X-ray crystal structure of rivoglitazone bound to PPARγ and PPAR subtype selectivity of TZDs

Thiazolidinedione (TZD) compounds targeting the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) demonstrate unique benefits for the treatment of insulin resistance and type II diabetes. TZDs include rosiglitazone, pioglitazone and rivoglitazone, with the latter being the most potent. The TZDs are only marginally selective for the therapeutic target PPARγ as they also activate PPARα and PPARδ homologues to varying degrees, causing off-target effects. While crystal structures for TZD compounds in complex with PPARγ are available, minimal structural information is available for TZDs bound to PPARα and PPARδ. This paucity of structural information has hampered the determination of precise structural mechanisms involved in TZD selectivity between PPARs. To help address these questions molecular dynamic simulations were performed of rosiglitazone, pioglitazone and rivoglitazone in complex with PPARα, PPARδ, and PPARγ in order to better understand the mechanisms of PPAR selectivity. The simulations revealed that TZD interactions with residues Tyr314 and Phe318 of PPARα and residues Phe291 and Thr253 of PPARδ as well as the omega loop, are key determinants of TZD receptor selectivity. Notably, in this study, we solve the first X-ray crystal structure of rivoglitazone bound to any PPAR. Rivoglitazone forms a unique hydrogen bond network with the residues of the PPARγ co-activator binding surface (known as AF2) and makes more extensive contacts with helix 3 and the β-sheet as compared to model TZD compounds such as rosiglitazone.     

Functional analysis of the chicken PPARγ gene 5'-flanking region and C/EBPα-mediated gene regulation

Peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α (C/EBPα) are the master regulators of adipogenesis. The regulatory mechanism of PPARγ and C/EBPα gene expression is clear in mammals, however, little is known in chicken. The aim of the present study was to characterize chicken PPARγ promoter and investigate whether PPARγ could be regulated by C/EBPα in chickens. A 2-kb nucleotide sequence upstream of the start codon of chicken PPARγ gene was cloned and characterized by using bioinformatics and experimental approaches. This 2-kb promoter region exhibited strong promoter activity in DF1 cells. The reporter gene assay showed that the chicken C/EBPα could activate PPARγ gene promoter. Further study by electrophoretic mobility shift assay and mutational analysis revealed that the chicken C/EBPα could directly bind to and regulate the PPARγ gene promoter. Our results demonstrate that PPARγ can be directly regulated by C/EBPα in chickens.     

N-Acetylfarnesylcysteine is a novel class of peroxisome proliferator-activated receptor γ ligand with partial and full agonist activity in vitro and in vivo

The thiazolidedione (TZD) class of drugs is clinically approved for the treatment of type 2 diabetes. The therapeutic actions of TZDs are mediated via activation of peroxisome proliferator-activated receptor γ (PPARγ). Despite their widespread use, concern exists regarding the safety of currently used TZDs. This has prompted the development of selective PPARγ modulators (SPPARMs), compounds that promote glucose homeostasis but with reduced side effects due to partial PPARγ agonism. However, this also results in partial agonism with respect to PPARγ target genes promoting glucose homeostasis. Using a gene expression-based screening approach we identified N-acetylfarnesylcysteine (AFC) as both a full and partial agonist depending on the PPARγ target gene (differential SPPARM). AFC activated PPARγ as effectively as rosiglitazone with regard to Adrp, Angptl4, and AdipoQ, but was a partial agonist of aP2, a PPARγ target gene associated with increased adiposity. Induction of adipogenesis by AFC was also attenuated compared with rosiglitazone. Reporter, ligand binding assays, and dynamic modeling demonstrate that AFC binds and activates PPARγ in a unique manner compared with other PPARγ ligands. Importantly, treatment of mice with AFC improved glucose tolerance similar to rosiglitazone, but AFC did not promote weight gain to the same extent. Finally, AFC had effects on adipose tissue remodeling similar to those of rosiglitazone and had enhanced antiinflammatory effects. In conclusion, we describe a new approach for the identification of differential SPPARMs and have identified AFC as a novel class of PPARγ ligand with both full and partial agonist activity in vitro and in vivo.     

Stable reporter cell lines for peroxisome proliferator-activated receptor γ (PPARγ)-mediated modulation of gene expression

Activation of peroxisome proliferator-activated receptor γ (PPARγ) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement of PPARγ agonist and antagonist activity. Two reporter gene assays, PPARγ1 CALUX and PPARγ2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPARγ1 or PPARγ2 and a pGL3–3xPPRE–tata-luc or pGL4–3xPPRE–tata-luc reporter construct, respectively. PPARγ1 CALUX and PPARγ2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPARγ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Δ^12,14-prostaglandin J₂. The potency to induce luciferase decreased in the following order: rosiglitazone > troglitazone = pioglitazone > netoglitazone > ciglitazone. A concentration-dependent decrease in the response to 50 nM rosiglitazone was observed on the addition of PPARγ antagonist GW9662 or T0070907 in both PPARγ1 CALUX and PPARγ2 CALUX cells. The PPARα agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPARγ agonists. In conclusion, PPARγ1 CALUX and PPARγ2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPARγ agonist or antagonist activity.     

