PPARγ Isoforms Research Articles

Anti-inflammatory and antiproliferative actions of PPAR-gamma agonists on T lymphocytes derived from MS patients.

Peroxisomal proliferator-activated receptors (PPARs) belong to a nuclear receptor superfamily of ligand-activated transcription factors. The PPAR-gamma isoform is expressed in human T lymphocytes, and oral administration of PPAR-gamma agonists ameliorates the clinical course and histopathological features in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, suggesting a potential role for PPAR-gamma agonists in the treatment of autoimmune diseases. To assess a potential therapeutic role of PPAR-gamma agonists in multiple sclerosis, we compared the immunomodulatory effects of the thiazolidinedione (TZD) drugs pioglitazone (PIO) and ciglitazone and the non-TZD PPAR-gamma agonist GW347845 on human T leukemia cells (Jurkat cells) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) derived from 21 multiple sclerosis patients and 12 healthy donors. PIO, ciglitazone, and GW347845 suppressed PHA-induced T cell proliferation by 40-50% and secretion of interferon-gamma and tumor necrosis factor alpha, by 30-50%. Inhibition of proliferation was increased to approximately 80% and that of proinflammatory cytokine secretion, to 80-90% when PBMCs were first preincubated with PPAR-gamma agonists and re-exposed at the time of PHA stimulation, indicating a sensitizing effect of PPAR-gamma agonists. Inhibition of proliferation was also observed in the tetanus toxoid-specific T cell line KHS.TT2, albeit to a lesser extent. The antiproliferative effects of PIO and GW347845 were accompanied by a decrease of cell viability. Electron microscopy and Western blot analysis revealed DNA condensation and down-regulation of bcl-2, suggesting the induction of apoptosis in activated T lymphocytes. In summary, the data support the potential use of PPAR-gamma agonists as immunomodulatory, therapeutic agents for autoimmune diseases.

Glitazones and heart failure: critical appraisal for the clinician.

Mr S. is a 46-year-old East-Indian male with typical manifestations of the cardiovascular dysmetabolic syndrome of insulin resistance (type II diabetes, obesity, dyslipidemia, hypertension, and elevated levels of high-sensitivity C-reactive protein). His medications include ramipril, simvastatin, enteric-coated aspirin, metformin, and glyburide. He has no symptoms of cardiac ischemia or congestive heart failure and has preserved left ventricular function. Over the past few months his glycemic control has been inadequate, and a decision to initiate an insulin sensitizer (glitazone) is made. Treatment is initiated with rosiglitazone 4 mg twice daily, with marked improvement in glycemic control and other components of the cardiovascular dysmetabolic syndrome. Approximately 6 months after initiation of therapy, your junior resident receives a phone call from Mr S, who is extremely anxious and distraught about the possibility of developing heart failure on glitazone therapy. The resident and the patient request your expert opinion about the effects of glitazones on cardiac function and associated hemodynamics. Insulin resistance has been increasingly recognized as a central metabolic disturbance predisposing a patient to hypertension, hyperlipidemia, premature atherosclerosis, left ventricular hypertrophy, and endothelial dysfunction.1 In addition to being a powerful risk marker for the development of cardiovascular disease, insulin resistance is also closely related to cardiac dysfunction and heart failure.2 Thiazolidinediones (TZD; glitazones) are peroxisome proliferator-activated receptor (PPAR) agonists that specifically augment insulin sensitivity and counter insulin resistance in patients with the cardiovascular dysmetabolic syndrome. Currently, there are 2 commercially available glitazones, rosiglitazone and pioglitazone. Troglitazone was withdrawn from the market because of hepatic side effects. The PPAR family is composed of 3 subtypes. TZDs bind with high affinity to the PPARγ isoform. The PPARγ isoform is found in the heart,3–6 endothelium, vascular smooth muscle (including atherosclerotic lesions and neointima formed after angioplasty), liver, skeletal muscle, monocytes/macrophages, and many other …

