Antiinflammatory effect of rosiglitazone via modulation of mRNA stability of interleukin 10 and cyclooxygenase 2 in astrocytes

Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E₂, PGE₂) and antiinflammatory marker (interleukin 10, IL-10) levels. The protein level of cyclooxygenase 2 (COX-2) was also increased. Rosiglitazone strengthened LPS-induced mRNA expression of COX-2 and IL-10 but not TNFα. Rosiglitazone is an agonist of nuclear receptor PPARγ, but its impact on IL-10 expression was not influenced by a PPARγ antagonist, GW9662, suggesting PPARγ-independent effect of rosiglitazone. The degradation of mRNA is one of the steps of inflammation regulation and might be affected by small molecules. In experiments with actinomycin D, we found that mRNA half-lives of IL-10, COX-2, and TNFα in naive astrocytes were 70, 44, and 19 min, respectively. LPS stimulation caused 2-fold increase in IL-10 and COX-2 mRNA decay rates, whereas addition of rosiglitazone restored them to the initial level. TNFα decay rate was not changed by these stimulations. This suggests that mRNA decay rate could be regulated by small molecules. Moreover, rosiglitazone could be used as a substance stimulating the resolution of inflammation without influence on proinflammatory signals. These results open new perspectives in the search for inflammation resolution modulators.     

Ligands of peroxisome proliferator-activated receptor-alpha promote glutamate transporter-1 endocytosis in astrocytes

Astrocytes, a stellate-shape glial population in the central nervous system (CNS), maintain glutamate homeostasis in adult CNS by undergoing glutamate uptake at the synapse through their glutamate transporter-1 (GLT-1). Peroxisome proliferator-activated receptor-α (PPARα) can be activated by endogenous saturated fatty acids to regulate astrocytic lipid metabolism and functions. However, it is unclear if PPARα can exert the regulatory action on GLT-1 expression in astrocytes. This study showed that treatment with palmitic acid (PA) and the other two PPARα agonists (GW 7647 and WY 14,643) caused no change in the morphology of astrocytes, whereas membranous GLT-1 protein levels in astrocytes were significantly decreased by PA and PPARα agonists. Through lentivirus-mediated overexpression of GLT-1 tagged with red fluorescent protein (GLT-1-RFP), we also observed that GLT-1-RFP puncta in the processes of astrocytes were inhibited by the PPARα agonists. This reduction was prevented by the addition of the PPARα antagonist, GW6471. GLT-1-RFP was co-localized to the early endosome marker–EEA1 in astrocytes treated with the PPARα agonists. Moreover, PPARα-induced inhibition in membranous GLT-1 expression was abolished by the addition of dynamin inhibitor (dynasore). Furthermore, the co-treatment of astrocytes with PPARα agonists and dynasore, or with PPARα agonists and protein kinase C (PKC) inhibitor bis-indolylmaleimide 1 (BIS1), prevented the endocytosis of GLT-1-RFP. Based on the results, we conclude that the PPARα agonists increased GLT-1 endocytosis in astrocytes possibly through the PKC signaling pathway. In addition, our findings provide important information of PPARα involvement in the downregulation of astrocytic glutamate uptake via the promoted GLT-1 endocytosis.     

Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO₂ and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

Differential responses of PPARalpha, PPARdelta, and PPARgamma reporter cell lines to selective PPAR synthetic ligands

