The ability of essentially all cells in the body to sense and respond to low oxygen(hypoxia) is crucial for survival, but also involved in most major human diseases.
The human body is able to sense and respond to changes in oxygen levels, most importantly hypoxia, to maintain oxygen homeostasis. These changes may be environmental, such as high altitude, or part of the normal developmental process. However, hypoxia is also an important factor in many major human diseases, such as heart attack and stroke, where blood flow is disrupted and consequently oxygen delivery is compromised. Here an urgent response to this localised hypoxia is crucial for minimising damage. In contrast, in cancer this process is exploited with hypoxic regions deep inside tumours stimulating the growth of new blood vessels and promoting tumour growth and metastasis. The Peet Laboratory is interested in characterising the molecular mechanisms by which cells are able to sense and respond to hypoxia in normal physiology and disease.
One crucial response to hypoxia involves regulating numerous genes to increase oxygen delivery and metabolically adapt to reduced oxygen availability. For example, genes such as erythropoietin (Epo) increase red blood cell production, vascular endothelial growth factor (VEGF) stimulates vascular development, and other genes increase glucose transport and glycolysis to produce energy during decreased oxidative phosphorylation.
Underlying hypoxic gene regulation are the hypoxia inducible transcription factors (HIFs). The HIFs consist of a dimer of HIF-alpha and HIF-beta subunits. The HIF-alpha subunits are regulated by oxygen, with the activity and abundance of the HIF-alpha subunit increased in hypoxia, whereas the HIF-beta subunit (better known as ARNT) is oxygen-independent (Figure1). Oxygen regulated control of HIF-alpha abundance is mediated by three homologous oxygen-dependent prolyl hydroxylases, PHD1-3. They modify distinct proline residues in the HIF-alpha proteins in normoxia resulting in the recruitment of the Von Hippel Lindau protein (pVHL), polyubuiquitylation and rapid proteosomal degradation of the HIF-alpha proteins. The control of HIF-alpha activity is mediated by asparaginyl hydroxylation by the oxygen-dependent asparaginyl hydroxylase, FIH (Factor Inhibiting HIF). Asparaginyl hydroxylation by FIH represses transcriptional activity by preventing the interaction of HIF-alpha proteins with transcriptional coactivators such as CBP/p300. When oxygen is limiting both prolyl and asparaginyl hydroxylases are unable to modify the HIFs, resulting in stable, transcriptionally active HIFs activating their target genes in response to hypoxia.Research by ourselves and others have demonstrated that these hydroxylases act as cellular oxygen sensors.
Figure 1: Oxygen-dependent regulation of gene expression mediated by the hypoxia inducible transcription factors (HIFs). The 3 HIF prolyl hydroxylases (PHD 1-3) and single HIF asparaginyl hydroxylase (factor inhibiting HIF, FIH) are oxygen dependent enzymes. The von hippel lindau protein (VHL) is part of an E3 ubiquitin ligase complex that promotes proteosomal degradation of HIF, and CBP and p300 are transcriptional coactivators. Examples of HIF target genes include erythropoietin (Epo), vascular endothelial growth factor (VEGF) and glucose transporter 1 (Glut1).
One major area of current research is characterizing the function of FIH, the novel oxygen-sensing asparaginyl hydroxylase. A second key area is understanding the different roles and mechanisms of regulation between HIF-1alpha and HIF-2alpha. This information is invaluable for our understanding of how the body is able to sense and respond to changes in oxygen, and the role of HIFs in major human diseases, and may also provide therapeutic targets for these same diseases. We currently use the latest techniques in molecular biology, cell culture and protein analysis, including PCR, cDNA cloning, inducible gene expression, RNAi, DNA microarrays, cell culture models of hypoxic response, transcription assays, animal models of development and disease, protein expression and purification, in vitro enzyme assays, immunoblotting, chromatography, and mass spectrometry. We also have close collaborations with a number of other international and national research groups who have complementary expertise and resources.