Designed transcription factors as tools for therapeutics and functional genomics

The paucity of tools that control expression of specific genes in vivo represents a major limitation of functional genomics in mammals; most available small-molecule regulators of transcription—e.g. histone deacetylase inhibitors—exert pan-genomic effects. Recent developments in understanding the role of chromatin in regulating the genome, and of protein–DNA interactions have allowed the development of designed transcription factors that regulate specific genes in vivo (Reik et al., Curr Opin Genet Dev 2002;12:233). These proteins contain two modules: (i) a zinc finger protein (ZFP)-based DNA-binding domain (DBD) designed to recognize a specific sequence (for example, a motif in the promoter of a certain gene); (ii) a functional module (for example, a transcriptional activation or repression domain). Recent data describe the use of such designed transcription factors to regulate a variety of clinically relevant gene targets in human cells: these include MDR1, erythropoietin, erbB-2 and erbB-3, VEGF, and PPARγ. In the case of VEGF (Liu et al., J Biol Chem 2001;276:11323), proportional upregulation by the designed transcription factor of all three distinct splice isoforms generated by this locus was observed, illuminating the utility of endogenous gene control in therapeutic settings (proper isoform ratio is essential for the proangiogenic function of VEGF). In the case of PPARγ, use of a transcriptional repressor designed to downregulate the expression of two PPARγ isoforms allowed “mutation-free reverse genetics” analysis that illuminated a unique role for the PPARγ2 isoform in adipogenesis (Ren et al., Genes Dev 2002;16:27). The ability to selectively activate or repress specific mammalian genes in vivo using designed transcription factors thus has considerable promise in clinical and in basic science settings.

Genetic analysis of four novel peroxisome proliferator activated receptor- [formula omitted] splice variants in monkey macrophages

Peroxisome proliferator activated receptor-γ (PPAR-γ) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-γ1, PPAR-γ2, and PPAR-γ3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-γ exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5 ′ terminal region to generate four novel PPAR-γ subtypes, PPAR-γ4, -γ5, -γ6, and -γ7. PPAR-γ4 and γ5 were detected only in macrophages whereas γ6 and γ7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-γ exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-γ1, PPAR-γ4, and PPAR-γ5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-γ expression in monkeys. RT-PCR showed a significant increase in total PPAR-γ and ABCA1 mRNA levels in macrophages of fat-fed monkeys ( n=7) compared to those maintained on a normal diet ( n=2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-γ4 and -γ5 in CHO cells. In these cells, expression of PPAR-γ5 but not -γ4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-γ isoforms may be responsible for unique tissue-specific biological effects and that PPAR-γ4 and -γ5 may modulate macrophage function and atherogenesis.

Generation and functional confirmation of a conditional null PPARgamma allele in mice.

Generation and functional confirmation of a conditional null PPARgamma allele in mice.

Peroxisome proliferator-activated receptor-gamma activity is associated with renal microvasculature.

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a nuclear transcription factor and the pharmacological target for antidiabetic thiazolidinediones (TZDs). TZDs ameliorate diabetic nephropathy and have direct effects on cultured mesangial cells (MCs); however, in situ hybridization failed to detect expression of PPAR gamma in glomeruli in vivo. The purpose of this study was to determine whether PPAR gamma is expressed in renal glomeruli. Two rabbit PPAR gamma isoforms were cloned. Nuclease protection assays demonstrate that both PPAR gamma isoforms are expressed in freshly isolated glomeruli. Treatment of rabbits with the TZD troglitazone selectively induced expression of an endogenous PPAR gamma target gene, adipocyte fatty acid-binding protein (A-FABP), in renal glomerular cells and renal medullary microvascular endothelial cells, demonstrated by both in situ hybridization and immunostain. Troglitazone also dramatically increased A-FABP expression in cultured MCs. Constitutive PPAR gamma expression was detected in cultured rabbit MCs. Endogenous MC PPAR gamma can also drive PPAR gamma reporter. Troglitazone and 15-deoxy-Delta 12,14 prostaglandin J(2) at low concentrations reduced mesangial cell [(3)H]thymidine incorporation without affecting viability. These data suggest that constitutive PPAR gamma activity exists in renal glomeruli in vivo and could provide a pharmacological target to directly modulate glomerular injury.

Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H 2 synthases by ibuprofen, naproxen, and indomethacin

A series of nonsteroidal anti-inflammatory drugs (NSAIDs) [ S(+)-naproxen, ibuprofen isomers, and indomethacin] were evaluated for their activation of peroxisome proliferator-activated receptor (PPAR) α and γ isoforms in CV-1 cells co-transfected with rat PPAR α and γ, and peroxisome proliferator response element (PPRE)-luciferase reporter gene plasmids, for stimulation of peroxisomal fatty acyl CoA β-oxidase activity in H4IIEC3 cells, and for comparative inhibition of ovine prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 and arachidonic acid-induced human platelet activation. Each drug produced a concentration-dependent activation of the PPAR isoforms and fatty acid β-oxidase activity, inhibition of human arachidonic acid-induced platelet aggregation and serotonin secretion, and inhibition of PGHS-1 and PGHS-2 activities. For PPARα activation in CV-1 and H4IIEC3 cells, and the stimulation of fatty acyl oxidase activity in H4IIEC3 cells, the rank order of stereoselectivity was S(+)- ibuprofen > R(−)-ibuprofen; S(+)-ibuprofen was more potent than indomethacin and naproxen on these parameters. On PPARγ, the rank order was S(+)-naproxen > indomethacin > S(+)-ibuprofen > R(−)-ibuprofen. Each drug inhibited PGHS-1 activity and platelet aggregation with the same rank order of indomethacin > S(+)-ibuprofen > S(+)-naproxen > R(−)-ibuprofen. Notably, the S(+)-isomer of ibuprofen was 32-, 41-, and 96-fold more potent than the R(−)-isomer for the inhibition of PGHS-1 activity, human platelet aggregation, and serotonin secretion, respectively. On PGHS-2, the ibuprofen isomers showed no selectivity, and indomethacin, S(+)-ibuprofen, and S(+)-naproxen were 6-, 27-, and 5-fold more potent as inhibitors of PGHS-1 than PGHS-2 activity. These results demonstrate that the mechanisms of action of NSAIDs on these cell systems are different, and we propose that the pharmacological effects of NSAIDs may be related to both their profile of inhibition of PGHS enzymes and the activation of PPARα and/or PPARγ isoforms.

Regulation of β-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor α

Amyloid deposition within the brains of Alzheimer's Disease patients results in the activation of microglial cells and the induction of a local inflammatory response. The interaction of microglia or monocytes with β-amyloid (Aβ) fibrils elicits the activation a complex tyrosine kinase-based signal transduction cascade leading to stimulation of multiple independent signaling pathways and ultimately to changes in proinflammatory gene expression. The Aβ -stimulated expression of proinflammatory genes in myeloid lineage cells is antagonized by the action of a family of ligand-activated nuclear hormone receptors, the peroxisome proliferator-activated receptors (PPARs). We report that THP-1 monocytes express predominantly PPARγ isoform and lower levels of PPARα and PPARδ isoforms. PPAR mRNA levels are not affected by differentiation of the cells into a macrophage phenotype, nor are they altered following exposure to the classical immune stimulus, lipopolysaccharide. Previous studies have found that PPARγ agonists act broadly to inhibit inflammatory responses. The present study explored the action of the PPARα isoform and found that PPARα agonists inhibited the Aβ-stimulated expression of TNFα and IL-6 reporter genes in a dose-dependent manner. Moreover, the PPARα agonist WY14643 inhibited macrophage differentiation and COX-2 gene expression. However, the PPARα agonists failed to inhibit Aβ-stimulated elaboration of neurotoxic factors by THP-1 cells. These findings demonstrate that PPARα acts to suppress a diverse array of inflammatory responses in monocytes.

Identification of a Novel Peroxisome Proliferator-Activated Receptor (PPAR) γ Promoter in Man and Transactivation by the Nuclear Receptor RORα1

PPARγ has been extensively studied for the past decade mainly due to its central role in promoting and maintaining the adipocyte phenotype. To date, three PPARγ isoforms have been described in man. Here we show the presence of a fourth PPARγ promoter with its cognate mRNA initiating at exon 1, as evidenced by primer extension analysis. The presence of a putative responsive element (RORE) for RORα, a representative of the ROR/RZR orphan receptor superfamily, in the novel promoter was investigated. By gelshift experiments and site-directed mutagenesis we show that this RORE specifically binds the RORα1 isoform. We further demonstrate that overexpression of RORα1, but not the RORα2 and RORα3 isoforms, induced a 40-fold increase in promoter activity in transient transfection assays in various cell lines. Considering the strong transcriptional activation it is likely that RORα1 forms a part of the multifactorial regulatory mechanisms that control expression of the human PPARγ gene.

