Hypoxia Proteins

 Hypoxia Proteins Background

Hypoxia in physiological processes

Oxygen plays a vital role in normal cellular and physiological processes. As the final electron acceptor in the mitochondrial electron transport chain, it is necessary for the generation of ATP via oxidative phosphorylation. Because of the absolute requirement for oxygen, cells and multi-cellular organisms have developed adaptive strategies in order to survive at varying levels of oxygen. Without oxygen, the cell must rely on anaerobic glycolysis to maintain energy production to power normal cellular functions. In a low oxygen environment, a cell not only switches to glycolysis to maintain ATP production, but it also reduces the use of non-essential machinery in the cell to decrease the demand for energy.

Hypoxia, or low oxygen, induces several well-studied physiological responses, which are best illustrated with high altitude physiology. Acute responses to the decreased oxygen tension at high altitude can be fatal in an unacclimatized person. During the acclimatization process, the human body responds in multiple ways to maximize oxygen intake and delivery to vital organs. An immediate response to hypoxia is an increase in ventilation. Along with breathing faster, pulmonary ventilation expands with utilization of greater lung volumes. The diffusing capacity of oxygen across the alveolar-capillary membranes is also increased. In addition, the capillary beds grow in number through a process called angiogenesis to supply vital organs such as the heart with adequate oxygen. After several months at high altitude, the hypoxic environment leads to increased production of red blood cells through the stimulation of erythropoietin as well as elevated hemoglobin levels and blood volume.

How oxygen is sensed and what signaling pathways mediate the adaptive cellular and physiologic responses to low oxygen are two related avenues of active research. Acute hypoxia induces the activation of oxygen-sensitive neurosecretory cells in chemoreceptor organs (the carotid and aortic bodies). Low oxygen inhibits potassium ion channels, leading to membrane depolarization, calcium influx, neurotransmitter release, and activation of afferent sensory fibers, all of which contribute to producing the cardiopulmonary effects of hypoxia. Chronic hypoxia induces expression of glycolytic enzymes and glucose transporters which support the increase in anaerobic respiration, vascular endothelial growth factor (VEGF) which stimulates angiogenesis, and erythropoietin (EPO) which stimulates red blood cell production.


Hypoxia in pathological processes

What causes pathological hypoxia? As we follow the flow of oxygen in an organism, potential causes of hypoxia emerge. Decreased oxygen tension from environmental stresses such as high altitude or impaired oxygen delivery to exchange membranes at the alveolar-capillary interface from neuromuscular and pulmonary diseases can cause hypoxia. Once the oxygen diffuses across this interface, it is transported by hemoglobin in red blood cells. Anemia or hemoglobinopathies by reducing oxygen carrying capacity can cause hypoxia as well. Pathology in circulation or hindrances to diffusion may result in poor perfusion and hence hypoxia of tissues or organs. In addition, veno-arterial shunts including right to left shunts of the heart, in which the oxygenated blood is mixed with de-oxygenated blood, are other potential causes of hypoxia. Finally, at the cellular level, hypoxia may result if the cell cannot utilize the oxygen, such as in cyanide poisoning.

The pathologic consequence of ischemia or hypoxia ranges from cell injury to cell death. Hypoxia as mentioned previously causes the switch from oxidative phosphorylation to anaerobic glycolysis for energy production. When a cell is exposed to diminishing levels of oxygen, which can be caused by any of the events mentioned above, mitochondria produce less ATP because of decreased oxidative phosphorylation and less efficient energy production by glycolysis. This scarcity of ATP leads to attenuated activity of all energy-requiring cellular functions, most importantly the sodium-potassium pump and protein synthesis. Morphologic changes of the cell reflect these biochemical events. Membranes depolarize and cells swell as the osmotic homeostasis is altered by the functional inactivity of the ATP-dependent ion pumps and channels. In addition, nuclear chromatin clumps because increased glycolysis leads to decreased pH in normal tissues. If the cell sustains injury to its cellular membrane, irreversible cell injury leading ultimately to cell death will occur.