To characterize the specificity of synthetic compounds for peroxisome proliferator-activated receptors (PPARs), three stable cell lines expressing the ligand binding domain (LBD) of human PPARalpha, PPARdelta, or PPARgamma fused to the yeast GAL4 DNA binding domain (DBD) were developed. These reporter cell lines were generated by a two-step transfection procedure. First, a stable cell line, HG5LN, expressing the reporter gene was developed. These cells were then transfected with the different receptor genes. With the help of the three PPAR reporter cell lines, we assessed the selectivity and activity of PPAR agonists GW7647, WY-14-643, L-165041, GW501516, BRL49653, ciglitazone, and pioglitazone. GW7647, L-165041, and BRL49653 were the most potent and selective agonists for hPPARalpha, hPPARdelta, and hPPARgamma, respectively. Two PPAR antagonists, GW9662 and BADGE, were also tested. GW9662 was a selective PPARgamma antagonist, whereas BADGE was a low-affinity PPAR ligand. Furthermore, GW9662 was a full antagonist on PPARgamma and PPARdelta, whereas it showed partial agonism on PPARalpha. We conclude that our stable models allow specific and sensitive measurement of PPAR ligand activities and are a high-throughput, cell-based screening tool for identifying and characterizing PPAR ligands.     

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.     

The involvement of peroxisome proliferator activated receptors (PPARs) in prostaglandin F2α production by porcine endometrium

In the present study, we investigated the in vitro effects of peroxisome proliferator activated receptor (PPAR) ligands on PGF_2α secretion and mRNA expression of prostaglandin F synthase (PGFS) in porcine endometrial explants collected on days 10–12 and 14–16 of the estrous cycle or pregnancy. The explants were incubated for 6 h with: PPARα ligands – WY-14643 (agonist) and MK 886 (antagonist); PPARβ ligands – l-165,041 (agonist) and GW 9662 (antagonist); PPARγ ligands – 15d-prostaglandin J₂ (PGJ₂, agonist), rosiglitazone (agonist) and T0070907 (antagonist). The expression of PGFS mRNA in the endometrium and the concentration of PGF_2α in culture media were determined by real time RT-PCR and radioimmunoassay, respectively. During the estrous cycle (days 10–12 and 14–16), the agonists – WY-14643 (PPARα), l-165,041 (PPARβ), PGJ₂ and rosiglitazone (PPARγ) – increased PGF_2α secretion but did not affect PGFS mRNA abundance. During pregnancy (days 10–12 and 14–16), PPARα and PPARγ ligands did not change PGF_2α release, whereas PPARβ agonist augmented PGF_2α release on days 14–16 of pregnancy. In addition, WY-14643 and l-165,041 increased PGFS mRNA level in both examined periods of pregnancy. PPARγ agonist (PGJ₂) and antagonist (T0070907) enhanced PGFS mRNA abundance in the endometrium on days 10–12 and 14–16 of pregnancy, respectively. The results indicate that PPARs are involved in the production of PGF_2α by porcine endometrium, and that the sensitivity of the endometrium to PPAR ligands depends on reproductive status of animals.     

Peroxisome proliferator-activated receptor α and γ agonists together with TGF-β convert human CD4+CD25- T cells into functional Foxp3+ regulatory T cells

Human peripheral CD4(+)CD25(-) T cells can be induced to express Foxp3 when activated in vitro by TCR stimulation with TGF-β and IL-2. However, these TGF-β-induced Foxp3(+) regulatory T cells (iTregs) lack a regulatory phenotype. From libraries of nuclear receptor ligands and bioactive lipids, we screened three peroxisome proliferator-activated receptor (PPAR)α (bezafibrate, GW7647, and 5,8,11,14-eicosatetraynoic acid) and two PPARγ agonists (ciglitazone and 15-deoxy-Δ-(12,14)-PG J(2)) as molecules that increased Foxp3 expression in human iTregs significantly compared with that in DMSO-treated iTregs (control). These PPARα and PPARγ agonist-treated iTregs maintained a high level of Foxp3 expression and had suppressive properties. There were no significant differences in the suppressive properties of iTregs treated with the three PPARα and two PPARγ agonists, and all of the treated iTregs increased demethylation levels of the Foxp3 promoter and intronic conserved noncoding sequence 3 regions. Furthermore, PPARα and PPARγ agonists, together with TGF-β, more strongly inhibited the expression of all three DNA methyltransferases (DNMTs) (DNMT1, DNMT3a, and DNMT3b) in activated CD4(+) T cells. These results demonstrate that PPARα and PPARγ agonists together with TGF-β elicit Foxp3 DNA demethylation through potent downregulation of DNMTs and induce potent and stable Foxp3 expression, resulting in the generation of functional iTregs. Moreover, trichostatin A and retinoic acid enhanced the generation of iTregs synergistically with PPARα and PPARγ agonists.     

