Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor alpha

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129mice were fed a control or WY-14,643-containing (1000ppm) diet for one week, five weeks or five months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at five months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparalpha-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of Pparalpha.     

Receptor interacting protein 140 regulates expression of uncoupling protein 1 in adipocytes through specific peroxisome proliferator activated receptor isoforms and estrogen-related receptor alpha

Expression of uncoupling protein 1 (Ucp1) mRNA is elevated in differentiated adipocytes derived from brown or white adipose tissue devoid of the nuclear receptor corepressor receptor interacting protein 140 (RIP140). Increased expression is mediated in part by the recruitment of peroxisome proliferator activated receptors alpha and gamma, together with estrogen-related receptor alpha, which functions through a novel binding site on the Ucp1 enhancer. This demonstrates that regulation of Ucp1 expression in the absence of RIP140 involves derepression of at least three different nuclear receptors. The ability to increase expression of Ucp1 by beta-adrenergic signaling is independent of RIP140, as shown by the action of the beta(3)-adrenergic agonist CL 316,243 to stimulate expression in both brown and white adipocytes in the presence and absence of the corepressor. Therefore, the expression of this metabolic uncoupling protein in adipose cells is regulated by inhibition as well as activation of distinct signaling pathways.     

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.     

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.     

CCR6 colocalizes with CD18 and enhances adhesion to activated endothelial cells in CCR6-transduced Jurkat T cells

CCR6 is expressed by memory T cells (mTC) and is a requirement for efficient arrest of a subset of mTC to activated human dermal microvascular endothelial cells (HDMEC) under physiologic shear stress. We now address whether CCR6 alone is sufficient to induce arrest of a model T cell line (Jurkat) that shows low expression of all CCRs tested (CCR1-10). Herein, we transduced Jurkat (JK) T cells expressing fucosyltransferase VII with a chimeric chemokine receptor consisting of CCR6 fused to enhanced green fluorescent protein. In contrast to the starting JK lines, the resulting cell line (JK fucosyltransferase VII-CCR6) migrated 6-fold better to CCL20 in chemotaxis assays, arrested in response to CCL20 that was immobilized to plastic, and demonstrated a 2.5-fold increase in adhesion to activated HDMEC (p = 0.001). Adhesion was blocked by anti-CD18 mAb (p = 0.005) but not by anti-CD49d mAb (p = 0.3). After arrest on recombinant substrates, CCR6 clustered on the surface as detected by real-time observation of enhanced green fluorescent protein fluorescence. Dual-label confocal microscopy revealed that LFA-1 (CD18 and CD11a), but not CXCR4, colocalized with clustered CCR6 in the presence of immobilized CCL20. Thus, the functional expression of CCR6 is sufficient to provide the chemokine signaling necessary to induce arrest of a JK T cell line to activated HDMEC. Clustering of CCR6 and coassociation with critical integrins may serve to strengthen adhesion between T cells and activated endothelial cells.     

Neuroprotective actions of noradrenaline: effects on glutathione synthesis and activation of peroxisome proliferator activated receptor delta

The endogenous neurotransmitter noradrenaline (NA) can protect neurons from the toxic consequences of various inflammatory stimuli, however the exact mechanisms of neuroprotection are not well known. In the current study, we examined neuroprotective effects of NA in primary cultures of rat cortical neurons. Exposure to oligomeric amyloid beta (Aβ) 1-42 peptide induced neuronal damage revealed by increased staining with fluorojade, and toxicity assessed by LDH release. Aβ-dependent neuronal death did not involve neuronal expression of the inducible nitric oxide synthase 2 (NOS2), since Aβ did not induce nitrite production from neurons, LDH release was not reduced by co-incubation with NOS2 inhibitors, and neurotoxicity was similar in wildtype and NOS2 deficient neurons. Co-incubation with NA partially reduced Aβ-induced neuronal LDH release, and completely abrogated the increase in fluorojade staining. Treatment of neurons with NA increased expression of γ-glutamylcysteine ligase, reduced levels of GSH peroxidase, and increased neuronal GSH levels. The neuroprotective effects of NA were partially blocked by co-treatment with an antagonist of peroxisome proliferator activated receptors (PPARs), and replicated by incubation with a selective PPARdelta (PPARδ) agonist. NA also increased expression and activation of PPARδ. Together these data demonstrate that NA can protect neurons from Aβ-induced damage, and suggest that its actions may involve activation of PPARδ and increases in GSH production.     

