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Event: 1864

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Stabilization of HIF-1 Alpha

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
HIF-1 Alpha Stabilization

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Inhibition of Hydroxylase leads to Breast Cancer KeyEvent Arthur Author (send email) Under development: Not open for comment. Do not cite

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
Homo sapiens Homo sapiens High NCBI
mammals mammals High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages High

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Mixed Moderate

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

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).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

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 1CLIN. 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.