Benzothiazole-based N-(phenylsulfonyl)amides as a novel family of PPARα antagonists

The discovery of PPAR antagonists is emerging as an useful tool for elucidating the biological role of the receptor. Here we report the identification of N-(phenylsulfonyl)amides containing the benzothiazole scaffold, a novel class of potent PPARα antagonists obtained from chemical modification of carboxylic acid agonists. In this work, a group of phenylsulfonamides were synthesized and in vitro evaluated against the agonistic effect of GW7647; they showed an inhibitory effect on PPARα activation, with best compounds revealing a dose-dependent antagonistic profile. Some of these antagonists showed also an inhibitory effect on CPT1A pattern expression.     

The PPARδ ligand L-165041 inhibits VEGF-induced angiogenesis, but the antiangiogenic effect is not related to PPARδ

Peroxisome proliferator-activated receptor (PPAR)δ is known to be expressed ubiquitously and involved in lipid and glucose metabolism. Recent studies have demonstrated that PPARδ is expressed in endothelial cells (ECs) and plays a potential role in endothelial survival and proliferation. Although PPARα and PPARγ are well recognized to play anti-inflammatory, antiproliferative, and antiangiogenic roles in ECs, the general effect of PPARδ on angiogenesis in ECs remains unclear. Thus, we investigated the effect of the PPARδ ligand L-165041 on vascular EC proliferation and angiogenesis in vitro as well as in vivo. Our data show that L-165041 inhibited VEGF-induced cell proliferation and migration in human umbilical vein ECs (HUVECs). L-165041 also inhibited angiogenesis in the Matrigel plug assay and aortic ring assay. Flow cytometric analysis indicated that L-165041 reduced the number of ECs in the S phase and the expression levels of cell cycle regulatory proteins such as cyclin A, cyclin E, CDK2, and CDK4; phosphorylation of the retinoblastoma protein was suppressed by pretreatment with L-165041. We confirmed whether these antiangiogenic effects of L-165041 were PPARδ-dependent using GW501516 and PPARδ siRNA. GW501516 treatment did not inhibit VEGF-induced angiogenesis, and transfection of PPARδ siRNA did not reverse this antiangiogenic effect of L-165041, suggesting that the antiangiogenic effect of L-165041 on ECs is PPARδ-independent. Together, these data indicate that the PPARδ ligand L-165041 inhibits VEGF-stimulated angiogenesis by suppressing the cell cycle progression independently of PPARδ. This study highlights the therapeutic potential of L-165041 in the treatment of many disorders related to pathological angiogenesis.     

PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687–694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER? patients.     

The potential signaling pathway between peroxisome proliferator-activated receptor gamma and retinoic acid receptor alpha in renal interstitial fibrosis disease

Abstract

Peroxisome proliferator-activated receptorγ (PPARγ) can regulate the process of cell apoptosis and is related to the progression of renal disorders. Retinoic acid receptor alpha (RARα) is one of the nuclear receptors involved in a variety of kidney diseases. Renal interstitial fibrosis (RIF) is a common denominator of chronic kidney disease (CKD). This study investigated whether a potential signaling pathway existed between PPARγ and RARα in RIF rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into four groups: a model group subjected to UUO (GU), and three other groups treated with rosiglitazone sodium (GRS), GW9662 and dimethyl sulfoxide (DMSO), n?=?40, respectively. Renal tissues were collected two and four weeks after post-surgery. The relevant indicators were detected. In comparison with the GU group, the expressions of PPARγ and RARα (protein and mRNA) were increased in the GRS group, and decreased in the GW9662 group (all p?<?0.01). The RIF index, mRNA and protein expression of transforming growth factor-β1 (TGF-β1), and the protein expressions of collagen-IV (Col-IV) and fibronectin (FN) in the GRS group were more markedly reduced than those in the GU group; their levels in the GW9662 group were elevated (all p?<?0.01). PPARγ or RARα was negatively correlated to the RIF index, TGF-β1, Col-IV and FN. PPARγ was positively correlated with RARα (all p?<?0.01). In conclusion, PPARγ agonist can elevate the expression of PPARγ or RARα in RIF rats. There might be a potential signaling pathway between PPARγ and RARα in RIF disease.     