Expression of rat intestinal fatty acid-binding protein in Escherichia coli. Purification and comparison of ligand binding characteristics with that of Escherichia coli-derived rat liver fatty acid-binding protein

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant, 15,124-Da polypeptide found in the cytosol of small intestinal epithelial cells (enterocytes). It is homologous to rat liver fatty acid-binding protein (L-FABP), a 14,273-Da cytosolic protein which is found in enterocytes as well as hepatocytes. It is unclear why the small intestinal epithelium contains two abundant fatty acid-binding proteins. A systematic comparative analysis of the ligand binding characteristics of the two FABPs has not been reported. To undertake such a study we expressed the coding region of a full length I-FABP cDNA in Escherichia coli and purified large quantities of the protein. We also purified rat L-FABP from a similar, previously described expression system (Lowe, J. B., Strauss, A. W., and Gordon, J. I. (1984) J. Biol. Chem. 259, 12696-12704). Analysis of fatty acids associated with each of the homogeneous E. coli-derived FABPs suggested that the two proteins differed in their ligand binding specificity and capacity. All of the fatty acids associated with I-FABP were saturated while 30% of the E. coli fatty acids bound to L-FABP were unsaturated (16:1, 18:1, 18:2). We directly analyzed the ability of I- and L-FABP to bind fatty acids of different chain length and degree of saturation using a hydroxyalkoxypropyl dextran-based assay. Scatchard analysis revealed that each mole of L-FABP can bind up to 2 mol of long chain fatty acid while each mole of I-FABP can bind only 1 mole of fatty acid. L-FABP exhibited a relatively higher affinity for unsaturated fatty acids (oleate, arachidonate) than for saturated fatty acid (palmitate). By contrast, we were not able to detect a significant difference in the affinity of I-FABP for palmitate, oleate, and arachidonate. Neither protein exhibited any appreciable affinity for fatty acids whose chain length was less than C16. The observed differences in ligand affinities and capacities suggest that these proteins may have distinct roles in metabolism and/or compartmentalization of fatty acids within enterocytes.     

Noradrenaline induces expression of peroxisome proliferator activated receptor gamma (PPARgamma) in murine primary astrocytes and neurons

Cerebral inflammatory events play an important part in the pathogenesis of Alzheimer's disease (AD). Agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear hormone receptor that mediates anti-inflammatory actions of non-steroidal anti-inflammatory drugs (NSAIDs) and thiazolidinediones, have been therefore proposed as a potential treatment of AD. Experimental evidence suggests that cortical noradrenaline (NA) depletion due to degeneration of the locus ceruleus (LC) - a pathological hallmark of AD - plays a permissive role in the development of inflammation in AD. To study a possible relationship between NA depletion and PPARgamma-mediated suppression of inflammation we investigated the influence of NA on PPARgamma expression in murine primary cortical astrocytes and neurons. Incubation of astrocytes and neurons with 100 micro m NA resulted in an increase of PPARgamma mRNA as well as PPARgamma protein levels in both cell types. These effects were blocked by the beta-adrenergic antagonist propranolol but not by the alpha-adrenergic antagonist phentolamine, suggesting that they might be mediated by beta-adrenergic receptors. Our results indicate for the first time that PPARgamma expression can be modulated by the cAMP signalling pathway, and suggest that the anti-inflammatory effects of NA on brain cells may be partly mediated by increasing PPARgamma levels. Conversely, decreased NA due to LC cell death in AD may reduce endogenous PPARgamma expression and therefore potentiate neuroinflammatory processes.     

Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor beta

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. Their regulation by post-translational modifications has been reported but is poorly understood. In the present study, we investigated whether ligand binding affects the turnover and ubiquitination of the PPARbeta subtype (also known as PPARdelta). Our data show that the ubiquitination and degradation of PPARbeta is not significantly influenced by the synthetic agonist GW501516 under conditions of moderate PPARbeta expression. By contrast, the overexpression of PPARbeta dramatically enhanced its degradation concomitant with its polyubiquitination and the formation of high molecular mass complexes containing multiple, presumably oligomerized PPARbeta molecules that lacked stoichiometical amounts of the obligatory PPARbeta dimerization partner, retinoid X receptor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARbeta, were strongly inhibited by GW501516. Our findings suggest that PPARbeta is subject to complex post-translational regulatory mechanisms that partly may serve to safeguard the cell against deregulated PPARbeta expression. Furthermore, our data have important implications regarding the widespread use of overexpression systems to evaluate the function and regulation of PPARs.     

