API

Event: 228

Key Event Title

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peroxisome proliferator activated receptor promoter demethylation

Short name

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demethylation, PPARg promoter

Key Event Component

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

Key Event Overview


AOPs Including This Key Event

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Stressors

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Level of Biological Organization

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Biological Organization
Molecular

Cell term

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Cell term
hepatocyte


Organ term

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Taxonomic Applicability

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Term Scientific Term Evidence Link
mice Mus sp. Strong NCBI
human Homo sapiens Moderate NCBI
rat Rattus norvegicus Moderate NCBI

Life Stages

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Sex Applicability

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How This Key Event Works

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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

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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.


Evidence Supporting Taxonomic Applicability

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


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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1.1 Binding and activation of receptor

PPARγ ligands thiazolidinediones (Rosiglitazone, Pioglitazone, Troglitazone) (Lehmann et al. 1995), (Forman et al. 1995), (Willson et al. 2000).

Phthalates

MEHP (CAS 4376-20-9) directly binds to PPARγ (Lapinskas et al. 2005), (ToxCastTM Data)in vitro and in silico (Feige et al. 2007), (Rotman et al. 2008), (Kaya et al. 2006) and activates this receptor in transactivation assays (Maloney & Waxman 1999), (Hurst & Waxman 2003b), (Venkata et al. 2006), (ToxCastTM Data). In summary, there is experimental in vitro evidence for binding and transcriptional activation of PPARγ. DEHP (CAS 117-81-7) was not found to bind and activate PPARγ (Lapinskas et al. 2005), (Maloney & Waxman 1999). However recent studies show activation of PPARγ by DEHP(ToxCastTM Data), (Pereira-Fernandes et al. 2013). DEHP was also found to increase the levels of PPARγ in vitro (Lin et al. 2011). Notably, PPARγ is responsive to DEHP in vitro and is translocated to the nucleus (in primary Sertoli cells) (Dufour et al. 2003), (Bhattacharya et al. 2005).

Parabens

Butylparaben was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) and mPPARγ in reporter gene assay (Taxvig et al., 2012).

Phenols

Bisphenol A was not found to bind to the PPARγ (ToxCastTM Data), but activated the human PPARγ (ToxCastTM Data), (Pereira-Fernandes et al. 2013) but not mouse PPARγ (Taxvig et al., 2012) in reporter gene assay. BPA was also reported to increase PPARγ (mRNA) in ovarian granulosa cell line and human luteinized granulosa cells (Kwintkiewicz, Nishi, Yanase, & Giudice, 2010).

Organotin

Tributyltin (TBT) activates all three heterodimers of PPAR with RXR, primarily through its interaction with RXR (le Maire et al. 2009).


1.2 Activation of target genes

MEHP activation of endogenous PPARγ target genes was evidenced by the stimulation of PPARγ-dependent adipogenesis in the 3T3-L1 cell differentiation model (Hurst & Waxman, 2003).



References

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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