Induction of cardiac Angptl4 by dietary fatty acids is mediated by peroxisome proliferator-activated receptor beta/delta and protects against fatty acid-induced oxidative stress.

RATIONALE: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart. OBJECTIVE: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences. METHODS AND RESULTS: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding. CONCLUSIONS: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Endothelial, but not smooth muscle, peroxisome proliferator-activated receptor β/δ regulates vascular permeability and anaphylaxis.

BACKGROUND: Remodeling of quiescent vessels with increases in permeability, vasodilatation, and edema are hallmarks of inflammatory disorders. Factors involved in this type of remodeling represent potential therapeutic targets. OBJECTIVES: We investigated whether the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) β/δ, a regulator of metabolism, fibrosis, and skin homeostasis, is involved in regulation of this type of remodeling. METHODS: Wild-type and various Pparb/d mutant mice were used to monitor dermal acute vascular hyperpermeability (AVH) and passive systemic anaphylaxis-induced hypothermia and edema. PPARβ/δ-dependent kinase activation and remodeling of endothelial cell-cell junctions were addressed by using human endothelial cells. RESULTS: AVH and dilatation of dermal microvessels stimulated by vascular endothelial growth factor A, histamine, and thrombin are severely compromised in PPARβ/δ-deficient mice. Selective deletion of the Pparb/d-encoding gene in endothelial cells in vivo similarly limits dermal AVH and vasodilatation, providing evidence that endothelial PPARβ/δ is the major player in regulating acute dermal microvessel remodeling. Furthermore, endothelial PPARβ/δ regulatory functions are not restricted to the skin vasculature because its deletion in the endothelium, but not in smooth muscle cells, also leads to reduced systemic anaphylaxis, the most severe form of allergic reaction, in which an acute vascular response plays a key role. PPARβ/δ-dependent AVH activation likely involves the activation of mitogen-activated protein kinase and Akt pathways and leads to downstream destabilization of endothelial cell-cell junctions. CONCLUSION: These results unveil not only a novel function of PPARβ/δ as a direct regulator of acute vessel permeability and dilatation but also provide evidence that antagonizing PPARβ/δ represents an important strategy to consider for moderating diseases with altered endothelial integrity, such as acute inflammatory and allergic disorders.

Nuclear receptor peroxisome proliferator activated receptor (PPAR) beta/delta in skin wound healing and cancer

We review the functions of peroxisome proliferator activated receptor (PPAR) beta/delta in skin wound healing and cancer. In particular, we highlight the roles of PPAR beta/delta in inhibiting keratinocyte apoptosis at wound edges via activation of the PI3K/PKB alpha/Akt1 pathway and its role during re-epithelialization in regulating keratinocyte adhesion and migration. In fibroblasts, PPAR beta/delta controls IL-1 signalling and thereby contributes to the homeostatic control of keratinocyte proliferation. We discuss its therapeutic potential for treating diabetic wounds and inflammatory skin diseases such as psoriasis and acne vulgaris. PPAR beta/delta is classified as a tumour growth modifier; it is activated by chronic low-grade inflammation, which promotes the production of lipids that, in turn, enhance PPAR beta/delta transcription activity. Our earlier,work unveiled a cascade of events triggered by PPAR beta/delta that involve the oncogene Src, which promotes ultraviolet-induced skin cancer in mice via enhanced EGFR/Erk1/2 signalling and the expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, PPAR beta/delta expression is correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma. Furthermore, there is a positive interaction between PPAR beta/delta, SRC, and TGF beta 1 at the transcriptional level in various human epithelial cancers. Taken together, these observations suggest the need for evaluating PPAR beta/delta modulators that attenuate or increase its activity, depending on the therapeutic target.

Developmental and tissue-specific involvement of peroxisome proliferator-activated receptor-alpha in the control of mouse uncoupling protein-3 gene expression.

