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Key Event Title
Stabilization of HIF-1 Alpha
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|All life stages||High|
Key Event Description
Oxygen is essential in the cells of all aerobic organisms. Oxygen is required for the production of ATP, as well as being a substrate for numerous enzyme activities. Hypoxia is defined as concentrations of oxygen below atmospheric (21%). Hypoxia occurs during tissue ischemia, where a region of tissues or cells in an organism is deprived of blood flow, and therefore adequate levels of oxygen (Semenza, 2000). In humans, hypoxia can be onset by numerous factors including abnormalities in the heart and lung, anemia, deficiency in red blood cells, and circulatory problems. Hypoxia can also have associated physiological stresses, such as oxidative stress. Under hypoxic conditions, an increase of reactive oxygen species (ROS) may occur, accumulate from hydrogen peroxide in the presence of trace amounts of metals (Hielscher & Gerecht, 2015). These ROS are highly reactive and are very short lived. It has been found that ROS causes DNA mutations which could further induce changes on oncogenic function, and potentially cause cancer (Tafani et al., 2016). Normally, most oncogenes undergo rapid apoptosis, but when there is a mutation, apoptosis can be inhibited. This results in these oncogenes expressed at high levels. The presence of ROS also has negative effects on proteins; more specifically by causing protein misfolding, disrupting protein-protein interactions and altering binding affinities to substrates (in the case of enzymes) and other proteins (Schieber & Chandel, 2014). This can have a negative impact on signal transduction.
Furthermore, the accumulation of the ROS under normoxic conditions can activate transcription factors known as Hypoxia-Inducible Factors (HIFs), and more specifically, HIF-1a; factors that are normally only found in the cell under hypoxic conditions (Brown et al., 2016). This is done by inactivating HIF-1a’s inhibitor; Prolyl Hydroxylase Domain (PHD) containing hydroxylases (Schieber & Chandel, 2014). PHD hydroxylases and HIFs can mediate transcriptional control of a number of target genes which contain a hypoxia response element (HREs) within their promoters. Under normoxic conditions (normal atmospheric oxygen levels), HIF-1a is rapidly hydroxylated at two proline residues and one asparagine residue by prolyl-hydroxylases (Maxwell et al., 1999). This allows the HIF to be targeted for ubiquitination by the von Hippel Lindau (VHL) containing E3 ubiquitin ligase, and further, degradation by the proteasomes.
Under hypoxic conditions, when oxygen is not present, the HIF-prolyl-hydroxylase is inhibited. Iron, molecular oxygen and alpha-ketoglutarate are all essential components necessary to hydroxylate these proline and asparagine residues. If oxygen is not present as a cofactor, then the mechanism cannot proceed, resulting in the inhibition of the hydroxylation. Similarly, when divalent metals such as Cobalt are introduced, they compete with the iron binding centre necessary for hydroxylation (Yuan et al., 2003). This response is known as a mimic to hypoxia which leads to the stabilization of HIF.
HIF is composed of an oxygen-sensitive HIF-a subunit and an oxygen-insensitive HIF-b subunit (Ozer & Bruick, 2007). Both subunits play an important role in DNA binding as the transcription factor cannot bind to the HRE without being a heterodimer. As mentioned above, HIF 1 alpha protein is stabile when hydroxylation is inhibited and is degraded when hydroxylation occurs correctly on the proline and asparagine residues of HIF. To expand on this topic a schematic is shown below.
Figure 1: Schematic of HIF-1 Alpha Broken Down with proper Hydroxylation vs the stabilization under Hydroxylation inhibition. (Willmore Lab Introductory Slides).
When iron is able to properly function in the hydroxylation of proline and asparagine residues it suppresses HIF transcriptional activity. Hydroxylation of proline residues at positions 405 and 531 on the HIF Alpha Subunit target it for rapid ubiquination by the von Hippel Lindau (VHL) containing E3 ubiquitin ligase (Strowitzki et al., 2019). The HIF alpha subunit is also hydroxylated in an asparagine residue (803) that blocks the binding of the p300 coactivator. These processes result in the degradation of HIF. When divalent metals, such as Cobalt compete for the iron coordination location, HIF alpha is stabilized. The alpha subunits are no longer hydroxylated at proline and asparagine residues and do not get degraded by proteosomes. Alpha subunits move to the nucleus and dimerize with beta subunits which recruit p300 for coactivation. This complex is now able to bind to HRE site and transactivate gene expression.
Figure 2: HIF heterodimer bind to HREs in the promoter regions of its target genes and recruits transcriptional coactivators p300. The targeted hydroxylated region of the HIF is also shown which can undergo ubiquitination (Ozer & Bruick, 2007).
How It Is Measured or Detected
The stabilization of HIF-1 Alpha can be determined quantitatively and semi-quantitatively. Common techniques for semi-quantitative analysis of HIF-1 Alpha could be Western Blot and immunocytochemistry (Karshovska et al., 2007). This method would determine the protein expression of HIF-1 Alpha against an antibody. HIF-1 Alpha levels can also be assessed at the genetic level using quantitative polymerase chain reaction (qPCR). This method would amplify DNA using HIF-1 Alpha primers and use thermal cycling to conduct multiple temperature dependent reactions (Saiki et al., 1988). Other more quantitative methods could include performing an enzyme-linked immunosorbent assay (ELISA) (Formento et al., 2005). In most cases, antigens from the sample are attached to a surface, then a matching antibody is applied over the surface so it can bind the antigen. In this case the antigen would be the protein HIF-1 Alpha.