PPARγ Agonist Suppresses TLR4 Expression and TNF-α Production in LPS Stimulated Monocyte Leukemia Cells

It has been well established that inflammation plays a critical role in cancer. Chronic inflammation promotes tumorgenesis and metastasis, which suggests that anti-inflammation drugs could act as a tumor suppressor. It is known that the peroxisome proliferator-activated receptor γ (PPARγ) has been implicated in anti-inflammatory responses; however, the anti-tumor effects of PPARγ have not been intensively investigated. In this study, we examined the effects of PPARγ in cancer. We show that the activation of PPARγ by its agonist rosiglitazone (RGZ) reduces cell proliferation rate in inflammatory and tumor-derived U937 cells. Treatment of RGZ suppresses the expression Toll-like receptor 4 (TLR4) and decreases the production of TNF-α in LPS treated U937 cells. This suggests that NF-κB signaling may be involved in anti-tumor effect of RGZ. Our results demonstrate a role of PPARγ in regulation of NF-κB signaling by modulating TLR4 expression and TNF-α production.     

The neuroprotective role of rosiglitazone in advanced glycation end product treated human neural stem cells is PPARgamma-dependent

Hyperglycemia is accompanied by an accelerated formation rate of advanced glycation end products (AGEs), which is associated with the pathogenesis of diabetic neuronal deficits. Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors and its ligands are known to control many physiological, pathological and inflammatory pathways. Weinvestigated the hypothesis that the PPARγ agonist (rosiglitazone) would abrogate AGEs-mediated neurotoxic effects on human neural stem cells (hNSCs), by whichAGEs may play a role in diabetic-related neuronal impairment. Here, we show that rosiglitazone treatment increases cell viability of hNSCs via downregulation of caspase 3 activity. These rescue effects were extended in our studies showingrosiglitazone-mediated activation of PPARγ reversed the expression levels of two neuroprotective factors (Bcl-2 and PGC1α) that were downregulated in hNSCs exposed to AGEs alone. The stimulation of mitochondrial function and anti-oxidative stress by rosiglitazone was associated with activation of the PGC1α pathway by up-regulation of mitochondrial (NRF-1 and Tfam) and oxidative defense (SOD1, SOD2 and Gpx1) genes. Moreover, rosiglitazone significantly normalized the inflammatory responses (TNF-α and IL-1β), NF-κB (p65), and inflammatory genes (iNOS and COX-2) in the hNSCs treated with AGEs. This neuroprotective effect of rosiglitazone was effectively blocked by PPARγ-specific antagonist (GW9662), demonstrating that the action of rosiglitazone was mediated by at PPARγ-dependent pathway. Collectively, these novel findings show AGEs induce neurotoxic effects in hNSCs, and provide important mechanistic insights that may explain the increased risk of neuronal impairment deficits in diabetic patients. More importantly, these data show rosiglitazone-mediated activation of PPARγ-dependent signaling is neuroprotective in AGE-treated hNSCs, and suggests PPARγ ligands may be useful in the therapeutic management of patients with neurodegenerative diseases     

罗格列酮对猪脂肪前体细胞分化过程中聚脂相关基因表达模式的影响

为了解罗格列酮对猪脂肪前体细胞诱导分化过程的影响, 利用胶原酶消化法分离猪皮下脂肪前体细胞, 采用含50 nmol/L胰岛素、100 nmol/L地塞米松及0.25 mmol/L 3-异丁基-1-甲基黄嘌呤的分化培养液Ⅰ(对照组)和在分化培养液Ⅰ中添加100 nmol/L罗格列酮的分化培养液Ⅱ(实验组)两种诱导分化方法对脂肪前体细胞进行诱导分化, 借助实时定量RT-PCR方法检测了细胞分化过程中聚脂相关基因的表达。结果显示: 罗格列酮对PPARγ、C/EBPα、FABP4、FASN和GPAT基因的表达有显著的上调作用, 而对PPARα有一定的下调作用。试验组中PPARα、PPARγ、C/EBPα、FABP4、FASN和GPAT等基因分别于48 h、48 h、48 h、108 h、60 h和24 h达到表达高峰, 此时的表达量分别是诱导前的1.7、48、3.3、487.5、5.8和3.6倍, GPAT同PPARα和FASN基因表达量间均达到显著相关(P<0.05); 而对照组中PPARα、PPARγ、C/EBPα、FABP4、FASN和GPAT等基因分别于84 h、96 h、48 h、96 h、36 h和36 h达到表达高峰, 此时的表达量分别是诱导前的2.1、11、1.6、216.5、3.5和2.8倍, GPAT同PPARα和FASN基因表达量间均达到极显著相关(P<0.01)。本实验结果表明: 罗格列酮不仅可以极大的促进PPARγ和C/EBPα基因的表达, 还能让其协同达到表达高峰; PPARγ和C/EBPα可能是调控猪脂肪前体细胞分化的关键转录因子; 在脂肪形成过程中, 甘油脂类的生物合成可能发生较早, 同时PPARα可能主要参与甘油脂类生物合成的调控。     

