Activation of PPAR delta Regulates Ywhae (Encoding 14-3-3 epsilon Protein), a Gene Required for Ventricular Morphogenesis

Cardiac ventricular morphogenesis is a key developmental stage during which the ventricles grow considerably in size, but the transcriptional pathways controlling this process remains poorly understood. 14-3-3_ is a member of a conserved protein family that regulates a wide range of processes such as transcription, apoptosis and proliferation by binding to the phospho-serine/threonine residues of its target proteins. We found that deletion of the Ywhae gene (encoding 14-3-3_) in mice leads to abnormal ventricular morphogenesis and an embryonic cardiomyopathy (Cieslik KA et al, Circ. Res. 2008, abstract). Interestingly, we recently showed in cultured cells that the Ywhae gene is regulated directly by peroxisome proliferator-activated receptor _ (PPAR_) (Brunelli L et al, Circ. Res. 2007), a ligand-inducible nuclear receptor that controls energy metabolism and development. Postnatal cardiac-specific deletion of the Ppard gene in mice causes a lethal dilated cardiomyopathy, but it is still unknown whether PPAR_ regulates genes involved in heart development. We hypothesized that the expression of the Ywhae gene is responsive to PPAR_ during heart development. We confirmed that PPAR_ is expressed in the heart during development, and found higher expression at E10.5 compared to later gestational ages. We showed by immunofluorescence that a PPAR_ agonist (50 _M L-165,041 for 24 hr) upregulates 14-3-3_ in primary cardiomyocytes. We showed that when P19CL6 cells are driven towards cardiomyocyte lineage by dimethyl sulfoxide (DMSO), 14-3-3_ levels increase 4-fold, while L-165,041 treatment increases levels by an additional 50%. Based on previous work in mice (Leibowitz MD et al, FEBS Lett. 2000; Letavernier E et al, J. Am. Soc. Nephrol. 2005), we tested the response of Ywhae to PPAR_ in vivo . We fed 30 mg/kg/day L-165,041 to 14-3-3__/_ adult pregnant mice for 3 days starting at E9.5 and assessed Ywhae mRNA levels in embryonic hearts at E12.5. Baseline mRNA levels in Ywhae_/_ hearts were double that of Ywhae_/ hearts, while L-165,041 upregulated Ywhae mRNA levels in both Ywhae_/_ and Ywhae_/ hearts by 65%. These results indicate that Ywhae responds to PPAR_ in vivo, and suggest that PPAR_ regulates Ywhae during ventricular morphogenesis.

Advanced QSAR Studies on PPAR delta Ligands Related to Metabolic Diseases

PPAR delta is a nuclear receptor that, when activated, regulates the metabolism of carbohydrates and lipids and is related to metabolic syndrome and type 2 diabetes. To understand the main interactions between ligands and PPAR delta, we have constructed 2D and 3D QSAR models and compared them with HOMO, LUMO and electrostatic potential maps of the compounds studied, as well as docking results. All QSAR models showed good statistical parameters and prediction outcomes. The QSAR models were used to predict the biological activity of an external test set, and the predicted values are in good agreement with the experimental results. Furthermore, we employed all maps to evaluate the possible interactions between the ligands and PPAR delta. These predictive QSAR models, along with the HOMO, LUMO and MEP maps, can provide insights into the structural and chemical properties that are needed in the design of new PPAR delta ligands that have improved biological activity and can be employed to treat metabolic diseases.

Ligand- and Structure-Based Drug Design Strategies and PPAR delta/alpha Selectivity

Peroxisome-proliferator-activated receptors are a class of nuclear receptors with three subtypes: a, ? and d. Their main function is regulating gene transcription related to lipid and carbohydrate metabolism. Currently, there are no peroxisome-proliferator-activated receptors d drugs being marketed. In this work, we studied a data set of 70 compounds with a and d activity. Three partial least square models were created, and molecular docking studies were performed to understand the main reasons for peroxisome-proliferator-activated receptors d selectivity. The obtained results showed that some molecular descriptors (log P, hydration energy, steric and polar properties) are related to the main interactions that can direct ligands to a particular peroxisome-proliferator-activated receptors subtype.

Structural Insights into Human Peroxisome Proliferator Activated Receptor Delta (PPAR-Delta) Selective Ligand Binding

Peroxisome proliferator activated receptors (PPARs delta, alpha and gamma) are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPAR delta more than 300-fold more tightly than PPAR alpha or PPAR gamma but the structural basis of PPAR delta: GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPAR delta:GW0742 complex. Comparisons of the PPAR delta:GW0742 complex with published structures of PPARs in complex with alpha and gamma selective agonists and pan agonists suggests that two residues (Val312 and Ile328) in the buried hormone binding pocket play special roles in PPAR delta selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPAR alpha and gamma ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPAR delta ligand design.

Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics.

Advances in wound care are of great importance in clinical injury management. In this respect, the nuclear receptor peroxisome proliferator-activated receptor (PPAR)beta/delta occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPARbeta/delta expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-beta1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPARbeta/delta accelerate wound closure. The results reveal a dual role of TGF-beta1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPARbeta/delta expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-beta1 at wound sites.

Transcriptional profiling reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse liver.

Little is known about the role of the transcription factor peroxisome proliferator-activated receptor (PPAR) beta/delta in liver. Here we set out to better elucidate the function of PPARbeta/delta in liver by comparing the effect of PPARalpha and PPARbeta/delta deletion using whole genome transcriptional profiling and analysis of plasma and liver metabolites. In fed state, the number of genes altered by PPARalpha and PPARbeta/delta deletion was similar, whereas in fasted state the effect of PPARalpha deletion was much more pronounced, consistent with the pattern of gene expression of PPARalpha and PPARbeta/delta. Minor overlap was found between PPARalpha- and PPARbeta/delta-dependent gene regulation in liver. Pathways upregulated by PPARbeta/delta deletion were connected to innate immunity and inflammation. Pathways downregulated by PPARbeta/delta deletion included lipoprotein metabolism and various pathways related to glucose utilization, which correlated with elevated plasma glucose and triglycerides and reduced plasma cholesterol in PPARbeta/delta-/- mice. Downregulated genes that may underlie these metabolic alterations included Pklr, Fbp1, Apoa4, Vldlr, Lipg, and Pcsk9, which may represent novel PPARbeta/delta target genes. In contrast to PPARalpha-/- mice, no changes in plasma free fatty acid, plasma beta-hydroxybutyrate, liver triglycerides, and liver glycogen were observed in PPARbeta/delta-/- mice. Our data indicate that PPARbeta/delta governs glucose utilization and lipoprotein metabolism and has an important anti-inflammatory role in liver. Overall, our analysis reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse liver.

Activation of peroxisome proliferator-activated receptor-β/-δ (PPAR-β/-δ) ameliorates insulin signaling and reduces SOCS3 levels by inhibiting STAT3 in interleukin-6-stimulated adipocytes.

OBJECTIVE It has been suggested that interleukin (IL)-6 is one of the mediators linking obesity-derived chronic inflammation with insulin resistance through activation of STAT3, with subsequent upregulation of suppressor of cytokine signaling 3 (SOCS3). We evaluated whether peroxisome proliferator-activated receptor (PPAR)-β/-δ prevented activation of the IL-6-STAT3-SOCS3 pathway and insulin resistance in adipocytes. RESEARCH DESIGN AND METHODS First, we observed that the PPAR-β/-δ agonist GW501516 prevented both IL-6-dependent reduction in insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. In addition, this drug treatment abolished IL-6-induced SOCS3 expression in differentiated 3T3-L1 adipocytes. This effect was associated with the capacity of the drug to prevent IL-6-induced STAT3 phosphorylation on Tyr(705) and Ser(727) residues in vitro and in vivo. Moreover, GW501516 prevented IL-6-dependent induction of extracellular signal-related kinase (ERK)1/2, a serine-threonine-protein kinase involved in serine STAT3 phosphorylation. Furthermore, in white adipose tissue from PPAR-β/-δ-null mice, STAT3 phosphorylation (Tyr(705) and Ser(727)), STAT3 DNA-binding activity, and SOCS3 protein levels were higher than in wild-type mice. Several steps in STAT3 activation require its association with heat shock protein 90 (Hsp90), which was prevented by GW501516 as revealed in immunoprecipitation studies. Consistent with this finding, the STAT3-Hsp90 association was enhanced in white adipose tissue from PPAR-β/-δ-null mice compared with wild-type mice. CONCLUSIONS Collectively, our findings indicate that PPAR-β/-δ activation prevents IL-6-induced STAT3 activation by inhibiting ERK1/2 and preventing the STAT3-Hsp90 association, an effect that may contribute to the prevention of cytokine-induced insulin resistance in adipocytes.

Peroxisome proliferator-activated receptor beta/delta exerts a strong protection from ischemic acute renal failure.

