Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl 2 ) in Hep3B cells. However, neither the mechanism of cellular O 2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O 2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O 2 ) or CoCl 2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA ( 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) 0 cells increase ROS generation in response to CoCl 2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl 2 in wild-type cells, and abolish the response to CoCl 2 in °cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl 2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

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-1␣ stabilization during hypoxia.Hypoxia initiates transcription of a number of gene products that help to sustain the supply of O 2 to tissues and to enhance cell survival during severe O 2 deprivation. Gene products that augment O 2 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 (1-3). At the cellular level, gene products that enhance survival during hypoxia include the glycolytic enzymes and the glucose transporters Glut1 and Glut3 (4). The induction of these genes is mediated by hypoxia-inducible factor-1 (HIF-1) (5-7), a heterodimeric transcription factor consisting of HIF-1␣ and the aryl hydrocarbon nuclear translocator (ARNT or HIF-1␤) subunits (7-9). 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-1␣-or ARNT-deficient murine embryonic stem cells (10 -12).The mechanism by which HIF-1 activation is initiated during hypoxia remains unclear. Both HIF-1␣ and ARNT mRNAs are constitutively expressed, indicating that functional activity of the HIF-1␣⅐ARNT complex is regulated by post-transcriptional events. ARNT levels are not significantly affected by [O 2 ], whereas HIF-1␣ protein is rapidly degraded under normoxic conditions by the ubiquitin-proteasome system (13,14). Hypoxia enhances HIF-1␣ 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-1␣⅐ARNT complex is primarily regulated by the abundance of the HIF-1␣ 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 [O 2 ] and initiate the stabilization of HIF-1␣ is not known. Presently, four diverse O 2 -sensing mechanisms have been proposed to mediate the transcriptional response to hypoxia (15). 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-1␣ under normoxic conditions (16). 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 f...

Multicellular organisms initiate adaptive responses when oxygen (O(2)) availability decreases, but the underlying mechanism of O(2) sensing remains elusive. We find that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1 alpha and HIF-2 alpha and that an increase in reactive oxygen species (ROS) links this complex to HIF-alpha stabilization. Using RNAi to suppress expression of the Rieske iron-sulfur protein of complex III, hypoxia-induced HIF-1 alpha stabilization is attenuated, and ROS production, measured using a novel ROS-sensitive FRET probe, is decreased. These results demonstrate that mitochondria function as O(2) sensors and signal hypoxic HIF-1 alpha and HIF-2 alpha stabilization by releasing ROS to the cytosol.

SUMMARY It is widely appreciated that T cells increase glycolytic flux during activation, however the role of mitochondrial flux is unclear. Here we have shown that mitochondrial metabolism, in the absence of glucose metabolism, was sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs−/− mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, however Uqcrfs1−/− T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1−/− T cells were not lacking bioenergetically, but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through production of complex III ROS.

Mitochondrial physiology is disrupted in either apoptosis or necrosis. Here, we report that a wide variety of apoptotic and necrotic stimuli induce progressive mitochondrial swelling and outer mitochondrial membrane rupture. Discontinuity of the outer mitochondrial membrane results in cytochrome c redistribution from the intermembrane space to the cytosol followed by subsequent inner mitochondrial membrane depolarization. The mitochondrial membrane protein Bcl-xL can inhibit these changes in cells treated with apoptotic stimuli. In addition, Bcl-xL-expressing cells adapt to growth factor withdrawal or staurosporine treatment by maintaining a decreased mitochondrial membrane potential. Bcl-xL expression also prevents mitochondrial swelling in response to agents that inhibit oxidative phosphorylation. These data suggest that Bcl-xL promotes cell survival by regulating the electrical and osmotic homeostasis of mitochondria.

Mitochondria cooperate with their host cells by contributing to bioenergetics, metabolism, biosynthesis, and cell death or survival functions. Reactive oxygen species (ROS) generated by mitochondria participate in stress signalling in normal cells but also contribute to the initiation of nuclear or mitochondrial DNA mutations that promote neoplastic transformation. In cancer cells, mitochondrial ROS amplify the tumorigenic phenotype and accelerate the accumulation of additional mutations that lead to metastatic behaviour. As mitochondria carry out important functions in normal cells, disabling their function is not a feasible therapy for cancer. However, ROS signalling contributes to proliferation and survival in many cancers, so the targeted disruption of mitochondria-to-cell redox communication represents a promising avenue for future therapy.

Reactive oxygen species and tumor biology are intertwined in a complex web, making it difficult to understand which came first, whether oxidants are required for tumor cell growth, and whether oxidant stress can be exploited therapeutically. Evidence suggests that transformed cells use ROS signals to drive proliferation and other events required for tumor progression. This confers a state of increased basal oxidative stress, making them vulnerable to chemotherapeutic agents that further augment ROS generation or that weaken antioxidant defenses of the cell. In this respect, it appears that tumor cells may die by the same systems they require.

Parkinson's disease (PD) is a pervasive, aging-related neurodegenerative disease whose cardinal motor symptoms reflect the loss of a small group of neurons – dopaminergic neurons in the substantia nigra pars compacta (SNc)1. Mitochondrial oxidant stress is widely viewed as responsible for this loss2, but why these particular neurons should be stressed is a mystery. Using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, it was discovered that the unusual engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. This stress engaged defenses that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, antagonists of cloned uncoupling proteins. Knocking out DJ-1, a gene associated with an early onset form of PD, down-regulated the expression of two uncoupling proteins (UCP4, 5), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of PD.