PPARgamma Transcriptional Activity Research Articles

Thiazolidinediones Inhibit Growth and Invasiveness of the Human Adrenocortical Cancer Cell Line H295R

Thiazolidinediones (TZDs) are a new class of antidiabetic drugs that have also been shown to possess antitumoral properties in different human cancers. TZDs bind and activate the peroxisome proliferator-activated receptor (PPAR)-gamma, which is a nuclear receptor acting as a transcription factor in several tissues. In the present study, we evaluated PPARgamma mRNA and protein expression in tissue samples of human adrenocortical carcinomas (ACCs), normal adrenal glands, and the human ACC cell line H295R. PPARgamma mRNA was expressed in six of eight ACC, two of three normal adrenal glands and the H295R cells. These results were confirmed by immunohistochemistry. PPARgamma transcriptional activity in H295R cells, monitored by a reporter gene assay, was induced 2- to 3-fold by TZDs, such as rosiglitazone (RGZ) and pioglitazone, whereas in PPARgamma-transfected cells RGZ alone or RGZ plus 9-cis retinoic acid further increased reporter activity. TZDs inhibited both the proliferation and invasiveness of H295R cells in a dose-dependent manner. Thymidine incorporation was reduced by about 60% by 20 mum of both TZDs. Cotreatment with the retinoic X receptor ligand 9-cis retinoic acid had an additive effect. TZDs increased the number of cells in the G(0)/G(1) phase and decreased them in the S phase. Western blot analysis showed that TZDs increased the expression of the cell cycle inhibitors p21 and p27 and reduced the expression of cyclin D1. Twenty micromoles of RGZ and pioglitazone reduced H295R invasiveness through Matrigel by about 85%. Zymography and ELISA tests showed that TZD inhibited metalloproteinase-2 secretion by H295R cells in a dose-dependent manner. These data suggest that TZDs reduce the malignant potential of the H295R ACC cell line and, therefore, might potentially constitute a novel tool in the medical treatment of human ACCs.

Ligand-independent Activation of Peroxisome Proliferator-activated Receptor-γ by Insulin and C-peptide in Kidney Proximal Tubular Cells: DEPENDENT ON PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY

Peroxisome proliferator-activated receptor gamma (PPARgamma) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPARgamma transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPARgamma transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPARgamma transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPARgamma. GW9662, a PPARgamma antagonist, blocked PPARgamma activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPARgamma transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPARgamma transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPARgamma transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPARgamma was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPARgamma transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPARgamma-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPARgamma activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in regulation of PPARgamma-related cell functions.

Peroxisome proliferator‐activated receptor γ‐dependent and ‐independent growth inhibition of gastrointestinal tumour cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) acts as a ligand-activated transcription factor. Although ligand-induced cellular differentiation and growth inhibition have been mostly studied on human cancers expressing PPARgamma, it is unclear if the transcriptional activation of PPARgamma is the main mechanism of growth inhibition. In this study, we investigated whether there is a link between growth inhibitory effect and transcriptional activation of PPARgamma in several gastrointestinal tumour cell lines. The transcriptional activation potential of PPARgamma was assessed by reporter gene assay employing a PPRE-luciferase vector, and growth inhibitory effect of PPARgamma was investigated by (3)H-thymidine incorporation assay, in the presence or absence of thiazolidinedione ligands, rosiglitazone and troglitazone. As expected, in the case of cell lines positive for the transcriptional activation potential of PPARgamma (T.Tn, MKN-45 and LoVo), both the ligands induced growth inhibition. However, in case of some other cell lines negative for the transcriptional activation potential of PPARgamma (TT, AGS and HCT-15), troglitazone still showed a growth inhibitory effect. Administration of the PPARgamma antagonist GW9662 did not reverse this growth inhibitory activity of troglitazone. The introduction of dominant negative mutants of PPARgamma did not suppress the activity either. These observations suggest that while rosiglitazone inhibits cellular growth predominantly through transcriptional activation of PPARgamma, troglitazone can induce it both in PPARgamma-dependent and -independent pathways.

The balance between concurrent activation of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.

