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Key Event Title
Decreased, PPARalpha transactivation of gene expression
|Level of Biological Organization|
Key Event Components
|receptor transactivation||peroxisome proliferator-activated receptor alpha||decreased|
Key Event Overview
AOPs Including This Key Event
|Not Otherwise Specified||Not Specified|
Key Event Description
PPARα acts as a nuclear signaling element that controls the transcription of a variety of genes involved in lipid catabolism and energy production pathways (Desvergne and Wahli 1999, Kersten 2014). Fatty acids serve as the natural ligands that stimulate PPARα nuclear signaling where the fatty acids (likely in association with fatty acid binding proteins) bind to the ligand binding domain of PPARα along with co-activators to the PPARα regulatory complex at promoter regions of PPARα-regulated genes (termed PPAR response elements, PPREs) initiating the transcription of genes that metabolize the fatty acids (Ahmed et al 2007, Wolfrum et al. 2001, Desvergne and Wahli 1999, Kersten 2014, Xu et al 2001, Janssen et al 2015).
Specifically, PPARα contains both a ligand-binding domain that binds fatty acids and a DNA-binding domain that initiates binding to PPREs in the promoter regions of PPARα-regulated genes (Ahmed et al 2007, Hihi et al 2002). Binding of the fatty acid ligands to PPARα facilitates heterodimeric binding with another ligand-activated nuclear receptor, the retinoid X receptor (RXR), forming an activated PPAR-RXR transcriptional regulator complex (DiRenzo et al 1997, Ahmed et al 2007). PPAR competes for binding to RXR with retinoic acid receptors (RARs) where the RAR/RXR heterodimer inhibits transcription of genes downstream of PPREs (DiRenzo et al 1997). Transcriptional regulation activity of the PPAR/RXR complex is also influenced by the binding and release of accessory molecules that act as coactivators such as steroid receptor co-activator 1 (SRC-1) or as corepressors such as nuclear receptor corepressor (N-CoR, DiRenzo et al 1997, Ahmed et al 2007, Xu et al 2002, Liu et al 2008). Such binding of the co-repressor N-CoR to the PPARα/RXRα complex has been demonstrated to inhibit transcriptional transactivation (Xu et al 2002, Liu et al 2008). The exact mechanisms by which the PPAR/RXR complex facilitate transcription are still not well understood. It has been observed that RXR contains a highly conserved motif at the C-terminal end of the ligand-binding domain known as activating function 2 (AF2) which undergoes conformational changes allowing interaction with coactivators / corepressors, the former of which is hypothesized to recruit the components of the transcriptional machinery necessary to transcribe the downstream gene (DiRenzo et al 1997). Even the basal transcriptional machinery itself is recognized to vary across cell types and the prototypical preinitiation complex (PIC) is inherently highly flexible, confirmationally diverse including multi-faceted interactions of activators, core promotion factors, the RNA polymerase II enzyme, elongation factors, and chromatin remodeling complexes all combined at the promoter to facilitate gene transcription (Levine et al 2014). Additionally, recent transcriptomic research coupling transcriptomic expression and chromatin immunoprecipitation (ChIP) sequencing to identify PPARα binding to PPREs suggests PPARα may exert transcriptional regulation beyond its direct genomic targets via secondary signaling networks including various kinases (McMullen et al 2014). Given the current KE (KE2, PPARalpha transactivation of gene expression, Decreased), a variety of upstream influences may impair the function of the PPARα-RXRα heterodimer and/or affect coactivator / corepressor binding leading to decreased PIC competence resulting in impaired transcription of downstream genes as well as secondary signaling networks.
PPARα regulates expression of genes encoding nearly every enzymatic step of fatty acid catabolism including fatty acid uptake into cells, fatty acid activation to acyl-CoAs, and the release of cellular energy from fatty acids through the oxidative breakdown of acyl-CoAs to acetyl-CoA, and in starvation conditions, the repackaging of Acetyl-CoA substrates into ketone bodies via ketogenesis pathways (Kersten 2014, Desvergne and Wahli 1999, Evans et al 2004). A pathway-level schematic for PPARα transactivation is illustrated in KEGG Pathway map03320 providing the specific gene targets and associated functional responses that are transcriptionally regulated by PPARα. It should be noted that there are species-specific differences in PPARα transactivation of gene expression among mice and humans which are explained in the “Evidence Supporting Taxonomic Applicability” section, below.
How It Is Measured or Detected
X-ray crystallography was used to describe the ligand binding domain (fatty acid binding domain) of PPARα and demonstrate the binding complex of PPARα, the artificial ligand (GW6471), and the co-repressor silencing mediator for retinoid and thyroid hormone receptors (SMRT, Xu et al 2001, 2002). Fold activation of the PPARα-GW6471-SMRT transcriptional regulatory complex was measured in mammalian two-hybrid assays (Xu et al 2001). PPAR-RXR and RAR-RXR heterodimerization and activity were quantified using expression vectors for murine PPARα, RAR and RXR in CV-1 cells transfected with 1ug of reporter plasmid and 50-200ng of expression plasmid (DiRenzo et al 1997). DNA-dependent radioligand binding assays were conducted to quantify ligand binding in the assays described in DiRenzo et al (1997). X-ray crystallography has been used with limited success to describe the PIC while high-resolution electron microscopy is providing additional insights into the fully functionalized PIC when bound to the promotor and including all accessory molecules (Levine et al 2014). Effects of PPARα transactivation on expression of downstream genes been examined by a variety of methods, especially RT-qPCR (Kersten et al 2014). As a recommendation for investigating specific genes regulated by PPARα, as part of this KE, see the KEGG pathway for PPAR Signaling (map03320). In McMullen et al (2014), transcriptomic expression was investigated in human primary hepatocytes in time and dose series exposures to the PPARα agonist GW7647 where transcriptomic expression was measured using Affimetrix microarrays and ChIPseq was conducted after completing immunoprecipitation and quantified using Illumina HiSeq 2000 sequencing to find binding to PPRE within 50K bp of differentially expressed genes.