Peroxisome proliferator-activated receptor (PPAR)-γ expression in human vascular smooth muscle cells: inhibition of growth, migration, and c-fos expression by the peroxisome proliferator-activated receptor (PPAR)-γ activator troglitazone

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ); whether the thiazolidinedione troglitazone, a ligand for PPARγ, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARγ protein and mRNA for both PPARγ isoforms, PPARγ1 and PPARγ2. Immunocytochemical staining localized the PPARγ protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor–induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARγ in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor–induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARγ receptor.

Expression of Peroxisome Proliferator Activated Receptor mRNA in Normal and Tumorigenic Rodent Mammary Glands

The peroxisome proliferator activated receptors (PPARs) α, β/δ, and γ are novel nuclear hormone receptors activated by long chain fatty acids and synthetic ligands and which regulate lipid metabolism. Recent studies have detected PPARγ mRNA in human mammary tumor cell lines. The current study examined the expression profile of PPAR mRNAs in normal and malignant rodent mammary tissues. Virgin murine mammary glands contained PPAR α, β/δ, and γ mRNAs based on northern blot analysis. The PPARγ isoform was predominantly γ2 based on quantitative PCR analysis. During pregnancy and lactation, the PPARα and γ mRNAs decreased while the PPAR β/δ mRNA remained relatively unchanged. NMuMG cells, an epithelial line derived from normal murine mammary gland, expressed PPAR α, β/δ, and γ mRNAs, independent of the presence or absence of compounds modifying PPAR activity. In rats, the physiologic expression pattern of PPARγ mRNA paralleled the murine model; levels were detected in virgin but not lactating mammary glands. In addition, the PPARγ mRNA was not detected in several histologically distinct 7,12-dimethylbenz(a)anthracene induced mammary tumors. These findings suggest that PPARs may regulate mammary epithelial and stromal cell function in response to physiologic or pathologic stimuli that profoundly alter lipid metabolism.

PPARγ3 mRNA: a distinct PPARγ mRNA subtype transcribed from an independent promoter

PPARγ is a member of the peroxisome proliferator activated receptors (PPAR) subfamily of nuclear receptors. So far two PPARγ isoforms, PPARγ1 and PPARγ2, were known in mammals. We describe the structure of a novel human PPARγ subtype, PPARγ3. The PPARγ3 mRNA is transcribed from a novel promoter localized 5′ of exon A2. PPARγ3 mRNA expression was restricted to adipose tissue and large intestine. Similar to human PPARγ1 and -2, PPARγ3 is activated by thiazolidinediones and prostaglandin J derivatives and binds with high affinity to a PPRE.

Molecular Cloning, Expression and Characterization of Human Peroxisome Proliferator Activated Receptors γ1 and γ2

We describe the molecular cloning and expression of cDNAs encoding human PPARγ1 and PPARγ2. Our sequences are distinct from the published sequence at 3 positions, resulting in nonconservative amino acid substitutions. In humans, PPARγ mRNA is expressed in spleen, bone marrow, liver, testis, skeletal muscle and brain, in addition to fat. Three thiazolidinediones were found to 1) displace a radiolabeled thiazolidinedione from both receptors with essentially the same IC50s and 2) to transactivate both PPARγ isoforms with similar EC50s in transient cotransfection assays utilizing the adipocyte-specific aP2 promoter. Saturating concentrations of these 3 thiazolidinediones altered the conformation ofin vitrosynthesized PPARγ protein producing a 27 kDa protease-resistant fragment. These results indicate that the antidiabetic effects of thiazolidinediones in humans are likely to be mediated via binding to and transactivation of PPARγ1 and γ2.