Ischemic acute renal failure is characterized by damages to the proximal straight tubule in the outer medulla. Lesions include loss of polarity, shedding into the tubule lumen, and eventually necrotic or apoptotic death of epithelial cells. It was recently shown that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases keratinocyte survival after an inflammatory reaction. Therefore, whether PPARbeta/delta could contribute also to the control of tubular epithelium death after renal ischemia/reperfusion was tested. It was found that PPARbeta/delta+/- and PPARbeta/delta-/- mutant mice exhibited much greater kidney dysfunction and injury than wild-type counterparts after a 30-min renal ischemia followed by a 36-h reperfusion. Conversely, wild-type mice that were given the specific PPARbeta/delta ligand L-165041 before renal ischemia were completely protected against renal dysfunction, as indicated by the lack of rise in serum creatinine and fractional excretion of Na+. This protective effect was accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation. On the basis of in vitro studies, PPARbeta/delta ligands seem to exert their role by activating the antiapoptotic Akt signaling pathway and, unexpectedly, by increasing the spreading of tubular epithelial cells, thus limiting potentially their shedding and anoikis. These results point to PPARbeta/delta as a remarkable new target for preconditioning strategies.

Crosstalk between peroxisome proliferator-activated receptor delta and VEGF stimulates cancer progression.

Peroxisome proliferator-activated receptor (PPAR) delta is a member of the nuclear hormone receptor superfamily. PPARdelta may ameliorate metabolic diseases such as obesity and diabetes. However, PPARdelta's role in colorectal carcinogenesis remains controversial. Here, we present genetic and pharmacologic evidence demonstrating that deletion of PPARdelta decreases intestinal adenoma growth in Apc(Min/+) mice and inhibits tumor-promoting effects of a PPARdelta agonist GW501516. More importantly, we found that activation of PPARdelta up-regulated VEGF in colon carcinoma cells. VEGF directly promotes colon tumor epithelial cell survival through activation of PI3K-Akt signaling. These results not only highlight concerns about the use of PPARdelta agonists for treatment of metabolic disorders in patients who are at high risk for colorectal cancer, but also support the rationale for developing PPARdelta antagonists for prevention and/or treatment of cancer.

Peroxisome proliferator-activated receptor beta/delta as a therapeutic target for metabolic diseases.

The peroxisome proliferator-activated receptor (PPAR) family comprises three distinct isotypes: PPARalpha, PPARbeta/delta and PPARgamma. PPARs are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. Until recently, the characterisation of the important role of PPARalpha in fatty acid oxidation and of PPARgamma in lipid storage contrasted with the sparse information concerning PPARbeta/delta. However, evidence is now emerging for a role of PPARbeta/delta in tissue repair and energy homeostasis. Experiments with tissue-specific overexpression of PPARbeta/delta or treatment of mice with selective PPARbeta/delta agonists demonstrated that activation of PPARbeta/delta in vivo increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. PPARbeta/delta activation also prevents the development of obesity and improves cholesterol homeostasis in obesity-prone mouse models. These new insights into PPARbeta/delta functions suggest that targeting PPARbeta/delta may be helpful for treating disorders associated with the metabolic syndrome. Although these perspectives are promising, several independent and contradictory reports raise concerns about the safety of PPARbeta/delta ligands with respect to tumourigenic activity in the gut. Thus, it appears that further exploration of PPARbeta/delta functions is necessary to better define its potential as a therapeutic target.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

The PPARbeta/delta agonist GW0742 relaxes pulmonary vessels and limits right heart hypertrophy in rats with hypoxia-induced pulmonary hypertension.

BACKGROUND: Pulmonary vascular diseases are increasingly recognised as important clinical conditions. Pulmonary hypertension associated with a range of aetiologies is difficult to treat and associated with progressive morbidity and mortality. Current therapies for pulmonary hypertension include phosphodiesterase type 5 inhibitors, endothelin receptor antagonists, or prostacyclin mimetics. However, none of these provide a cure and the clinical benefits of these drugs individually decline over time. There is, therefore, an urgent need to identify new treatment strategies for pulmonary hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the PPARbeta/delta agonist GW0742 induces vasorelaxation in systemic and pulmonary vessels. Using tissue from genetically modified mice, we show that the dilator effects of GW0742 are independent of the target receptor PPARbeta/delta or cell surface prostacyclin (IP) receptors. In aortic tissue, vascular relaxant effects of GW0742 were not associated with increases in cGMP, cAMP or hyperpolarisation, but were attributed to inhibition of RhoA activity. In a rat model of hypoxia-induced pulmonary hypertension, daily oral dosing of animals with GW0742 (30 mg/kg) for 3 weeks significantly reduced the associated right heart hypertrophy and right ventricular systolic pressure. GW0742 had no effect on vascular remodelling induced by hypoxia in this model. CONCLUSIONS/SIGNIFICANCE: These observations are the first to show a therapeutic benefit of 'PPARbeta/delta' agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.

Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.

The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration.

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARbeta/delta-/- mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations.