Effect of peroxisome proliferator-activated receptor gamma on thromboxane A(2) and prostaglandin E(2) production in macrophage cell lines

We studied the effect of peroxisome proliferator-activated receptor gamma (PPARgamma) activation on thromboxane A(2)(TXA(2)) and prostaglandin E(2)(PGE(2)) production in monocyte/macrophage cell lines. In present experiment, we used human peripheral blood monocyte (PBMC), monocyte-cell line THP-1 and mouse macrophage-like cell line RAW264.7. The expression of PPARgamma is reported in PBMC and THP-1. Synthetic PPARgamma ligands (troglitazone or BRL49653) inhibited TXA(2) production and enhanced PGE(2) production of PBMC and THP-1. When treated with 0.5-10 microM of troglitazone, there were no significant changes of TXA(2) and PGE(2) production of RAW264.7 cells, which express very low levels of PPARgamma. When RAW264.7 cells was transfected with PPARgamma expression plasmid and treated with troglitazone, PPARgamma was activated in a dose-dependent manner. In PPARgamma-transfected RAW264.7, TXA(2) production was decreased and PGE(2) production was increased by troglitazone treatment. But it needs high concentration of troglitazone (10 microM) for increasing PGE(2) production. These results suggest that PPARgamma may have negative effect on TXA(2) production, and also have slightly positive effect on PGE(2) production of macrophage.     

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.     

Pro-MMP-2 activation by the PPARgamma agonist, ciglitazone, induces cell invasion through the generation of ROS and the activation of ERK

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor modulating a variety of biological functions including cancer cell proliferation and differentiation. However, the role of PPARgamma and its ligands in tumor invasion is unclear. To evaluate a possible role for PPARgamma ligands in tumor invasion, we examined whether PPARgamma agonists including pioglitazone, troglitazone, rosiglitazone, and ciglitazone could affect the activity of matrix metalloproteinases (MMPs) in the HT1080 cell line, a well-studied and well-characterized cell line for MMP research. The gelatin zymography assay showed that ciglitazone activated pro-MMP-2 significantly. In addition, ciglitazone increased the expression of MMP-2, which was accompanied by an increase of membrane type 1-MMP (MT1-MMP) expression. The PPARgamma antagonist, GW9662 attenuated the ciglitazone-induced PPARgamma activation but it did not affect the pro-MMP2 activation by ciglitazone, suggesting that the action of ciglitazone on the pro-MMP-2 activation bypassed the PPARgamma pathway. Antioxidants and various inhibitors of signal transduction were used to investigate the mechanism of ciglitazone-induced pro-MMP-2 activation. We found that the sustained production of reactive oxygen species (ROS) was required for pro-MMP-2 activation by ciglitazone. We also found that PB98059, an inhibitor of MEK-ERK, significantly blocked ciglitazone-induced pro-MMP-2 activation and that extracellular signal-regulated kinase (ERK) was hyperphosphorylated by ciglitazone. Moreover, cell invasion was significantly increased by ciglitazone in the HT1080 cell lines, whereas cell motility was not affected. This study suggests that ciglitazone-induced pro-MMP-2 activation increases PPARgamma-independent tumor cell invasion through ROS production and ERK activation in some types of cancer cells.     

Regulation of prostaglandin endoperoxide synthase-2 and IL-6 expression in mouse bone marrow-derived mast cells by exogenous but not endogenous prostanoids.

Mouse bone marrow-derived mast cells (BMMC), stimulated with stem cell factor, IL-1beta, and IL-10, secrete IL-6 and demonstrate a delayed phase of PGD(2) generation that is dependent upon the induced expression of PG endoperoxide synthase (PGHS)-2. We have examined the potential for exogenous prostanoids, acting in a paracrine fashion, and endogenous prostanoids, acting in an autocrine fashion, to regulate PGHS-2 induction and IL-6 secretion in mouse BMMC. Exogenous PGE(2), which acts through G protein-coupled receptors, and 15-deoxy-Delta(12,14)-PGJ(2), which is a ligand for peroxisome proliferator-activated receptor (PPAR)gamma, elicited a 2- to 3-fold amplification of PGHS-2 induction, delayed-phase PGD(2) generation, and IL-6 secretion in response to stem cell factor, IL-1beta, and IL-10. The effect of PGE(2) was reproduced by the E prostanoid (EP)1 receptor agonist 17-trinor-PGE(2), and the EP1/EP3 agonist, sulprostone, but not the EP2 receptor agonist, butaprost. Although BMMC express PPARgamma, the effects of 15-deoxy-Delta(12,14)-PGJ(2) were not reproduced by the PPARgamma agonists, troglitazone and ciglitazone. PGHS-2 induction, but not IL-6 secretion, was impaired in cPLA(2)-deficient BMMC. However, there was no impairment of PGHS-2 induction in BMMC deficient in hematopoietic PGD synthase or PGHS-1 in the presence or absence of the PGHS-2 inhibitor, NS-398. Thus, although exogenous prostanoids may contribute to amplification of the inflammatory response by augmenting PGD(2) generation and IL-6 secretion from mast cells, endogenous prostanoids do not play a role.     

