By Sashini Ranawana
Over the years, the topics of oxygen metabolism and respiration have been noticeably associated with the names of Nobel Laureates. In 1931, Otto Warburg received the prize for his work on the mechanism of action of cytochrome C oxidase, a crucial enzyme in the mitochondrial electron transport chain. He observed a decreased reliance on this oxygen-dependent process in tumour cells, an unusual phenomenon called aerobic glycolysis, which provided important insights into the link between respiration and oncogenesis.1,2 In 1938, Corneille Heymans was acknowledge with the prize for elucidating the role of chemoreceptors of the aortic and carotid bodies in measuring arterial oxygen levels.1 Hans Krebs, in 1953, became a Laureate after establishing the tricarboxylic acid cycle, which helped to demonstrate the intricate relationships between aerobic respiration and other important metabolic processes within cells.3 Such findings have been crucial to our overall understanding of how oxygen is used in physiologically normal cells, and how its abnormal regulation can lead to cellular dysfunction.
The seminal work of Peter Ratcliffe, Gregg Semenza and William Kaelin, which culminated in their 2019 Nobel Prize, integrated the actions of specific genes and proteins into a single molecular machinery used by cells to sense and respond to alterations in oxygen levels.4,5 In doing so, they helped explain how large-scale processes such as angiogenesis (new blood vessel formation), immune system function and red blood cell production could differ in areas of normal compared to low oxygen concentrations (hypoxic environments).5 The transcription factor hypoxia-inducible factor (HIF-1α) and the Von Hippel-Lindau tumour suppressor protein mediate these hypoxia-stimulated changes by controlling the production of proteins such as erythropoietin (EPO), vascular endothelial growth factor (VEGF) and lysyl oxidase (LOX), along with the expression of genes such as Oct-4.4,6,7 These upregulated molecules elevate red blood cell levels, stimulate blood vessel growth into oxygen-starved regions, enable vasodilation and increase cell proliferation.4,6 The similarities to cancer hallmarks are also prominently clear.
The genomic region of the EPO gene was the starting point in a series of experiments used by Semenza et al. to determine how transcriptional regulation of the gene is controlled in areas of hypoxia.8 Expression of the EPO gene and its 3’ and 5’ flanking sequences was induced in mouse liver cells before treatment with the DNase I enzyme. Two DNase I hypersensitive sites were found solely in the liver cells of these transgenic mice, indicating a lack of chromatin packing and an increased accessibility to the DNA sequence at the corresponding positions downstream of the EPO transgene. By subjecting this region to further DNase I digestion in the presence of protein extracts from the nucleus of anaemic mouse cells, Semenza et al. found that certain DNA sequences were more protected from cleavage. It was apparent that specific nuclear factors triggered by the hypoxic conditions in these anaemic mice were binding to this EPO enhancer element. A protein complex named HIF-1 was ultimately identified as the predominant nuclear transcription factor controlling this genomic sequence, when its activity and capacity to bind DNA were found to significantly correlate with EPO expression.
A part of the oxygen sensing machinery had now been revealed, but the regulation of one subunit of the HIF-1 transcription factor, HIF-1α, was still unclear. To explain this, Ratcliffe et al. and Kaelin et al. turned to the Von Hippel-Lindau (VHL) genetic disease, a condition where an inherited mutation results in inactivated or absent pVHL.9,10 Patients lacking this crucial protein have an increased risk of developing vascularized benign tumours, and a higher transcription level of hypoxia-induced genes. In renal carcinoma cell lines RCC4 and 786-O, without VHL expression, the levels of HIF-1α constantly remained high and didn’t fluctuate with exposure to varying oxygen levels. By re-expressing a wild-type VHL gene in these cells, they were able to restore the sensitivity of HIF-1α to oxygen.9 Subsequently, the precise function of pVHL was deduced. By analysing an eight amino-acid peptide in the HIF-1 molecule, they discovered an oxygen- and iron-dependent mechanism of proline hydroxylation. This hydroxyl group enables an E3 ubiquitin ligase protein complex containing pVHL to target HIF-1α for ubiquitin-mediated degradation.10,11
Combining the conclusions from these multiple studies into a cohesive whole allowed the researchers to understand how the concentration of oxygen sensed by a cell determines which set of reactions are initiated. Under normoxic conditions, oxygen initiates the hydroxylation of two proline residues in the oxygen-dependent degradation domain of the HIF-1α peptide chain, and enables pVHL to bind to the transcription factor. The consequent addition of a ubiquitin chain to HIF-1α marks it as a molecule needing to be destroyed by the proteasome. In hypoxic conditions, the failure of pVHL to bind to un-hydroxylated proline residues allows the stabilisation of HIF-1α. This functional form of the transcription factor can then bind to the enhancer elements of multiple hypoxia-associated genes, bringing about physiological changes that increase the supply of oxygenated blood to the affected cells and tissues.
The relative simplicity of this system can be misleading, especially when considering its essential function in maintaining tissue homeostasis, controlling cellular metabolic rate and refining appropriate immunological responses. The role of such a central pathway in a number of diseases has also identified it as a therapeutic target.5 In the hypoxic tumour microenvironment, blocking components of the oxygen sensing machinery can exacerbate oxygen starvation and facilitate cancer cell death by apoptosis or necrosis. In patients with chronic renal failure, HIF-1α could be continuously stabilised by inhibiting the binding of pVHL to hydroxylated proline residues, to increase the transcription of EPO. The severe anaemia caused as a consequence of the disease could therefore be treated. The expression levels of HIF-1α can also provide an indirect indication of the levels of deoxygenated and oxygenated haemoglobin in certain tissue regions, allowing it to be used in diagnostic methods such as functional magnetic resonance imaging.4 It is clear that even in the few years after its discovery, this ubiquitous molecular pathway has had an impact in all aspects of healthcare.
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