If the adaptive strategies mentioned above fail to adequately compensate for the decreased level of oxygen, pathology occurs. Several pathological processes result from the lack of oxygen delivered to vital organs. The leading causes of death in the United States are heart attacks and strokes, both of which result from ischemia due to occluded blood vessels which in turn lead to cellular injury and death and organ dysfunction. Tumors, on the other hand, use hypoxia as a selective advantage. The recent discovery of the molecular pathways by which hypoxia activates the aforementioned physiological processes have led to several new and innovative therapeutic interventions for these pathological processes.

In cancer, hypoxic micro-environments in solid tumors give tumor cells a selective advantage by protecting them against conventional radiation therapy and most chemotherapy, suppressing apoptosis, stimulating angiogenesis, and promoting malignant progression. In one model of solid tumors, which comprise 90% of all tumors, a tumor grows around a blood vessel. Those cells closest to the vessel are well oxygenated whereas those that are distant from the vessel, because of the diffusion limit of oxygen (approximately 100 micrometers), are hypoxic, as well as nutrient-deprived. In addition, solid tumors often have abnormal vasculature and disturbed microcirculation resulting in areas of transient hypoxia. In the early 1970's, it was shown that hypoxic cells required three times more radiation treatment than aerobic cells to cause the same amount of cell killing. Because radiation therapy kills cells primarily by damaging DNA which is enhanced by oxygen radicals, without the presence of oxygen to form the radicals, less DNA damage and subsequent cell killing occur in a hypoxic environment. For the delivery of chemotherapeutic drugs, most of which are delivered intravenously, the hypoxia cells that are furthest away from the blood vessels are not exposed to the drugs. In addition, because a majority of chemotherapeutic drugs are most effective in highly proliferating cells, hypoxic cells which divide less rapidly and often are arrested in the cell cycle are resistant to conventional chemotherapeutic agents.

Tumor hypoxia is an adverse prognostic factor, and the severity of hypoxia correlates with a poorer prognosis. Not only are hypoxic tumor cells resistant to radiation therapy and chemotherapy, but they also tend to acquire a more aggressive and malignant phenotype. Several studies have shown that the hypoxic fraction of a tumor predicts for poor therapeutic outcome after both chemo-radiation therapy as well as surgery. These studies introduced and supported the concept that hypoxia selects for more malignant cells that are resistant to apoptotic signals and that can induce the production of angiogenic growth factors. The resulting neovascularization around the tumor provides it with a means to grow and a route to metastasize. Tumor cells which manage to grow in a hypoxic microenvironment have acquired in their arsenal several weapons to ensure their growth and survival, by inducing angiogenesis, avoiding cell death, and promoting metastasis. As the degree of hypoxia is more severe in tumor cells than in normal tissue, this unique environment serves as a selective target for cancer therapeutics.


Hypoxia inducible factor

The hypoxia inducible factor was first identified after studies on transcriptional control of the erythropoietin gene. It was known that hypoxia stimulated erythropoietin gene expression, and through investigation of the transcription factors binding to the promoter region of this gene, the hypoxia inducible factor (HIF) was identified. HIF consists of 2 subunits, both of which exist in multiple forms, with molecular masses of 120-130 kDa and 91-94 kDa and designated HIFα and HIFβ respectively (also known as the Aryl hydrocarbon Receptor Nuclear Translocator, or ARNT). The HIF subunits contain basic helix loop helix (bHLH) and Per Arnt Sim (PAS) domains, which control DNA binding and dimerisation. expression of the HIFα and HIFβ subunits is constitutive; however, HIFα protein is rapidly degraded in normoxia.