Peroxisome proliferator-activated receptor (PPAR)gamma is expressed by human spermatozoa: its potential role on the sperm physiology

The peroxisome proliferation-activated receptor gamma (PPARgamma) is mainly expressed in the adipose tissue and integrates the control of energy, lipid, and glucose homeostasis. The present study, by means of RT-PCR, Western blot, and immunofluorescence techniques, demonstrates that human sperm express the PPARgamma. The functionality of the receptor was evidenced by 15-deoxy-12,14-prostaglandin J(2) (PGJ2) and rosiglitazone (BRL) PPARgamma-agonists that were tested on capacitation, acrosome reaction, and motility. Both treatments also increase AKT phosphorylations and influence glucose and lipid metabolism in sperm. The specificity of PGJ2 and BRL effects through PPARgamma on human sperm was confirmed by an irreversible PPARgamma antagonist, GW9662. Our findings provide evidence that human sperm express a functional PPARgamma whose activation influences sperm physiology. In conclusion, the presence of PPARgamma in male gamete broadens the field of action of this nuclear receptor, bringing us to look towards sperm as an endocrine mobile unit independent of the systemic regulation.     

Effect of PPAR activators on cytokine-stimulated cyclooxygenase-2 expression in human colorectal carcinoma cells.

Cyclooxygenase-2 (COX-2) expression is up-regulated in colorectal cancer tissue. Peroxisome proliferator-activated receptors (PPARs) are expressed in human colorectal tissue and activation of PPARs can alter COX-2 expression. In macrophages, activation of PPARs down-regulates COX-2 expression. We examined the effect of PPARalpha and PPARgamma ligands on untreated and TNF-alpha-induced COX-2 expression in the human colorectal epithelial cell line HT-29. The expression of PPARalpha and PPARgamma was confirmed in these cells. TNF-alpha, an inflammatory cytokine, increased COX-2 expression via the NFkappaB pathway. In the absence of TNF-alpha, WY14643 (PPARalpha activator) caused an increase, while BRL49653 (PPARgamma activator) did not alter COX-2 expression. When HT-29 cells were incubated with TNF-alpha and WY14643, a further increase in COX-2 expression was detected. Incubation with TNF-alpha and BRL49653 caused an additional twofold increase in COX-2 expression. Our results suggest that both PPARalpha signaling and TNF-alpha signaling increase COX-2 expression by independent pathways, while PPARgamma stimulates COX-2 expression by up-regulation of the TNF-alpha pathway.     

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.     

Proline oxidase, a proapoptotic gene, is induced by troglitazone: evidence for both peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms

Proline oxidase (POX) is a redox enzyme localized in the mitochondrial inner membrane. We and others have shown that POX is a p53-induced gene that can mediate apoptosis through generation of reactive oxygen species (ROS). The peroxisome proliferator-activated receptor gamma (PPARgamma) ligand troglitazone was found to activate the POX promoter in colon cancer cells. PPARgamma ligands have been reported to induce apoptosis in a variety of cancer cells. In HCT116 cells expressing a wild-type PPARgamma, troglitazone enhanced the binding of PPARgamma to PPAR-responsive element in the POX promoter and increased endogenous POX expression. Blocking of PPARgamma activation either by antagonist GW9662 or deletion of PPAR-responsive element in the POX promoter only partially decreased the POX promoter activation in response to troglitazone, indicating also the involvement of PPARgamma-independent mechanisms. Further, troglitazone also induced p53 protein expression in HCT116 cells, which may be the possible mechanism for PPARgamma-independent POX activation, since POX has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15 cells, with both mutant p53 and mutant PPARgamma, there was no effect of troglitazone on POX activation, whereas in HT29 cells, with a mutant p53 and wild type PPARgamma, increased activation was observed by ligand stimulation, indicating that both PPARgamma-dependent and -independent mechanisms are involved in the troglitazone-induced POX expression. A time- and dose-dependent increase in POX catalytic activity was obtained in HCT116 cells treated with troglitazone with a concomitant increase in the production of intracellular ROS. Our results suggest that the induction of apoptosis by troglitazone may, at least in part, be mediated by targeting POX gene expression for generation of ROS by POX both by PPARgamma-dependent and -independent mechanisms.     

A novel method for analysis of nuclear receptor function at natural promoters: peroxisome proliferator-activated receptor gamma agonist actions on aP2 gene expression detected using branched DNA messenger RNA quantitation

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.