THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.283,NO.16,pp.10892–10903,April18,2008
Received for publication, January 4, 2008, and in revised form, February 8, 2008 Published, JBC Papers in Press, February 15, 2008, DOI 10.1074/jbc.M800102200
Huafeng Zhang‡§, Marta Bosch-Marce‡§, Larissa A. Shimoda¶, Yee Sun Tan‡§, Jin Hyen Baek‡§, Jacob B. Wesley‡§, Frank J. Gonzalez , and Gregg L. Semenza‡§¶**‡‡§§1
From the ‡ Vascular Program, Institute for Cell Engineering, §McKusick-Nathans Institute of Genetic Medicine, and ¶ Department of Medicine, **Pediatrics, ‡‡Oncology,
and §§Radiation Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and Laboratory of Metabolism,
NCI, National Institutes of Health, Bethesda, Maryland 20892
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 (1). Although oxidative phosphorylation is more efficient than glycolysis in generating ATP, it carries the inherent risk of generating reactive oxygen species (ROS)2 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 (2, 3). HIF-1 is a heterodimer composed of a constitutively expressed HIF-1β, subunit and an O2-regulated HIF-1α subunit (4). 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 (5–8). Under hypoxic conditions the rate of hydroxylation declines, either as a result of inadequate substrate (O2) or as a result of hypoxia-induced mitochondrial ROS production, which may oxidize Fe (II) in the catalytic center of the hydroxylase (9, 10). Hydroxylated HIF-1α is bound by the von Hippel-Lindau protein, which recruits a ubiquitin protein ligase complex that targets HIF-1α for proteasomal degradation (11–14).
HIF-1 regulates the transcription of hundreds of genes in response to hypoxia (15, 16), including the EPO (17) and VEGF (18) genes that encode proteins required for erythropoiesis and angiogenesis, respectively, which serve to increase O2 delivery. In addition, HIF-1 controls a series of molecular mechanisms designed to maintain energy and redox homeostasis. First, HIF-1 coordinates a switch in the composition of cytochrome c oxidase (mitochondrial elec-tron-transport chain complex IV) from COX4-1 to COX4-2 subunit utilization, which increases the efficiency of cytochrome c oxidase under hypoxic conditions (19). 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 (20, 21). Third, HIF-1 activates transcription of genes encoding glucose transporters and glycolytic enzymes to increase flux from glucose to lactate (22–24). Fourth, HIF-1 represses mitochondrial biogenesis and respiration (25). Interference with the HIF-1-dependent regulation of mitochondrial respiration under conditions of prolonged hypoxia (≥24 h) leads to increased ROS levels and increased apoptosis (18, 20, 25).
Mitochondria are replaced every 2–4 weeks in rat brain, heart, liver, and kidney (26). The destruction of mitochondria is believed to occur via the process of autophagy, in which parts of the cytoplasm, including organelles, are equestered in double-membrane autophagic vacuoles or autophagosomes (27, 28). In addition to providing a mecha-nism for disposing of damaged mitochondria, autophagy is induced by environmental stress stimuli such as nutrient deprivation (29, 30). Autophagy is induced in hearts subjected to hypoxic or ischemic conditions and has been proposed by various investigators to play either a protective or pathogenic role in heart disease (30–33).
We hypothesized that induction of mitochondrial autophagy, in concert with inhibition of mitochondrial biogenesis (25), represents a critical adaptive mechanism to maintain oxygen homeostasis under hypoxic conditions. To test this hypothesis, we performed experiments to establish conditions under which hypoxia was a sufficient stimulus to induce mitochondrial autophagy, to determine whether this response was HIF-1-dependent, and to investigate whether mitochondrial autophagy was specifically required for the maintenance of redox homeostasis and the survival of hypoxic cells.