The protective role of peroxisome proliferator-activated receptor gamma in lipotoxic podocytes

Podocytes are specialized epithelial cells that maintain the glomerular filtration barrier. These cells are susceptible to lipotoxicity in the obese state and irreversibly lost during kidney disease leading to proteinuria and renal injury. PPARγ is a nuclear receptor whose activation can be renoprotective. This study examined the role of PPARγ in the lipotoxic podocyte using a PPARγ knockout (PPARγKO) cell line and since the activation of PPARγ by Thiazolidinediones (TZD) is limited by their side effects, it explored other alternative therapies to prevent podocyte lipotoxic damage.Wild-type and PPARγKO podocytes were exposed to the fatty acid palmitic acid (PA) and treated with the TZD (Pioglitazone) and/or the Retinoid X receptor (RXR) agonist Bexarotene (BX).It revealed that podocyte PPARγ is essential for podocyte function. PPARγ deletion reduced key podocyte proteins including podocin and nephrin while increasing basal levels of oxidative and ER stress causing apoptosis and cell death. A combination therapy of low-dose TZD and BX activated both the PPARγ and RXR receptors reducing PA-induced podocyte damage. This study confirms the crucial role of PPARγ in podocyte biology and that their activation in combination therapy of TZD and BX may be beneficial in the treatment of obesity-related kidney disease.     

Prothymosin-α interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders.     

A PPARgamma agonist inhibits aldosterone-induced mesangial cell proliferation by blocking ROS-dependent EGFR intracellular signaling

Mesangial cell (MC) proliferation is a key feature in the pathogenesis of a number of renal diseases. Peroxisome proliferator-activated receptor-γ (PPARγ) has attracted considerable attention for its effects on stimulating cell differentiation and on inducing cell cycle arrest. We previously showed that aldosterone (Aldo) stimulates MC proliferation via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, which was dependent on reactive oxygen species (ROS)-mediated epithelial growth factor receptor (EGFR) transactivation (Huang S, Zhang A, Ding G, and Chen R. Am J Physiol Renal Physiol 296: F1323-F1333, 2009). In this study, we examined whether the PPARγ agonist rosiglitazone inhibited Aldo-induced MC proliferation by modulating ROS-dependent EGFR intracellular signaling. Rosiglitazone at 1-10 μM dose dependently inhibited Aldo-induced MC proliferation of cultured mouse MCs. The inhibitory effect was blocked by the PPARγ antagonist PD-68235, indicating that the rosiglitazone effect acted through PPARγ activation. Rosiglitazone also arrested Aldo-induced cell cycle progression and suppressed expression of cyclins D1 and A. Moreover, rosiglitazone dose dependently blocked Aldo-induced ROS production, EGFR phosphorylation, and PI3K/Akt activation. These results suggest that the PPARγ agonist rosiglitazone may inhibit Aldo-induced MC proliferation directly, by affecting ROS/EGFR/PI3K/Akt signaling pathways and cell cycle-regulatory proteins. PPARγ might be a novel therapeutic target against glomerular diseases.     

Rational design of a pirinixic acid derivative that acts as subtype-selective PPARγ modulator

Peroxisome proliferator-activated receptor γ (PPARγ) is involved in glucose and lipid homeostasis. PPARγ agonists are in clinical use for the treatment of type 2 diabetes. Lately, a new class of selective PPARγ modulators (SPPARγMs) was developed, which are believed to show less side effects than full PPARγ agonists. We have previously shown that α-substitution of pirinixic acid, a moderate agonist of PPARα and PPARγ, leads to low micromolar active balanced dual agonists of PPARα and PPARγ. Herein we present modifications of pirinixic acid leading to subtype-selective PPARγ agonists and furthermore the development of a selective PPARγ modulator guided by molecular docking studies.