The soy phytoestrogen daidzein has biphasic dose responses, but the underlying mechanisms are not yet clear. Transcriptional and biochemical data show that PPARs, in addition to ERs, are molecular targets of daidzein, which divergently regulates osteogenesis and adipogenesis. Dose responses are the result of a balance among PPARs and between ERs and PPARs. Soy phytoestrogens have been used for the purposes of treatment and prevention of osteoporosis. Biphasic dose responses of daidzein, one of the main soy phytoestrogens, have long been recognized, but the underlying molecular mechanisms of action are not yet clear. Mouse bone marrow cells and mouse osteoprogenitor KS483 cells that concurrently differentiate into osteoblasts and adipocytes were cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, RT-PCR, and gene reporter assays were used in this study. Daidzein, one of the major soy phytoestrogens, had biphasic effects on osteogenesis and adipogenesis. Daidzein stimulated osteogenesis (ALP activity and nodule formation) and decreased adipogenesis (the number of adipocytes) at concentrations below 20 microM, whereas it inhibited osteogenesis and stimulated adipogenesis at concentrations higher than 30 microM. When estrogen receptors (ERs) were blocked by ICI182,780, daidzein-induced effects were not biphasic. A decrease in osteogenesis and an increase in adipogenesis were observed at the concentrations higher than 20 and 10 microM, respectively. In addition to ERs, daidzein transactivated not only peroxisome proliferator-activate receptor gamma (PPARgamma), but also PPARalpha and PPARdelta at micromolar concentrations. Activation of PPARalpha had no direct effects on osteogenesis and adipogenesis. In contrast, activation of PPARdelta stimulated osteogenesis but had no effects on adipogenesis, whereas PPARgamma inhibited osteogenesis and stimulated adipogenesis. Transfection experiments show that an activation of PPARalpha or PPARgamma by daidzein downregulated its estrogenic transcriptional activity, whereas activation of PPARdelta upregulated its estrogenic transcriptional activity. Activation of ERalpha or ERbeta by daidzein downregulated PPARgamma transcriptional activity but had no influence on PPARalpha or PPARdelta transcriptional activity. Daidzein at micromolar concentrations concurrently activates different amounts of ERs and PPARs, and the balance of the divergent actions of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.

PPARgamma and bone metabolism

PPARgamma is a ligand-dependent nuclear receptor and regulates adipogenesis and associates with diabetes, obesity. Recently, relationship between PPARgamma and bone metabolism was investigated. Here, we report the molecular mechanism of PPARgamma transcriptional activity and the suppression of PPARgamma activity by cytokines in bone marrow-derived mesenchymal stem cells.

Peroxisome proliferator-activated receptor-gamma activation inhibits tumor progression in non-small-cell lung cancer.

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors and a crucial regulator of cellular differentiation. Differentiation-inducing and antiproliferative effects of PPAR-gamma suggest that PPAR-gamma agonists might be useful as effective anticancer agents. Few studies have examined the efficacy of these agonists in animal models of tumorigenesis, and their mechanism(s) of action are still not clear. Our studies indicate higher PPAR-gamma expression in primary tumors from non-small-cell lung cancer (NSCLC) patients when compared to normal surrounding tissue. The expression of PPAR-gamma was also observed in several NSCLC lines. The treatment of lung adenocarcinoma cells (A549) with troglitazone (Tro), a PPAR-gamma ligand, enhanced PPAR-gamma transcriptional activity and induced a dose-dependent inhibition of A549 cell growth. The observed growth arrest was predominantly due to the inhibition of cell proliferation without significant induction of apoptosis. Cell cycle analysis of Tro-treated cells revealed a cell cycle arrest at G(0)/G(1) with concomitant downregulation of G(0)/G(1) cyclins D and E. In addition, Tro treatment stimulated sustained Erk1/2 activation in A549 cells, suggesting the activation of a differentiation-inducing pathway. Furthermore, treatment of A549 tumor-bearing SCID mice with Tro or Pio inhibited primary tumor growth by 66.7% and significantly inhibited the number of spontaneous lung metastatic lesions. Collectively, our data demonstrate that activation of PPAR-gamma impedes lung tumor progression and suggest that PPAR-gamma ligands may serve as potential therapeutic agents for NSCLC.

Peroxisome proliferator-activated receptor-gamma activator 15-deoxy-Delta12,14-prostaglandin J2 inhibits neuroblastoma cell growth through induction of apoptosis: association with extracellular signal-regulated kinase signal pathway.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been demonstrated to inhibit growth of several cancer cells. Here, we investigated whether one of the PPAR-gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (15-deoxy-PGJ2) inhibits cell growth of two human neuroblastoma cells (SK-N-SH and SK-N-MC) in a PPAR-gamma-dependent manner. PPAR-gamma was expressed in these cells, and 15-deoxy-PGJ2 increased expression, DNA binding activity, and transcriptional activity of PPAR-gamma. 15-Deoxy-PGJ2 also inhibited cell growth in time- and dose-dependent manners in both cells. Cells were arrested in G2/M phase after 15-deoxy-PGJ2 treatment with concomitant increase in the expression of G2/M phase regulatory protein cyclin B1 but decrease in the expression of cdk2, cdk4, cyclin A, cyclin D1, cyclin E, and cdc25C. Conversely, related to the growth inhibitory effect, 15-deoxy-PGJ2 increased the induction of apoptosis in a dose-dependent manner. Consistent with the induction of apoptosis, 15-deoxy-PGJ2 increased the expression of proapoptotic proteins caspase 3, caspase 9, and Bax but down-regulated antiapoptotic protein Bcl-2. 15-Deoxy-PGJ2 also activated extracellular signal-regulated kinase (ERK) 2. In addition, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor PD98059 (2'-amino-3'-methoxyflavone) decreased 15-deoxy-PGJ2-induced ERK2 activation, and expression of PPAR-gamma, capase-3, and cyclin B1. Moreover, MEK1/2 inhibitor PD98059 significantly prevented against the 15-deoxy-PGJ2-induced cell growth inhibition. We also found that PPAR-gamma antagonist GW9662 (2-chloro-5-nitro-N-phenylbenzamide) reversed the 15-deoxy-PGJ2-induced cell growth inhibition, PPAR-gamma expression, and activation of ERK2. These results demonstrate that 15-deoxy-PGJ2 inhibits growth of human neuroblastoma cells via the induction of apoptosis in a PPAR-gamma-dependent manner through activation of ERK pathway and suggest that 15-deoxy-PGJ2 may have promising application as a therapeutic agent for neuroblastoma.

Coactivator PRIP, the Peroxisome Proliferator-activated Receptor-interacting Protein, Is a Modulator of Placental, Cardiac, Hepatic, and Embryonic Development

Nuclear receptor coactivator PRIP (peroxisome proliferator-activated receptor (PPAR gamma)-interacting protein) and PRIP-interacting protein with methyltransferase activity, designated PIMT, appear to serve as linkers between cAMP response element-binding protein-binding protein (CBP)/p300-anchored and PBP (PPAR gamma-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. To assess the biological significance of PRIP, we disrupted the PRIP gene in mice by homologous recombination. Mice nullizygous for PRIP died between embryonic day 11.5 and 12.5 (postcoitum) due in most part to defects in the development of placenta, heart, liver, nervous system, and retardation of embryonic growth. Transient transfection assays using fibroblasts isolated from PRIP(-/-) embryos revealed a significant decrease in the capacity for ligand-dependent transcriptional activation of retinoid X receptor alpha and to a lesser effect on PPAR gamma transcriptional activity. These observations indicate that PRIP like PBP, CBP, and p300 is an essential and nonredundant coactivator.

Hepatic stellate cells as a target for TZD: Can we treat liver fibrosis and type II diabetes at the same time?

