed in at O2, that protein may be of autophagy in expression of the protein for autophagy at O2 be the HIF-1 to the expression of Ratcliffe P.J. P. A.L. Cancer Res. 2001; Google Scholar, D.J. Rowan A. Lavoie T. Natarajan L. Kelly B.D. Ye S.Q. Garcia J.G. Semenza G.L. Blood. 2005; 105: 659-669Crossref PubMed Scopus (904) Google Scholar), Kim K. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar), and K. J. 2004; PubMed Scopus Google Scholar). of proteins in cells to hypoxia-induced cell death. are required to the role of proteins in this in the protein to of the K. Schumacker P. Mol. Cell. Biol. 2007; 27: PubMed Scopus Google Scholar). BNIP3 required for autophagy induced by and O2 or by with or of prolyl the role of HIF-1 in this process and autophagy as an in the process of cell death. In that autophagy is an adaptive with cell the of In the study we the that in mitochondrial are to the mechanisms and of hypoxia-induced of demonstrate that mitochondrial autophagy is an adaptive metabolic response that the survival of cells under conditions of prolonged hypoxia process requires the of BNIP3 and the autophagy as by Beclin-1 and and the of protein we demonstrate that HIF-1 regulates mitochondrial under conditions, as of HIF-1α a on BNIP3 expression and mitochondrial in the of to are with the of mitochondrial autophagy as an of the utilized by HIF-1 to maintain O2 homeostasis. with (19Fukuda R. Zhang H. Kim J.W. Shimoda L. Dang C.V. Semenza G.L. Cell. 2007; 129: 111-122Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, J.W. Tchernyshyov I. Semenza G.L. Dang C.V. Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2646) Google Scholar, H. Gao P. Fukuda R. Kumar G. Krishnamachary B. Zeller K.I. Dang C.V. Semenza G.L. Cancer Cell. 2007; 11: Full Text Full Text PDF PubMed Scopus Google Scholar), the that O2, and redox homeostasis are and that the of an their is to cell the that which of adaptive metabolic to hypoxia are utilized by cell in and are in preventing cell a for L. and R. for providing the and Dang for of the oxygen with