This Event is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

Event: 228

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

peroxisome proliferator activated receptor promoter demethylation

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. More help

demethylation, PPARg promoter

Explore in a Third Party Tool

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help

Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.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. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Cell term
hepatocyte

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). 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 signaling 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. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
peroxisome proliferator activated receptor signaling pathway	peroxisome proliferator-activated receptor gamma	increased

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
LXR Activation to Liver Steatosis	MolecularInitiatingEvent	Agnes Aggy (send email)	Not under active development
NR1I3 suppression to steatosis	MolecularInitiatingEvent	Allie Always (send email)	Under Development: Contributions and Comments Welcome
PPARG mod to adipogenesis	MolecularInitiatingEvent	Cataia Ives (send email)	Under Development: Contributions and Comments Welcome
Demethylation of PPAR promotor leading to vascular disrupting effects	MolecularInitiatingEvent	Allie Always (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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
mice	Mus sp.	High	NCBI
human	Homo sapiens	Moderate	NCBI
rat	Rattus norvegicus	Moderate	NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Sex Applicability

An indication of the the relevant sex for this KE. More help

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. More help

Biological state

The Peroxisome Proliferator Activated receptor γ (PPARγ) belongs to Peroxisome Proliferator Activated receptors (PPARs; NR1C) steroid/thyroid/retinoid receptor superfamily of transcription factors, which respond to specific ligands by altering gene expression in a cell-specific manner. The PPARγ gene contains three promoters that yield three isoforms, namely, PPAR-γ1, 2 and 3. PPAR-γ1 and γ3 RNA transcripts translate into the identical PPAR-γ1 protein.

Biological compartments

PPARγ is abundantly expressed in adipose tissue, promoting adipocyte differentiation, but is also present in various cells and tissues, for review see (Braissant et al. 1996). PPARγ expression is tissue dependent (L Fajas et al. 1997), (Lluis Fajas, Fruchart, and Auwerx 1998). PPARγ is most highly expressed in white adipose tissue and brown adipose tissue, where it is a master regulator of adipogenesis as well as a potent modulator of whole-body lipid metabolism and insulin sensitivity (Evans, Barish, and Wang 2004), (Tontonoz and Spiegelman 2008). Whereas PPARγ1 is expressed in many tissues, the expression of PPARγ2 is restricted to adipose tissue under physiological conditions but can be induced in other tissues by a high-fat diet (Saraf et al. 2012).

General role in biology

PPARγ is activated after the binding of natural ligands such as polyunsaturated fatty acids and prostaglandin metabolites. It can also be activated by synthetic ligands such as thiazolidinediones (TZDs) (rosiglitazone, pioglitazone or troglitazone) (Lehmann et al., 1995). PPARγ controls many vital processes such as glucose metabolism and inflammation as well as variety of developmental programs(Wahli & Desvergne, 1999), (Rotman et al., 2008), (Wahli & Michalik, 2012). This receptor itself is essential for developmental processes since targeted disruption of this gene results in embryo lethality, due in part to defective placental development, therefore modulation of PPARγ activity may impact endocrine regulated processes during development as well as later in life.

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?

Binding of ligands to PPARγ is measured using binding assays in vitro and in silico, whereas the information about functional activation is derived from the transactivation using e.g. reporter assay with a reporter gene that demonstrates functional activation of a nuclear receptor by a specific compound. Binding of agonists within the ligand-binding site of PPARs causes a conformational change promoting binding to transcriptional coactivators. Conversely, binding of antagonists results in a conformation that favours the binding of corepressors (Yu & Reddy, 2007) (Viswakarma et al., 2010. Transactivation assays are performed using the transient or stably transfected cells with the PPARγ expression plasmid and a reporter plasmid, correspondingly. There are also other methods that have been used to measure PPARγ activity, such as the Electrophoretic Mobility Shift Assay (EMSA) or commercially available PPARγ transcription factor assay kits, see Table 1. The transactivation (stable transfection) assay provides the most applicable OECD Level 2 assay aimed at identifying the initiating event leading to adverse outcome (LeBlanc, Norris, & Kloas, 2011). Currently no internationally validated assays are available.