Domain of Applicability
Aspects of PPARα signaling have been observed to differ comparing human and rodent responses as described for Mus musculus (Kersten 2014) and Homo sapiens in clinical observations (Kersten 2014) and in in vitro assays (reviewed in Kersten 2014). Microarray-based comparative transcriptomic expression among mouse and human primary hepatocyte samples exposed to the PPARα agonist Wy14643 showed minor overlap in individual gene-level expression, however substantial overlap was observed at the pathway level (Rakshanderhroo et al 2009). In that study, most of the genes that were differentially expressed in common among human and mouse were involved in lipid metabolism, including CPT1A, HMGCS2, FABP1, ACSL1 and ADFP. Thus, in Rakshanderhroo et al (2009), evaluation of PPARα transactivation differences among human and mouse suggest expression for gene-transcripts involved in lipid metabolism tended to be the most conserved among species. (IMPORTANT NOTE: The results from Rakshanderhroo et al (2009) should be viewed with caution given that primary hepatocytes were obtained from 1 mouse per strain, only.) Feige et al (2010) found that mice with humanized PPARα were insensitive to the PPARα agonist pollutant, diethylhexyl phthalate (DEHP), where mRNA expression (measured by reverse transcriptase-qPCR) for genes involved in fatty-acid metabolism were not induced relative to wild type mice. (IMPORTANT NOTE: the results from Fiege et al (2010) show that DEHP is a weak partial agonist of PPARα. Additionally, the use of wild type versus mice with humanized PPARα for extrapolating species-to-species differences should be viewed with caution. Humanized receptor is not likely to interact with the same cofactors in mice relative to humans and the regulatory grammars may differ between among species that may further complicate the biochemistry.)
Evidence for Perturbation by Stressor
Ahmed, W., Ziouzenkova, O., Brown, J., Devchand, P., Francis, S., Kadakia, M., Kanda, T., Orasanu, G., Sharlach, M., Zandbergen, F., Plutzky, J., 2007. PPARs and their metabolic modulation: new mechanisms for transcriptional regulation? J. Intern. Med. 262, 184-198.
Desvergne B, Wahli W (1999) Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 20(5): 649-688.
DiRenzo, J., Soderstrom, M., Kurokawa, R., Ogliastro, M.H., Ricote, M., Ingrey, S., Horlein, A., Rosenfeld, M.G., Glass, C.K., 1997. Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors. Mol. Cell. Biol. 17, 2166-2176.
Evans RM, Barish GD, Wang YX: PPARs and the complex journey to obesity. Nat Med 2004, 10(4):355-361.
Feige, J.N., Gerber, A., Casals-Casas, C., Yang, Q., Winkler, C., Bedu, E., Bueno, M., Gelman, L., Auwerx, J., Gonzalez, F.J., Desvergne, B., 2010. The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms. Environ. Health Perspect. 118, 234-241.
Hihi, A.K., Michalik, L., Wahli, W., 2002. PPARs: transcriptional effectors of fatty acids and their derivatives. Cell Molec Life Sci 59, 790-798.
Janssen, A.W., Betzel, B., Stoopen, G., Berends, F.J., Janssen, I.M., Peijnenburg, A.A., Kersten, S., 2015. The impact of PPARalpha activation on whole genome gene expression in human precision cut liver slices. BMC Genomics 16, 760.
Kersten S. 2014. Integrated physiology and systems biology of PPARalpha. Molecular Metabolism 2014, 3(4):354-371.
Levine, M., Cattoglio, C., Tjian, R., 2014. Looping Back to Leap Forward: Transcription Enters a New Era. Cell 157, 13-25.
Liu, M.H., Li, J., Shen, P., Husna, B., Tai, E.S., Yong, E.L., 2008. A natural polymorphism in peroxisome proliferator-activated receptor-alpha hinge region attenuates transcription due to defective release of nuclear receptor corepressor from chromatin. Mol. Endocrinol. 22, 1078-1092.
McMullen, P.D., Bhattacharya, S., Woods, C.G., Sun, B., Yarborough, K., Ross, S.M., Miller, M.E., McBride, M.T., LeCluyse, E.L., Clewell, R.A., Andersen, M.E., 2014. A map of the PPARalpha transcription regulatory network for primary human hepatocytes. Chem. Biol. Interact. 209, 14-24.
Rakhshandehroo, M., Hooiveld, G., Muller, M., Kersten, S., 2009. Comparative analysis of gene regulation by the transcription factor PPARalpha between mouse and human. PLoS One 4, e6796.
Wolfrum C, Borrmann CM, Borchers T, Spener F (2001) Fatty acids and hypolipidemic drugs regulate peroxisome proliferator-activated receptors alpha - and gamma-mediated gene expression via liver fatty acid binding protein: a signaling path to the nucleus. Proc Natl Acad Sci USA 98(5):2323-2328.
Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gampe RT, McKee DD et al: Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proceedings of the National Academy of Sciences 2001, 98(24):13919-13924.
Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT et al: Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR[alpha]. Nature 2002, 415(6873):813-817.