Background & Aims: The ligand-dependent transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in hepatic stellate cells (HSC), and its transcriptional activity is reduced during cell transdifferentiation in culture. PPARgamma transcriptional activation decreases platelet-derived growth factor-induced proliferation and inhibits alpha-smooth muscle actin expression in cultured HSC. The aim of our study was to evaluate whether oral administration of synthetic PPARgamma ligands, thiazolidinediones (TZD), might affect collagen deposition in animal models of liver fibrosis. Methods: The effect of 2 TZD (pioglitazone or rosiglitazone) was tested on liver fibrosis induced in rats by either toxin administration (dimethylnitrosamine or carbon tetrachloride) or bile duct ligation. In vivo PPARgamma activation was evaluated by gel shift assay using nuclear extracts from HSC isolated from control and treated rats. Results: Oral administration of TZD reduced extracellular matrix deposition and HSC activation in both toxic and cholestatic models of liver fibrosis. PPARgamma-specific DNA binding was significantly impaired in nuclear extracts of HSC isolated from fibrotic rats compared with HSC from control rats. TZD administration restored PPARgamma DNA binding in HSC nuclei. In vitro, TZD-induced PPARgamma activation inhibited collagen and fibronectin synthesis induced by transforming growth factor (TGF)-betal in human HSC, as measured by enzyme-linked immunosorbent assay and Northen blotting. TZD also reduced the TGF-beta1-induced activity of a 3.5-kilobase procollagen type I promoter transfected in human HSC. conclusions: These findings indicate that PPARgamma activation in HSC retards fibrosis in vivo and suggest the use of TZD for the treatment of liver fibrosis.

A novel potent antagonist of peroxisome proliferator-activated receptor gamma blocks adipocyte differentiation but does not revert the phenotype of terminally differentiated adipocytes.

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Their antihyperglycemic effects seem to be linked to the regulation of PPARgamma-responsive genes. Here, we report the characterization of a specific PPARgamma antagonist that blocks several of the biological activities of the PPARgamma agonist rosiglitazone. PD068235 inhibited rosiglitazone-dependent PPARgamma transcriptional activity with an IC(50) of 0.8 microM and rosiglitazone-stimulated in vitro coactivator association. The role of PPARgamma in the initiation of differentiation is well documented. In this study, we used PD068235 as a tool to evaluate the functional role of PPARgamma in the maintenance of the terminally differentiated state. Treatment of confluent, growth-arrested 3T3-L1 preadipocytes with PD068235 blocked adipocyte differentiation induced by the standard adipogenic hormonal mixture (insulin/dexamethasone/isobutylmethylxanthin) and fully antagonized rosiglitazone-induced adipogenesis. In contrast, long-term treatment of terminally differentiated 3T3-L1 adipocytes with PD068235 did not induce any obvious morphological changes and had no effect on basal lipolysis rates. In addition, in fully differentiated adipocytes PD068235 did not alter the basal expression of PPARgamma target genes aP2 and CAP, but it effectively blocked rosiglitazone-induced expression of both genes. These results suggest that in terminally differentiated adipocytes, the PPARgamma activity is minimal and may not be required for the maintenance of PPARgamma target gene expression.

Peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity is inhibited by acetaldehyde in human hepatic stellate cells (HSC)

Peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity is inhibited by acetaldehyde in human hepatic stellate cells (HSC)

Peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity is inhibited by acetaldehyde in human hepatic stellate cells (HSC)

Peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity is inhibited by acetaldehyde in human hepatic stellate cells (HSC)

The role of PPARgamma as a thrifty gene both in mice and humans.

The biological role of peroxisome proliferator-activated receptor gamma (PPARgamma) was investigated by gene targeting and case-control study of the Pro12Ala PPARgamma2 polymorphism. Homozygous PPARgamma-deficient embryos died at 10.5-11.5 days post conception (dpc) due to placental dysfunction. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet, whose phenotypes were abrogated by PPARgamma agonist treatment. Heterozygous PPARgamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPARgamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPARgamma. A Pro12Ala polymorphism has been detected in the human PPARgamma2 gene. Since this amino acid substitution may cause a reduction in the transcriptional activity of PPARgamma, this polymorphism may be associated with decreased insulin resistance and decreased risk of type 2 diabetes. To investigate this hypothesis, we performed a case-control study of the Pro12Ala PPARgamma2 polymorphism. In an obese group, subjects with Ala12 were more insulin sensitive than those without. The frequency of Ala12 was significantly lower in the diabetic group, suggesting that this polymorphism protects against type 2 diabetes. These results revealed that in both mice and humans, PPARgamma is a thrifty gene mediating type 2 diabetes.

Differential activation of peroxisome proliferator-activated receptor-gamma by troglitazone and rosiglitazone.