Key event	PPARγ activation
	What is measured?	Ligand Binding		Transcriptional activity
Method/test category	molecular modelling	binding assay	transactivation reporter gene assay			transcription factor assay
Method/test name	molecular modelling; docking	Scintillation proximity binding assay	luciferase reporter gene assay			PPARγ (mouse/rat) Reporter Assay Kit	Electrophoretic Mobility Shift Assay (EMSA)
Test environment	In silico	In vitro	In vitro			In vitro, ex vivo
Test principle	Computational simulation of a candidate ligand binding to a receptor, Predicts the strength of association or binding affinity.	direct binding indicating the mode of action for PPARα/γ	Quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPAR functional activity.			PPARγ once activated by a ligand, the receptor binds to a promoter element in the gene for target gene and activates its transcription. The bound (activated) to DNA PPAR is measured.
Test outcome	A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. If the protein is a receptor, ligand binding may result in agonism or antagonism	Assess the ability of compounds to bind to PPARγ. Identifies the modulators of PPARγ.	The changes in activity of reporter gene levels functionally linked to a PPAR-responsive element/promoter gives information about the activity of the PPAR activation.			Protein: DNA binding, DNA binding activity
Test background	Predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using, for example, scoring functions.	This assay determines whether compounds interact directly with PPARγ.	PPARγ COS-1cell transactivation assay (transient transfection with human or mouse PPARγ expression plasmid and pHD(x3)-Luc reporter plasmid	(PPRE)3- luciferase reporter construct C2C12	Proprietary rodent cell line expressing the mouse/rat PPARγ	Transcriptional activity of PPARγ can be assessed using commercially available kits like e.g. PPARγ transcription factor assay kit (Abcam, Cambridge, USA or Cayman Chemical, USA).	Gene regulation and determining protein: DNA interactions are the detected by the EMSA. EMSA can be used qualitatively to identify sequence-specific DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences within a given genes upstream regulatory region. EMSA can also be utilized quantitatively to measure thermodynamic and kinetic parameters.
Assay type	Quantitative	Qualitative	Quantitative	Quantitative	Quantitative	Quantitative	Quantitative
Application domain	Virtual screening	In vitro screening	In vitro Screening, functional studies activity (reported use: agonist)		In vitro Screening functional activity (antagonist/agonist)	Functional studies	Functional studies
Source	Research/commercial	Research	Research	Research	commercial	commercial	Research/commercial
Ref	(Feige et al., 2007), (Kaya, Mohr, Waxman, & Vajda, 2006)	(Lapinskas et al., 2005), (Wu, Gao, & Wang, 2005)	(Maloney & Waxman, 1999)	(Feige et al., 2007)	Cayman, (Gijsbers et al. 2013)	Abcam[1]

Table 1 Summary of the chosen methods to measure the PPARγ activation.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

PPARγ have been identified in frog (Xenopus laevis), mouse, human, rat, fish, hamster and chicken (Wahli & Desvergne, 1999).

References

List of the literature that was cited for this KE description. More help

Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., … Evans, R. M. (1999). PPAR gamma is required for placental, cardiac, and adipose tissue development. Molecular Cell, 4(4), 585–95.

Braissant, O., Foufelle, F., Scotto, C., Dauça, M., & Wahli, W. (1996). Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat. Endocrinology, 137(1), 354–66.

Burns, K. A., & Vanden Heuvel, J. P. (2007). Modulation of PPAR activity via phosphorylation. Biochimica et Biophysica Acta, 1771(8), 952–60. doi:10.1016/j.bbalip.2007.04.018

Fajas, L., Auboeuf, D., Raspé, E., Schoonjans, K., Lefebvre, A. M., Saladin, R., … Auwerx, J. (1997). The organization, promoter analysis, and expression of the human PPARgamma gene. The Journal of Biological Chemistry, 272(30), 18779–89.

