IntroductionConsiderable progress has been made in understanding mechanisms that underlie the regulation of mammalian gene expression by oxygen. A heterodimeric DNA binding complex, hypoxia-inducible factor (HIF), consisting of 2 basic-helix-loop-helix PAS proteins, termed HIF-␣ and HIF-␤, plays a critical role in a widespread transcriptional response induced by hypoxia. [1][2][3] First discovered in the context of erythropoietin regulation, 4 it is now recognized that HIF is central to the regulation of genes involved in a diverse range of functions in mammalian physiology, including angiogenesis, glucose and energy metabolism, iron metabolism, vasomotor regulation, and regulation of apoptosis and cell proliferation (for review, see 5,6 ). The regulation of HIF itself occurs mainly through modifications of its ␣ subunit, whereas the ␤ subunit is a constitutive nuclear protein. The HIF-␣ subunit is rapidly degraded in normoxia by a ubiquitinproteasome pathway, involving the von Hippel-Lindau tumor suppressor protein. [7][8][9][10][11] In hypoxia this pathway is inhibited and HIF-␣ is stabilized. Nuclear translocation, coactivator recruitment, dimerization, and DNA binding follow in a multistep process that results in transactivation of target genes 12,13 (for review, see 14 ). A substantial body of evidence has implicated partially reduced reactive oxygen species (ROS) in the sensing and transduction mechanism, 7,15-19 but knowledge of the source, precise species, and mode of interaction of such molecules with the transcriptional system are limited, and the experimental findings are conflicting.The mitochondrion is the major oxygen-consuming organelle and, at least in some circumstances, is a major producer of oxygen radical species. 20,21 As such it might be expected to play a central role in oxygen-sensitive processes, and evidence has suggested such a role in HIF regulation. 18,19 Chandel et al 18,19 found impairment of HIF activation by hypoxia in cells treated with pharmacologic inhibitors of complex I of the electron chain and in cells lacking mitochondrial DNA. They proposed that complex III of the mitochondrial respiratory chain acts as an oxygen sensor and is a source of increased ROS in hypoxia that have a stabilizing effect on HIF-1␣. In this model, complex I inhibitors were proposed to act by blocking the electron flow proximally, so as to ablate such a signal. Mitochondrial function has also been implicated in other oxygen-sensing systems. For instance, Baysal et al 22 recently demonstrated that germline mutations in the gene that encodes the small subunit of cytochrome b (cybS) in complex II predisposes to hereditary paraganglioma of the carotid body. Chronic hypoxia predisposes to similar tumors, leading those researchers to suggest that the genetic defect might mimic the normal hypoxic signal and that cybS is a critical component of the oxygen-sensing system of paraganglionic tissue. However, no specific model was proposed, and despite implicating the mitochondrion, the findings are not easy to r...

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Hypoxia-inducible factor (HIF) mediates a widespread transcriptional response to hypoxia through binding to cis-acting DNA sequences termed hypoxia response elements (HREs). Activity of the transcriptional complex is suppressed in the presence of oxygen by processes that include the targeting of HIF-␣ subunits for ubiquitin-mediated proteolysis. To provide further insights into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antigens and used these cells in genetic screens for mutants that demonstrated constitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increased HIF-1␣ protein levels in normoxic culture. Transfection and cell fusion studies suggested that these cells possess recessive defects that affect one or more pathways involved in HIF-␣ proteolysis. Two lines were demonstrated to harbor truncating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1␣ in vitro could be complemented by wild type pVHL, and re-expression of a wild type VHL gene restored a normal pattern of HIF/ HRE activity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIF-1␣ normally, suggesting that they contain defects at other points in the oxygen-regulated processing of HIF-␣ subunits. Hypoxia-inducible factor (HIF)1 is a DNA binding complex that plays a central role in oxygen homeostasis (reviewed in Refs. 1-3). Transcriptional targets include genes that function in angiogenesis, matrix metabolism, erythropoiesis, glucose metabolism, vasomotor control, and the regulation of cell proliferation/apoptosis (1-3). The HIF complex binds DNA sequences within hypoxia response elements (HREs) as a heterodimer of two basic-helix-loop-helix PAS proteins, termed alpha and beta subunits (4). HIF-␣ subunits exist as at least two isoforms, HIF-1␣ and HIF-2␣, each of which can form heterodimers with HIF-␤. Although HIF-␤ subunits are constitutive nuclear proteins, both HIF-␣ subunits are strongly induced by hypoxia in a similar manner. Studies of HIF-␣ induction have defined a number of distinct activation mechanisms, including inhibition of oxygen-dependent proteolysis, co-activator recruitment, and nuclear localization (5-10).In oxygen-replete cells HIF-␣ subunits are targeted for ubiquitin-mediated proteasomal destruction. Insights into the mechanisms regulating oxygen-dependent proteolysis have been provided by studies of the von Hippel-Lindau tumor suppressor protein (pVHL). In the presence of oxygen, pVHL binds and targets HIF-␣ subunits to the ubiquitin-proteasome pathway (11). This process involves interaction between conserved subsequences within the oxygen-dependent degradation domains of HIF-␣ subunits and the ␤-domain of pVHL, with pVHL acting as the re...