We demonstrate that mice lacking the oxysterol receptor, LXR alpha, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXR alpha (-/-) mice fail to induce transcription of the gene encoding cholesterol 7alpha-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXR alpha (-/-) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXR alpha as the major sensor of dietary cholesterol.

The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators.Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O 2 to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway. Received March 14, 2002; revised version accepted April 30, 2002. Almost all mammalian cells possess the ability to recognize changes in the local availability of oxygen. When oxygen levels are low (hypoxia), a conserved hypoxic response pathway is activated. At the center of this pathway lies the ubiquitously expressed transcription factor hypoxia-inducible factor (HIF) (Semenza 1999). HIF is a heterodimer composed of an alpha subunit, HIF-1␣ or the paralogs HIF-2␣ or HIF-3␣ (Tian et al. 1997;Gu et al. 1998;O'Rourke et al. 1999;Srinivas et al. 1999), and the HIF-1␤ subunit, also known as the aryl hydrocarbon receptor nuclear translocator (ARNT) (Wang et al. 1995). Whereas HIF-1␤ expression and activity levels remain largely unaffected by changes in oxygen levels, the HIF-␣ subunit is strongly induced following exposure to hypoxic conditions. Two primary mechanisms by which HIF-␣ activity is regulated by oxygen have been identified. Under normoxic conditions, the oxygen-dependent degradation domain (ODD) within the HIF-␣ subunit is recognized by the product of the von-Hippel Lindau tumor suppressor gene (pVHL) (Maxwell et al. 1999). pVHL is a component of a protein-ubiquitin ligase complex that targets the alpha subunit for degradation by the proteasome (Maxwell et al. 1999;Cockman et al. 2000;Ohh et al. 2000;Tanimoto et al. 2000). pVHL recognition of HIF-␣ is dependent on hydroxylation of proline residues within the ODD (Ivan et al. 2001;Jaakkola et al. 2001;Yu et al. 2001). Under hypoxic conditions, prolyl hydroxylation is blocked, resulting in increased HIF-␣ stability and accumulation (Ivan et al. 2001;Jaakkola et al. 2001;Yu et al. 2001). This posttranslational modification is carried out by a family of prolyl hydroxylase enzymes that bear structural and functional similarities to previously characterized hydroxylases (Bruick and McKnight 2001;Epstein et al. 2001). Like these enzymes, the HIF prolyl hydroxylase enzymes use Fe(II) to bind O 2 to hydroxylate both 2-oxoglutarate and the target proline residue (Bruick and McKnight 2001;Epstein et al. 2001). Because these enzymes bind oxygen directly, it has been speculated that they may be critical oxygen sensors within the hypoxic response pathway.In addition to inducing HIF stability, hypoxic conditions promote the ...

gene regulation ͉ hydroxylation ͉ signal transduction R educed oxygen levels (hypoxia) lead to a set of cellular adaptations, including increased angiogenesis and erythropoiesis and a switch to glycolytic metabolism. The cellular machinery that senses hypoxia is composed of several proteins. A critical component is the transcription factor hypoxiainducible factor 1␣ (HIF-1␣) (1). The level and activity of HIF-1␣ are controlled by oxygen-dependent prolyl (PHD) and asparaginyl factor-inhibiting HIF-1␣ (FIH-1)] hydroxylases. PHDs hydroxylate two proline residues in the degradation domain of HIF-1␣ in normoxia, which makes HIF-1␣ a substrate for the von Hippel-Lindau E3 ubiquitin ligase and proteasomal degradation. After stabilization in hypoxia, HIF-1␣ interacts with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to hypoxia response elements (HREs)

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SUMMARY Factor Inhibiting HIF-1α (FIH) is an asparaginal hydroxylase. Hydroxylation of HIF-α proteins by FIH blocks association of HIFs with the transcriptional co-activators CBP/p300, thus inhibiting transcriptional activation. We have created mice with a null mutation in the FIH gene, and found that it has little or no discernable role in mice in altering classical aspects of HIF function, e.g., angiogenesis, erythropoiesis, or development. Rather, it is an essential regulator of metabolism: mice lacking FIH exhibit reduced body weight, elevated metabolic rate, hyperventilation, improved glucose and lipid homeostasis, and are resistant to high fat diet-induced weight gain and hepatic steatosis. Neuron-specific loss of FIH phenocopied some of the major metabolic phenotypes of the global null animals: those mice have reduced body weight, increased metabolic rate, enhanced insulin sensitivity, and are also protected against high fat diet-induced weight gain. These results demonstrate that FIH acts to a significant degree through the nervous system to regulate metabolism.

Mammalian basic HLH (helix-loop-helix)-PER-ARNT-SIM (bHLH-PAS) proteins are heterodimeric transcription factors that sense and respond to environmental signals (such as pollutants) or to physiological signals (for example, hypoxia and circadian rhythms) through their two PAS domains. PAS domains form a generic three-dimensional fold, which commonly contains an internal cavity capable of small-molecule binding and outer surfaces adept at protein-protein interactions. These proteins are important in several pro-tumour and antitumour pathways and their activities can be modulated by both natural metabolites and oncometabolites. Recently determined structures and successful small-molecule screening programmes are now providing new opportunities to discover selective agonists and antagonists directed against this multitasking family of transcription factors.

Oxygen depravation in mammals leads to the transcriptional induction of a host of target genes to metabolically adapt to this deficiency, including erythropoietin and vascular endothelial growth factor. This response is primarily mediated by the hypoxia-inducible factors (HIFs) which are members of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) transcription factor family. The HIFs are primarily regulated via a two-step mechanism of HIF post-translational modification, increasing both protein stability and transactivation capacity. This review aims to summarise our current understanding of these processes, and discuss the important role of the HIFs in the pathophysiology of many human diseases.