Uncoupling protein-3 (UCP3) is a member of the mitochondrial carrier family expressed preferentially in skeletal muscle and heart. It appears to be involved in metabolic handling of fatty acids in a way that minimizes excessive production of reactive oxygen species. Fatty acids are powerful regulators of UCP3 gene transcription. We have found that the role of peroxisome proliferator-activated receptor-α (PPARα) on the control of UCP3 gene expression depends on the tissue and developmental stage. In adults, UCP3 mRNA expression is unaltered in skeletal muscle from PPARα-null mice both in basal conditions and under the stimulus of starvation. In contrast, UCP3 mRNA is down-regulated in adult heart both in fed and fasted PPARα-null mice. This occurs despite the increased levels of free fatty acids caused by fasting in PPARα-null mice. In neonates, PPARα-null mice show impaired UCP3 mRNA expression in skeletal muscle in response to milk intake, and this is not a result of reduced free fatty acid levels. The murine UCP3 promoter is activated by fatty acids through either PPARα or PPARδ but not by PPARγ or retinoid X receptor alone. PPARδ-dependent activation could be a potential compensatory mechanism to ensure appropriate expression of UCP3 gene in adult skeletal muscle in the absence of PPARα. However, among transcripts from other PPARα and PPARδ target genes, only those acutely induced by milk intake in wild-type neonates were altered in muscle or heart from PPARα-null neonates. Thus, PPARα-dependent regulation is required for appropriate gene regulation of UCP3 as part of the subset of fatty-acid-responsive genes in neonatal muscle and heart.

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.

Influence of pregnancy on the adipocytokine and peroxisome proliferator-activated receptor pathways in peripheral blood mononuclear cells from healthy donors and rheumatoid arthritis patients

To identify candidate genes that are regulated by human pregnancy and have the potential to modulate rheumatoid arthritis (RA) disease activity.

Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma

Agonist ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ have been shown to induce terminal differentiation of normal preadipocytes and human liposarcoma cells in vitro. Because the differentiation status of liposarcoma is predictive of clinical outcomes, modulation of the differentiation status of a tumor may favorably impact clinical behavior. We have conducted a clinical trial for treatment of patients with advanced liposarcoma by using the peroxisome proliferator-activated receptor-γ ligand troglitazone, in which extensive correlative laboratory studies of tumor differentiation were performed. We report here the results of three patients with intermediate to high-grade liposarcomas in whom troglitazone administration induced histologic and biochemical differentiation in vivo. Biopsies of tumors from each of these patients while on troglitazone demonstrated histologic evidence of extensive lipid accumulation by tumor cells and substantial increases in NMR-detectable tumor triglycerides compared with pretreatment biopsies. In addition, expression of several mRNA transcripts characteristic of differentiation in the adipocyte lineage was induced. There was also a marked reduction in immunohistochemical expression of Ki-67, a marker of cell proliferation. Together, these data indicate that terminal adipocytic differentiation was induced in these malignant tumors by troglitazone. These results indicate that lineage-appropriate differentiation can be induced pharmacologically in a human solid tumor.

Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ in RAW 264.7 macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis.

A peroxisome proliferator-activated receptor γ ligand inhibits adipocyte differentiation

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARγ subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2,4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPARγ ligand that was a weak partial agonist of PPARγ transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.

Effects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancer

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that plays a key role in the differentiation of adipocytes. Activation of this receptor in liposarcomas and breast and colon cancer cells also induces cell growth inhibition and differentiation. In the present study, we show that PPARγ is expressed in human prostate adenocarcinomas and cell lines derived from these tumors. Activation of this receptor with specific ligands exerts an inhibitory effect on the growth of prostate cancer cell lines. Further, we show that prostate cancer and cell lines do not have intragenic mutations in the PPARγ gene, although 40% of the informative tumors have hemizygous deletions of this gene. Based on our preclinical data, we conducted a phase II clinical study in patients with advanced prostate cancer using troglitazone, a PPARγ ligand used for the treatment of type 2 diabetes. Forty-one men with histologically confirmed prostate cancer and no symptomatic metastatic disease were treated orally with troglitazone. An unexpectedly high incidence of prolonged stabilization of prostate-specific antigen was seen in patients treated with troglitazone. In addition, one patient had a dramatic decrease in serum prostate-specific antigen to nearly undetectable levels. These data suggest that PPARγ may serve as a biological modifier in human prostate cancer and its therapeutic potential in this disease should be further investigated.