Domain of Applicability
Stabilization of the HIF-1 Alpha protein can be categorized as Term Homo sapiens for Taxonomic applicability. The evidence for this statement is high as it is a human HIF1A gene which encodes for the transcription factor hypoxia inducible factor (HIF, NCBI). HIF1 Alpha functions as a master transcription regulator of the adaptive response to hypoxia in the Taxonomic class Homo sapiens (Kim et al., 2008).
Many studies have shown that HIF-1 Alpha stabilization can occur at all life stages (Madan et al., 2002). HIF-1 A stabilization is also seen in human fetus at all organs and at various stages of gestation. HIF-1 Alpha has also been shown to play a key role in regulation of human metabolism (Formenti et al., 2010).
Studies have shown that HIF-1 Alpha can stabilize in multiple different cell lines. These include HCT116, HEK-293T, Hela and many others. In this case sex applicability does not apply. Some evidence suggest that females have been reported for reduced risk of cardiovascular disease (Bohuslavová et al., 2010). This could have some effect on HIF-1 Alpha stabilization. Other studies have shown that HIF-1 alpha expression in the hearts of female mice is higher then that of males (Zampino et al., 2006).
Bohuslavová, R., Kolář, F., Kuthanová, L., Neckář, J., Tichopád, A. and Pavlinkova, G. (2010). Gene expression profiling of sex differences in HIF1-dependent adaptive cardiac responses to chronic hypoxia. Journal of Applied Physiology 109, 1195–1202.
Brown, D. I., Willis, M. S. and Berthiaume, J. M. (2016). Chapter 11 - Influence of Ischemia-Reperfusion Injury on Cardiac Metabolism. In The Scientist’s Guide to Cardiac Metabolism (ed. Schwarzer, M.) and Doenst, T.), pp. 155–167. Boston: Academic Press.
Formenti, F., Constantin-Teodosiu, D., Emmanuel, Y., Cheeseman, J., Dorrington, K. L., Edwards, L. M., Humphreys, S. M., Lappin, T. R. J., McMullin, M. F., McNamara, C. J., et al. (2010). Regulation of human metabolism by hypoxia-inducible factor. Proc Natl Acad Sci USA 107, 12722–12727.
Formento, J. L., Berra, E., Ferrua, B., Magne, N., Simos, G., Brahimi-Horn, C., Pouyssegur, J. and Milano, G. (2005). Enzyme-Linked Immunosorbent Assay for Pharmacological Studies Targeting Hypoxia-Inducible Factor 1␣. CLIN. DIAGN. LAB. IMMUNOL. 12, 5.
Hielscher, A. and Gerecht, S. (2015). Hypoxia and free radicals: role in tumor progression and the use of engineering-based platforms to address these relationships. Free Radical Biological Medicine. 0, 281–291.
"HIF1A". National Center for Biotechnology Information.
Karshovska, E., Zernecke, A., Sevilmis, G., Millet, A., Hristov, M., Cohen, C., Schmid, H., Krotz, F., Sohn, H., Klauss, V., et al. (2007) Expression of HIF-1 alpha in injured arteries controls SDF-1 alpha-mediated neointima formation in apolipoprotein E-deficient mice. Arteriosclerosis Thrombosis and Vascular Biology. 27, 2540–2547.
Kim, E.-J., Yoo, Y.-G., Yang, W.-K., Lim, Y.-S., Na, T.-Y., Lee, I.-K. and Lee, M.-O. (2008). Transcriptional Activation of HIF-1 by RORα and its Role in Hypoxia Signaling. ATVB 28, 1796–1802.
Madan, A., Varma, S. and Cohen, H. J. (2002). Developmental Stage-Specific Expression of the α and β Subunits of the HIF-1 Protein in the Mouse and Human Fetus. Molecular Genetics and Metabolism 75, 244–249.
Maxwell, P. H., Wiesener, M. S., Chang, G.-W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R. and Ratcliffe, P. J. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 399, 271.
Ozer, A. and Bruick, R. K. (2007). Non-heme dioxygenases: cellular sensors and regulators jelly rolled into one? Nature Chemical Biology. 3, 144–153.
Saiki, R., Gelfand, D., Stoffel, S., Scharf, S., et al. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–91.
Schieber, M. and Chandel, N. S. (2014). ROS Function in Redox Signaling and Oxidative Stress. Current Biology. 24, R453–R462.
Semenza, G. L. (2000). HIF-1: mediator of physiological and pathophysiological responses to hypoxia. Journal Applied Physiology. 88, 1474–1480.
Strowitzki, M., Cummins, E. and Taylor, C. (2019). Protein Hydroxylation by Hypoxia-Inducible Factor (HIF) Hydroxylases: Unique or Ubiquitous? Cells 8, 384.
Tafani, M., Sansone, L., Limana, F., Arcangeli, T., De Santis, E., Polese, M., Fini, M. and Russo, M. A. (2016). The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in Cancer initiation and progression. Oxidative Medicine Cellular Longevity 2016.
Yuan, Y., Hilliard, G., Ferguson, T. and Millhorn, D. E. (2003). Cobalt Inhibits the Interaction between Hypoxia-inducible Factor-α and von Hippel-Lindau Protein by Direct Binding to Hypoxia-inducible Factor-α. Journal of Biological Chemistry 278, 15911–15916.
Zampino, M., Yuzhakova, M., Hansen, J., McKinney, R. D., Goldspink, P. H., Geenen, D. L. and Buttrick, P. M. (2006). Sex-related dimorphic response of HIF-1α expression in myocardial ischemia. American Journal of Physiology-Heart and Circulatory Physiology 291, H957–H964.