Down-regulation of apolipoprotein M expression is mediated by phosphatidylinositol 3-kinase in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein present mostly in high-density lipoprotein (HDL) in human plasma. In the present study, we demonstrate that insulin, insulin-like growth factor I (IGF-I), and IGF-I potential peptide (IGF-IPP) significantly inhibits apoM expression, in a dose- and a time-dependent manner, in the human hepatoma cell line, HepG2 cells. Insulin-induced down-regulation of apoM was blocked by AG1024 (a specific insulin receptor inhibitor) and LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), which indicates that it is mediated via the activation of PI3K pathway. In contrast, PD98059 (a MAP kinase inhibitor) did not influence insulin-induced down-regulation of apoM expression, and activation of neither PPAR-alpha agonist (GW7647) nor PPAR-gamma agonist (GW1929) influences apoM expression in HepG2 cells, which indicates that regulation of apoM expression is not related to the activation of PPAR-alpha and PPAR-gamma in hepatic cells, whereas, both PPAR-alpha and PPAR-gamma agonists could inhibit apoB expression. Moreover, in the present study, we demonstrated that PPAR beta/delta agonist (GW501516) could inhibit both apoM and apoB expression in the HepG2 cells. In conclusion, this study shows that apoM expression is regulated by PI3-kinase in HepG2-cells.     

Endothelial surface N-glycans mediate monocyte adhesion and are targets for anti-inflammatory effects of peroxisome proliferator-activated receptor γ ligands

Endothelial-monocyte interactions are regulated by adhesion molecules and key in the development of vascular inflammatory disease. Peroxisome proliferator-activated receptor (PPAR) γ activation in endothelial cells is recognized to mediate anti-inflammatory effects that inhibit monocyte rolling and adhesion. Herein, evidence is provided for a novel mechanism for the anti-inflammatory effects of PPARγ ligand action that involves inhibition of proinflammatory cytokine-dependent up-regulation of endothelial N-glycans. TNFα treatment of human umbilical vein endothelial cells increased surface expression of high mannose/hybrid N-glycans. A role for these sugars in mediating THP-1 or primary human monocyte rolling and adhesion was indicated by competition studies in which addition of α-methylmannose, but not α-methylglucose, inhibited monocyte rolling and adhesion during flow, but not under static conditions. This result supports the notion that adhesion molecules provide scaffolds for sugar epitopes to mediate adhesion with cognate receptors. A panel of structurally distinct PPARγ agonists all decreased TNFα-dependent expression of endothelial high mannose/hybrid N-glycans. Using rosiglitazone as a model PPARγ agonist, which decreased TNFα-induced high mannose N-glycan expression, we demonstrate a role for these carbohydrate residues in THP-1 rolling and adhesion that is independent of endothelial surface adhesion molecule expression (ICAM-1 and E-selectin). Data from N-glycan processing gene arrays identified α-mannosidases (MAN1A2 and MAN1C1) as targets for down-regulation by TNFα, which was reversed by rosiglitazone, a result consistent with altered high mannose/hybrid N-glycan epitopes. Taken together we propose a novel anti-inflammatory mechanism of endothelial PPARγ activation that involves targeting protein post-translational modification of adhesion molecules, specifically N-glycosylation.