Daniel J. Peet

Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the Nuclear Oxysterol Receptor LXRα

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 LXRs: a new class of oxysterol receptors

The liver X receptors (LXRs) are a family of transcription factors that were first identified as orphan members of the nuclear receptor superfamily. The identification of a specific class of oxidized derivatives of cholesterol as ligands for the LXRs has been crucial to helping understand the function of these receptors in vivo and first suggested their role in the regulation of lipid metabolism. Confirmation of this role has come from the recent analysis of LXR-deficient mice, which has demonstrated the essential function of one of these receptors in the liver as a major sensor of dietary cholesterol.

The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses

AbstractOxygen 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.

Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways

Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1α (HIF-1α) becomes stabilized and directly activates transcription of downstream genes. In addition to this “canonical” response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1α can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N1945 and N2012) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo. FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1α to its target promoters and derepresses HIF-1α function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1α, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1α. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.

Turn me on: regulating HIF transcriptional activity.

The hypoxia-inducible factors (HIFs) are critical for cellular adaptation to limiting oxygen and regulate a wide array of genes when cued by cellular oxygen-sensing mechanisms. HIF is able to direct transcription from either of two transactivation domains, each of which is regulated by distinct mechanisms. The oxygen-dependent asparaginyl hydroxylase factor-inhibiting HIF-1α (FIH-1) is a key regulator of the HIF C-terminal transactivation domain, and provides a direct link between oxygen sensation and HIF-mediated transcription. Additionally, there are phosphorylation and nitrosylation events reported to modulate HIF transcriptional activity, as well as numerous transcriptional coactivators and other interacting proteins that together provide cell and tissue specificity of HIF target gene regulation.

The asparaginyl hydroxylase factor inhibiting HIF-1α is an essential regulator of metabolism

Factor inhibiting HIF-1alpha (FIH) is an asparaginyl hydroxylase. Hydroxylation of HIF-alpha proteins by FIH blocks association of HIFs with the transcriptional coactivators 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, and 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, and 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.

Cell-specific Regulation of Hypoxia-inducible Factor (HIF)-1α and HIF-2α Stabilization and Transactivation in a Graded Oxygen Environment

The hypoxia-inducible factor (HIF)-1α and HIF-2α are closely related, key transcriptional regulators of the hypoxic response, countering a low oxygen situation with the up-regulation of target genes associated with numerous processes, including vascularization and glycolysis. This involves a dual mechanism of control through both stabilization and transactivation, regulated via prolyl and asparaginyl hydroxylation. Despite high similarity with respect to protein sequence and activation pathway, a growing number of physiological and mechanistic differences between HIF-1α and HIF-2α are being reported. To further characterize this nonredundancy, the stabilization of endogenous proteins and regulation of the transactivation domains were compared in a graded oxygen environment across a series of cell lines. Although generally similar results were found, interesting and specific differences between the HIF-α proteins were observed within certain cell lines, such as rat adrenal PC12s, emphasizing the cell-specific nature of HIF-α regulation. We characterize a conserved amino acid substitution between HIF-1α and HIF-2α that contributes to the intrinsically higher FIH-1-mediated asparaginyl hydroxylation of HIF-1α and, hence, lower HIF-1α activity. In addition, our data demonstrate that the different cell lines can be classified into two distinct groups: those in which stabilization and transactivation proceed in conjunction (HeLa, 293T, and COS-1) and those cells in which HIF-α is stabilized prior to transactivation (PC12, HepG2, and CACO2). Interestingly, the initial stabilization of HIF-α prior to transactivation up-regulation predicted from in vitro derived hydroxylation data is only true for a subset of cells.

The emerging role of hypoxia, HIF-1 and HIF-2 in multiple myeloma

Hypoxia is an imbalance between oxygen supply and demand, which deprives cells or tissues of sufficient oxygen. It is well-established that hypoxia triggers adaptive responses, which contribute to short- and long-term pathologies such as inflammation, cardiovascular disease and cancer. Induced by both microenvironmental hypoxia and genetic mutations, the elevated expression of the hypoxia-inducible transcription factor-1 (HIF-1) and HIF-2 is a key feature of many human cancers and has been shown to promote cellular processes, which facilitate tumor progression. In this review, we discuss the emerging role of hypoxia and the HIFs in the pathogenesis of multiple myeloma (MM), an incurable hematological malignancy of BM PCs, which reside within the hypoxic BM microenvironment. The need for current and future therapeutic interventions to target HIF-1 and HIF-2 in myeloma will also be discussed.

Rare variants in single-minded 1 (SIM1) are associated with severe obesity

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.

bHLH-PAS proteins in cancer

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.