Identification of novel PPARγ target genes by integrated analysis of ChIP-on-chip and microarray expression data during adipocyte differentiation

PPARγ (peroxisome proliferator-activated receptor gamma) acts as a key molecule of adipocyte differentiation, and transactivates multiple target genes involved in lipid metabolic pathways. Identification of PPARγ target genes will facilitate to predict the extent to which the drugs can affect and also to understand the molecular basis of lipid metabolism. Here, we have identified five target genes regulated directly by PPARγ during adipocyte differentiation in 3T3-L1 cells using integrated analyses of ChIP-on-chip and expression microarray. We have confirmed the direct PPARγ regulation of five genes by luciferase reporter assay in NIH-3T3 cells. Of these five genes Hp, Tmem143 and 1100001G20Rik are novel PPARγ targets. We have also detected PPREs (PPAR response elements) sequences in the promoter region of the five genes computationally. Unexpectedly, most of the PPREs detected proved to be atypical, suggesting the existence of more atypical PPREs than previously thought in the promoter region of PPARγ regulated genes.     

Protein expression changes in the Sprague Dawley rat liver proteome following administration of peroxisome proliferator activated receptor alpha and gamma ligands

Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily and are intimately involved in lipid metabolism and energy homeostasis. Activation of these receptors in rodents can lead to hepatomegaly and ultimately hepatic carcinogenesis although the mechanisms by which these processes occur are poorly understood. To further our understanding of these processes and to discriminate between different PPAR mediated signalling pathways, a proteomic approach has been undertaken to identify changes in protein expression patterns in Sprague Dawley rat liver following dosing with a PPARalpha agonist (Wyeth 14643), a PPARgamma agonist (Troglitazone) and a compound with mixed PPARalpha/gamma agonist activity (SB-219994). Using one-and-two-dimensional electrophoresis of tissue lysates a diverse range of protein abundance changes was observed in these tissues. Whilst a number of these proteins have PPAR response elements (PPREs) in their respective promoters, another group was detected whose expression has been documented to be sensitive to peroxisome proliferator administration. Most notably within these groups, proteins involved in lipid catabolism displayed increased expression following drug administration. A further subset of proteins, with less obvious biological implications, also showed altered expression patterns. Where available, sequences upstream of the coding regions of genes not previously known to have PPREs were searched with positional consensus matrices for the presence of PPREs in an attempt to validate these changes. Using such an approach putative PPARgamma and PPARdelta response elements were discovered upstream of the tubulin beta coding region. There was limited overlap in observed protein abundance changes between the three groups, and where this was the case (cytosolic epoxide hydrolase, peroxisomal bifunctional enzyme, hydroxymethyl glutaryl CoA, synthase, long chain acyl-CoA thioesterase), expression of these proteins had previously been shown to be under the control of PPAR activity.     

The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine

In this study, we found that the mRNA level of peroxisome proliferator-activated receptor (PPAR) alpha, but not of PPARdelta, was elevated in the jejunum during the postnatal development of the rat. Moreover, we found that the expressions of PPAR-dependent genes, such as acyl-CoA oxidase, L-FABP, and I-FABP, were also increased during the postnatal development of the small intestine. Electrophoretic mobility shift assay revealed that both the PPARalpha-9-cis-retinoic acid receptor alpha (RXRalpha) heterodimer and the PPARdelta-RXRalpha heterodimer bound to the peroxisome proliferator response element (PPRE) of acyl-CoA oxidase and L-FABP genes. The binding of the PPARalpha-RXRalpha heterodimer to the PPREs of the various genes was enhanced by the addition of PPARalpha, with a concomitant reduction of the binding of PPARdelta-RXRalpha to the PPREs. Furthermore, the binding activity of PPARalpha-RXRalpha, but not PPARdelta-RXRalpha, to the PPREs was enhanced by the addition of a PPAR ligand, WY14,643. The GAL4-PPAR-chimera reporter assay showed that WY14,643 transactivated the reporter gene through action of PPARalpha, but not through PPARdelta, in Caco-2 cells. Furthermore, oral administration of a PPAR ligand, clofibrate, during 3 consecutive days of the weanling period caused a parallel increase in the mRNA levels of these PPAR-dependent genes. These results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.     

Sesquiterpene lactones from Tithonia diversifolia act as peroxisome proliferator-activated receptor agonists

Tithonia diversifolia is a well-known traditional Chinese medicine treating diabetes, hepatitis, and hepatocarcinoma but its molecular mechanism is not fully understood. Peroxisome proliferator-activated receptors (PPARs) α and γ are members of nuclear receptor superfamily. Their agonists are prescribed as antihyperlipidemic and antihyperglycemic drugs now. In this study, sesquiterpene lactones, tirotundin and tagitinin A, were isolated from T. diversifolia and evaluated for their activity against PPARs by the transient transfection reporter assay. Tirotundin and tagitinin A transactivated PPARγ dependent promoters including PPRE (PPARγ response element), SHP, and ABCA1 gene promoters in dose-dependent manner. Furthermore, the fluorescence polarization competitive binding assay showed that tirotundin (IC(50)=27 μM) and tagitinin A (IC(50)=55 μM) enhanced PPARγ transactivation activity by directly binding to PPARγ ligand binding domain. Additionally, they stimulated the transactivation of PPARα dependent SULT2A1 gene promoter by 2.3-fold of vehicle effect at 10 μM. These results highly indicated that tirotundin and tagitinin A are the active components of T. diversifolia to exert anti-diabetic effect through PPARγ pathway. Moreover, these sesquiterpene lactones behaved as PPARα/γ dual agonists so they might be useful as the potential herbal treatment for diabetes.     

Docosahexaenoic acid suppresses the activity of peroxisome proliferator-activated receptors in a colon tumor cell line

Fatty acids are generally considered as agonists for peroxisome proliferator-activated receptors (PPARs). Fatty acids have been shown to bind to and transactivate PPARs; it is not known whether fatty acids act as generalized agonists for PPARs in different cell types, and thus, stimulate the expression of PPAR-regulated target genes. Here, we investigated the potency of unsaturated fatty acids on transactivation of PPRE, DNA-binding activity of PPARs, and the expression of a PPAR-regulated gene product, CD36. Docosahexaenoic acid (DHA) suppressed the basal and PPAR agonist-induced transactivation of PPRE, and DNA binding of PPARs in colon tumor cells (HCT116). The suppression of PPAR transactivation by DHA leads to reduced expression of CD36 in HCT116 cells and human monocytic cells (THP-1) as determined by promoter reporter gene assay and flow cytometric analysis. Our results demonstrate that DHA and other unsaturated fatty acids act as antagonists instead of agonists for transactivation of PPRE and PPAR-regulated gene expression in the cell lines tested. These results suggest that PPAR-mediated gene expression and cellular responses can be dynamically modulated by different types of dietary fatty acids consumed.