Fajas, L., Fruchart, J.-C., & Auwerx, J. (1998). PPARγ3 mRNA: a distinct PPARγ mRNA subtype transcribed from an independent promoter. FEBS Letters, 438(1-2), 55–60. doi:10.1016/S0014-5793(98)01273-3

Feige, J. N., Gelman, L., Michalik, L., Desvergne, B., & Wahli, W. (2006). From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions. Progress in Lipid Research, 45(2), 120–59. doi:10.1016/j.plipres.2005.12.002

Feige, J. N., Gelman, L., Rossi, D., Zoete, V., Métivier, R., Tudor, C., … Desvergne, B. (2007). The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis. The Journal of Biological Chemistry, 282(26), 19152–66. doi:10.1074/jbc.M702724200

Gijsbers, Linda, Henriëtte D L M van Eekelen, Laura H J de Haan, Jorik M Swier, Nienke L Heijink, Samantha K Kloet, Hai-Yen Man, et al. 2013. “Induction of Peroxisome Proliferator-Activated Receptor Γ (PPARγ)-Mediated Gene Expression by Tomato (Solanum Lycopersicum L.) Extracts.” Journal of Agricultural and Food Chemistry 61 (14) (April 10): 3419–27. doi:10.1021/jf304790a.

Issemann, I., & Green, S. (1990). Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature, 347(6294), 645–650.

Kaya, T., Mohr, S. C., Waxman, D. J., & Vajda, S. (2006). Computational screening of phthalate monoesters for binding to PPARgamma. Chemical Research in Toxicology, 19(8), 999–1009. doi:10.1021/tx050301s

Lapinskas, P. J., Brown, S., Leesnitzer, L. M., Blanchard, S., Swanson, C., Cattley, R. C., & Corton, J. C. (2005). Role of PPARα in mediating the effects of phthalates and metabolites in the liver. Toxicology, 207(1), 149–163.

Le Maire, A., Grimaldi, M., Roecklin, D., Dagnino, S., Vivat-Hannah, V., Balaguer, P., & Bourguet, W. (2009). Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Reports, 10(4), 367–73. doi:10.1038/embor.2009.8

LeBlanc, G., Norris, D., & Kloas, W. (2011). Detailed Review Paper State of the Science on Novel In Vitro and In Vivo Screening and Testing Methods and Endpoints for Evaluating Endocrine Disruptors, (178).

Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., & Kliewer, S. A. (1995). An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor (PPAR ). Journal of Biological Chemistry, 270(22), 12953–12956. doi:10.1074/jbc.270.22.12953

Maloney, E. K., & Waxman, D. J. (1999). trans-Activation of PPARα and PPARγ by Structurally Diverse Environmental Chemicals. Toxicology and Applied Pharmacology, 161(2), 209–218.

Michalik, L., Zoete, V., Krey, G., Grosdidier, A., Gelman, L., Chodanowski, P., … Michielin, O. (2007). Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior. The Journal of Biological Chemistry, 282(13), 9666–77. doi:10.1074/jbc.M610523200

Morán-Salvador, E., López-Parra, M., García-Alonso, V., Titos, E., Martínez-Clemente, M., González-Périz, A., … Clària, J. (2011). Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts. FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 25(8), 2538–50. doi:10.1096/fj.10-173716

Pereira-Fernandes, A., Demaegdt, H., Vandermeiren, K., Hectors, T. L. M., Jorens, P. G., Blust, R., & Vanparys, C. (2013). Evaluation of a screening system for obesogenic compounds: screening of endocrine disrupting compounds and evaluation of the PPAR dependency of the effect. PloS One, 8(10), e77481. doi:10.1371/journal.pone.0077481

ToxCastTM Data, US Environmental Protection Agency. http://www.epa.gov/ncct/toxcast/data.html.

Vanden Heuvel, J. P. (1999). Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis. Toxicological Sciences : An Official Journal of the Society of Toxicology, 47(1), 1–8.

Viswakarma, N., Jia, Y., Bai, L., Vluggens, A., Borensztajn, J., Xu, J., & Reddy, J. K. (2010). Coactivators in PPAR-Regulated Gene Expression. PPAR Research, 2010. doi:10.1155/2010/250126

Wahli, W., & Desvergne, B. (1999). Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews, 20(5), 649–88.

Wu, B., Gao, J., & Wang, M. (2005). Development of a complex scintillation proximity assay for high-throughput screening of PPARgamma modulators. Acta Pharmacologica Sinica, 26(3), 339–44. doi:10.1111/j.1745-7254.2005.00040.x

Yu, S., & Reddy, J. K. (2007). Transcription coactivators for peroxisome proliferator-activated receptors. Biochimica et Biophysica Acta, 1771(8), 936–51. doi:10.1016/j.bbalip.2007.01.008