The mechanism by which HIFα degradation occurs has only recently been elucidated. Investigations into the hereditary cancer syndrome caused by germ line mutations in the Von Hippel Lindau (VHL) gene, associated with highly vascularised tumors, found transcription of hypoxia-associated genes was up-regulated. This led to the discovery that VHL protein (pVHL) interacts with the HIFα subunit; pVHL recognizes HIFα via 2 hydroxyproline residues in the oxygen-dependent degradation domain of the HIFα protein. Binding of pVHL to HIFα leads to polyubiquitination of the HIFα subunit and its subsequent degradation. This provides a mechanism by which oxygen directly modulates gene expression, since prolyl hydroxylases require molecular oxygen. Three conserved prolyl-4-hydroxylases have been identified which interact directly with HIFα. These are designated as prolyl hydroxylase domain containing 1, 2 and 3 (PHD1-3); their activity is regulated directly by oxygen tension. PHD1 is expressed constitutively, whereas PHD2 and PHD3 are induced by hypoxia.

HIFα is not only regulated by oxygen at the level of protein stability. Further hydroxylation occurs at an asparagine residue in the C-terminal transactivating domain (C-TAD) that disrupts HIF binding to its co-activator c300/CBP, leading to repression of transcriptional activity. This hydroxylation is performed by factor inhibiting HEF (FIH), an asparaginyl hydroxylase. Hence, oxygen is capable of regulating gene expression by acting as an essential substrate for enzymes that hydroxylate specific proline and asparagine residues within the HIFα peptide, leading to degradation and loss of activity in normoxia.

The initial identification of the HIF PHDs occurred in the nematode worm C. elegans, indicating that the HIF signaling system is highly conserved. The downstream effects of HIF activation are transcription of specific genes associated with cell activity and survival. In general, genes activated by HIF function to maintain cell energy supplies during hypoxia, e.g. glycolytic enzymes and glucose transporters, and to provide increased O2 delivery; examples include erythropoietin and vascular endothelial growth factor (VEGF). The effect of hypoxia on specific cell types is discussed in detail below.

Several HIFα subunits have been described, with HIF1α being the most studied to date. HIF1α is expressed ubiquitously throughout the body and is indispensable for survival. HIF1α knockout mice show abnormal vascular development, failure of neural tube closure, and extensive mesenchymal cell death leading to death by embryonic day 11. Further investigation in HIF1α deficient cell lines revealed that the glycolytic response to hypoxia appears to be regulated exclusively by HIF1α. HIF2α shares 48% overall amino acid identity with HIF1α; however, in the bHLH (DNA binding) and PAS (HIFP binding) domains that homology increases to 83% and 70% respectively. The critical proline residues in the oxygen-dependent degradation domain are also conserved, as are the terminal 50 amino acids that regulate p300 binding and transcriptional co-activation. HIF2α knockout animals show variable phenotypes, with some offspring surviving to term. Those animals surviving to term have multiple organ pathologies including retinopathy, hepatic steatosis, cardiac hypertrophy, and mitochondrial dysfunction as well as severe abnormalities in haematopoiesis. These studies indicate that the functions of HIF1α and HIF2α are non-redundant and do not overlap. A third variant, HIF3α, has also been described; it is induced by hypoxia and is capable of binding to HIFP, but lacks the N-terminal transactivation domain. Its role in hypoxia-induced signaling seems to be as a negative regulator of HIFα activity.


Hypoxia reference

1. Guyton, A. C. Textbook of medical physiology, 8th edition, 1991.

2. Cotran, R. S., Kumar, V., and Robbins, S. L. Robbins pathologic basis of disease, 5th edition, 1994.

3. Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis[J]. Nature, 1992, 359(6398): 843-845.

4. Semenza G L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1[J]. Annual review of cell and developmental biology, 1999, 15(1): 551-578.

5. Cockman M E, Masson N, Mole D R, et al. Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein[J]. Journal of Biological Chemistry, 2000, 275(33): 25733-25741.

6. Metzen E, Berchner-Pfannschmidt U, Stengel P, et al. Intracellular localisation of human HIF-1α hydroxylases: implications for oxygen sensing[J]. Journal of cell science, 2003, 116(7): 1319-1326.

7. Kotch L E, Iyer N V, Laughner E, et al. Defective vascularization of HIF-1α-null embryos is not associated with VEGF deficiency but with mesenchymal cell death[J]. Developmental biology, 1999, 209(2): 254-267.