Docking and molecular dynamics simulations of peroxisome proliferator activated receptors interacting with pan agonist sodelglitazar

PPAR (peroxisome proliferator-activated receptor) pan agonists play a critical role in treating metabolic diseases, especially the Type-2 diabetes mellitus (T2DM). GlaxoSmithKline's sodelglitazar (GW677954) is one of the potent PPAR pan agonists, which is currently being investigated in Phase II clinical trials for the treatment of T2DM and its complications. The present study was aimed at investigation into the effect of sodelglitazar at the binding pockets of PPARs. The Schrodinger Suite program (2009) was used for the molecular docking, while the GROMACS program used for the molecular dynamics (MD) simulations. The results thus obtained showed that sodelglitazar being docked well in the active site of PPARs. It was revealed by the MD simulations that the structures of the receptors remained quite stable during the simulations and that the important AF-2 helix showed less flexibility after binding with sodelglitazar. Also, it was observed that sodelglitazar could periodically form hydrogen bonds with the AF-2 helix of PPARs to stabilize the AF-2 helix in an active conformation. Our findings have confirmed that GlaxoSmithKline's sodelglitazar can activate the PPARs, which is quite consistent with the previous biological studies.     

Free fatty acid transfer from rat liver fatty acid-binding protein to phospholipid vesicles. Effect of ligand and solution properties.

Fatty acid binding proteins (FABP) are a family of 14-15-kDa proteins found in many mammalian cell types in high abundance. Although their precise physiological role remains hypothetical, the transfer of free fatty acids (ffa) to intracellular membrane sites is believed to be an important function of FABP. To better understand the role of FABP in this process, we have examined how the rate of ffa transfer from liver FABP (L-FABP) to model membranes is influenced by variations in ffa structure and properties of the aqueous phase. The rate of transfer of fluorescent anthroyloxy ffa to model acceptor membranes was monitored using a resonance energy transfer assay. The results show that a monounsaturated ffa transfers 2-fold more rapidly than a saturated ffa of equivalent chain length, and a two-carbon increase in acyl chain length results in a 3-fold decrease in transfer rate. The transfer rate decreases logarithmically with increasing ionic strength, suggesting that the aqueous solubility of the ffa is an important determinant of its dissociation rate from L-FABP. Fatty acid binding and the relative partition of n-(9-anthroloxy) ffa to L-FABP as compared with phospholipid membranes both decrease as pH decreases, indicating that ionized but not protonated ffa bind to L-FABP. The rate of ffa transfer from L-FABP to membranes increases approximately 4-fold with increasing pH, suggesting that ionization of the ffa carboxyl group is also an important determinant of the transfer process. Analysis of the dependence of the transfer rate on temperature demonstrates that the delta G++ of the activated state for ffa transfer arises from both enthalpic and entropic processes. These studies demonstrate that the rate of transfer of long chain ffa from L-FABP to membranes is substantially affected by aqueous phase variables as well as properties of the ffa ligand itself.     

Immunohistochemical distribution of hepatic fatty acid-binding protein in rat and human alimentary tract

Tissues from rat and human alimentary tract were immunostained with rabbit antibodies to fatty acid-binding protein (FABP) isolated from rat liver, since the precise immunohistochemical localization of the protein in gut has not been determined. The results obtained indicated that FABP immunoreactivity was found almost exclusively in intestinal absorptive cells, the sole exception being its presence in the cytoplasm of a few goblet cells. In small bowel, FABP-positive cells were most often found in the upper and middle segments, and less frequently in the lower to terminal portion. Immunoreactive cells were also found in large bowel of rat and human, but with differing patterns of distribution. In rat, positive cells were found mainly in the lower portion of the large intestine, whereas in human positive cells were present in all portions. Immunoreactive cells were detected in rat and human cecum, in the upper half of human rectum, and in human vermiform appendix. No such cells were found in esophageal and nonmetaplastic gastric mucosa or in pancreatic tissue, whereas they were present in great numbers in metaplastic gastric mucosa. The results of this study therefore suggest that FABP is a useful marker for research into the physiology or pathology of absorptive cells in the gastrointestinal tracts of both species.