The antidiabetic thiazolidinediones, which include troglitazone and rosiglitazone, are ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma and exert their antihyperglycemic effects by regulation of PPAR-gamma-responsive genes. We report here that PPAR-gamma activation by troglitazone depends on the experimental setting. Troglitazone acts as a partial agonist for PPAR-gamma in transfected muscle (C2C12) and kidney (HEK 293T) cells, producing a submaximal transcriptional response (1.8- to 2.5-fold activation) compared with rosiglitazone (7.4- to 13-fold activation). Additionally, troglitazone antagonizes rosiglitazone-stimulated PPAR-gamma transcriptional activity. Limited protease digestion of PPAR-gamma suggests conformational differences in the receptor bound to troglitazone versus rosiglitazone. Consistent with this finding, an in vitro coactivator association assay demonstrated that troglitazone-bound PPAR-gamma recruited the transcriptional coactivators p300 and steroid receptor coactivator 1 less efficiently than rosiglitazone-bound receptor. In contrast to these observations, troglitazone behaves as a full agonist of PPAR-gamma in 3T3L1 adipocytes. Two-dimensional protein gel electrophoresis demonstrated that troglitazone and rosiglitazone regulated distinct but overlapping sets of genes in several cell types. Thus, troglitazone may behave as a partial agonist under certain physiological circumstances and as a full agonist in others. These differences could be caused by variations in the amount of specific cofactors, differences in PPAR response elements, or the presence of different isoforms of PPAR-gamma.

A Potent Antidiabetic Thiazolidinedione with Unique Peroxisome Proliferator-activated Receptor γ-activating Properties

Thiazolidinediones (TZDs) constitute an exciting new class of antidiabetic compounds, which function as activating ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Until now, there has been an excellent correlation between in vivo hypoglycemic potency and in vitro binding and activation of PPARgamma by TZDs. We have characterized MCC-555, a novel thiazolidinedione ligand for PPARgamma with unique functional properties. The antidiabetic potency of this compound is greater than that of other TZDs, including BRL49653, yet its binding affinity for PPARgamma is less than (1)/(10) that of BRL49653. The effect of MCC-555 binding on PPARgamma transcriptional activity is highly context-specific such that it can function as a full agonist, partial agonist, or antagonist depending on the cell type or DNA binding site. These transcriptional properties are partly explained by unique partial agonism of coactivator recruitment to PPARgamma. The properties of MCC-555 are mechanistically distinct from those of the estrogen receptor partial agonist and antagonist tamoxifen because the N terminus of PPARgamma is not required for activation by MCC-555, and MCC-555 does not stimulate corepressor recruitment to PPARgamma. The context selectivity of MCC-555 may contribute to its enhanced hypoglycemic potency in vivo despite reduced affinity for PPARgamma relative to other TZDs.

Interdomain communication regulating ligand binding by PPAR-gamma.

Binding to receptors in the cell nucleus is crucial for the action of lipophilic hormones and ligands. PPAR-gamma (for peroxisome proliferator-activated receptor) is a nuclear hormone receptor that mediates adipocyte differentiation and modulates insulin sensitivity, cell proliferation and inflammatory processes. PPAR-gamma ligands have been implicated in the development of atherogenic foam cells and as potential cancer treatments. Transcriptional activity of PPAR-gamma is induced by binding diverse ligands, including natural fatty acid derivatives, antidiabetic thiazolidinediones, and non-steroidal anti-inflammatory drugs. Ligand binding by PPAR-gamma, as well as by the entire nuclear-receptor superfamily, is an independent property of the carboxy-terminal ligand-binding domain (LBD) of the receptor. Here we show that ligand binding by PPAR-gamma is regulated by intramolecular communication between its amino-terminal A/B domain and its carboxy-terminal LBD. Modification of the A/B domain, for example by physiological phosphorylation by MAP kinase, reduces ligand-binding affinity, thus negatively regulating the transcriptional and biological functions of PPAR-gamma. The ability of the A/B domain to regulate ligand binding has important implications for the evaluation and mechanism of action of potentially therapeutic ligands that bind PPAR-gamma and that are likely to extend to other members of the nuclear-receptor superfamily.