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.     

Regulation of peroxisome proliferator-activated receptor-gamma in liver fibrosis

The peroxisome proliferator-activated receptors (PPARs) impart diverse cellular effects in biological systems. Because stellate cell activation during liver injury is associated with declining PPARgamma expression, we hypothesized that its expression is critical in stellate cell-mediated fibrogenesis. We therefore modulated its expression during liver injury in vivo. PPARgamma was depleted in rat livers by using an adenovirus-Cre recombinase system. PPARgamma was overexpressed by using an additional adenoviral vector (AdPPARgamma). Bile duct ligation was utilized to induce stellate cell activation and liver fibrosis in vivo; phenotypic effects (collagen I, smooth muscle alpha-actin, hydroxyproline content, etc.) were measured. PPARgamma mRNA levels decreased fivefold and PPARgamma protein was undetectable in stellate cells after culture-induced activation. During activation in vivo, collagen accumulation, assessed histomorphometrically and by hydroxyproline content, was significantly increased after PPARgamma depletion compared with controls (1.28 +/- 0.14 vs. 1.89 +/- 0.21 mg/g liver tissue, P < 0.03). In isolated stellate cells, AdPPARgamma overexpression resulted in significantly increased adiponectin mRNA expression and decreased collagen I and smooth muscle alpha-actin mRNA expression compared with controls. During in vivo fibrogenesis, rat livers exposed to AdPPARgamma had significantly less fibrosis than controls. Collagen I and smooth muscle alpha-actin mRNA expression were significantly reduced in AdPPARgamma-infected rats compared with controls (P < 0.05, n = 10). PPARgamma-deficient mice exhibited enhanced fibrogenesis after liver injury, whereas PPARgamma receptor overexpression in vivo attenuated stellate cell activation and fibrosis. The data highlight a critical role for PPARgamma during in vivo fibrogenesis and emphasize the importance of the PPARgamma pathway in stellate cells during liver injury.     

Activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) by atorvastatin is mediated by 15-deoxy-delta-12,14-PGJ2

Several studies suggested that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) activate peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Atorvastatin (ATV) increases myocardial levels of prostaglandins (PG) by upregulating and activating cytosolic-phospholipase-A(2) and cycloxygenase-2 (COX2). We investigated whether ATV activates PPAR-gamma via 15-deoxy-delta-12,14-PGJ(2) (15DPGJ(2)) an endogenous ligand of PPAR-gamma and a product of PGD(2), and to compare the effects of pioglitazone (PIO), a known direct PPAR-gamma activator, to that of ATV. First we measured myocardial 15DPGJ(2) levels in the rat heart after a 3-day pretreatment with oral ATV (10 mg/(kg d)), PIO (10 mg/(kg d)), ATV+PIO, ATV+COX1 inhibitor, and ATV+COX2 inhibitor. We also assessed in human umbilical venous endothelial cells (HUVEC) whether ATV and PIO activate PPAR-gamma via 15DPGJ(2) using siRNA targeted to PGD(2) synthase. Both 15DPGJ(2) levels and PPAR-gamma activation were assessed. ATV and PIO increased myocardial 15DPGJ(2) levels in the rat myocardium and HUVEC. siRNA inhibited this increase in both groups. Both ATV and PIO augmented PPAR-gamma activation while co-treatment with siRNA completely blocked the ATV effect but only partially inhibited the PIO effect. In conclusion, both ATV and PIO activate PPAR-gamma and increase myocardial 15DPGJ(2) levels. Activation of PPAR-gamma by ATV is mediated solely by 15DPGJ(2), whereas PIO activates PPAR-gamma both directly and indirectly via 15DPGJ(2).     

Synergistic interactions between heregulin and peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist in breast cancer cells

Here, we demonstrate that troglitazone (Rezulin), a peroxisome proliferator-activated receptor agonist, acted in synergy with heregulin to induce massive cell death in breast cancer cells. Although the combination of heregulin and troglitazone (HRG/TGZ) induced both apoptosis and necrosis, the main mode of cell death was caspase-independent and occurred via necrosis. This combination increased generation of superoxide in mitochondria, which in turn destabilized mitochondria potential. Pretreatment with N-acetyl-l-cysteine and catalase expression ameliorated cell death induced by the combination treatment, indicating a role of oxidative stress in mediating HRG/TGZ-induced cell death. Notably, pretreatment with pyruvate significantly prevented the cell death, suggesting a potential mechanistic link between metabolic stress and HRG/TGZ-induced cell death. The activation of the HRG signaling axis has been considered as a poor prognostic factor in breast cancer and confers resistance to gefitinib (Iressa) and tamoxifen. However, our data presented here paradoxically suggest that HRG expression can actually be beneficial when it comes to treating breast cancer with peroxisome proliferator-activated receptor-γ ligands. Taken together, the combination of HRG and TGZ may provide a basis for the development of a novel strategy in the treatment of apoptosis-resistant and/or hormone-refractory breast cancer.     

Production of prostaglandin D2 by murine macrophage cell lines

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.     

Cyclooxygenase-2-derived prostaglandin D(2) is an early anti-inflammatory signal in experimental colitis

The ability of nonsteroidal anti-inflammatory drugs and cyclooxygenase-2 inhibitors to exacerbate inflammatory bowel disease suggests that prostaglandins are important anti-inflammatory mediators in this context. Prostaglandin D(2) has been suggested to exert anti-inflammatory effects. We investigated the possibility that prostaglandin D(2) derived from cyclooxygenase-2 plays an important role in downregulating colonic inflammation in rats. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various times thereafter (from 1 h to 7 days), colonic prostaglandin synthesis and myeloperoxidase activity (index of granulocyte infiltration) were measured. Prostaglandin D(2) synthesis was elevated >4-fold above controls within 1-3 h of induction of colitis, preceding significant granulocyte infiltration. Treatment with a selective cyclooxygenase-2 inhibitor abolished the increase in prostaglandin D(2) synthesis and caused a doubling of granulocyte infiltration. Colonic granulocyte infiltration was significantly reduced by administration of prostaglandin D(2) or a DP receptor agonist (BW-245C). These results demonstrate that induction of colitis results in a rapid increase in prostaglandin D(2) synthesis via cyclooxygenase-2. Prostaglandin D(2) downregulates granulocyte infiltration into the colonic mucosa, probably through the DP receptor.     

Microarray analysis of peroxisome proliferator-activated receptor-gamma induced changes in gene expression in macrophages

We used a combination of expression microarray and Northern blot analyses to identify target genes for peroxisome proliferator-activated receptor (PPAR) gamma in RAW264.7 macrophages. PPARgamma natural ligand 15-deoxy-Delta(12,14) prostaglandin and synthetic ligands ciglitazone and rosiglitazone increased the expression of scavenger receptor CD36 and ATP-binding cassette transporter A1, as well as adipophilin (a lipid droplet coating protein involved in intracellular lipid storage and transport), calpain (a protease implicated in ABCA1 protein degradation), and ADAM8 (a disintegrin and metalloprotease protein involved in cell adhesion). These findings are relevant to understanding the effect of PPARgamma activation on gene expression and cognate pathways in macrophages.     

Novel biphenylcarboxylic acid peroxisome proliferator-activated receptor (PPAR) δ selective antagonists

We designed and synthesized novel PPARδ antagonists based on the crystal structure of the PPARδ full agonist TIPP-204 bound to the PPARδ ligand-binding domain, in combination with our nuclear receptor helix 12 folding modification hypothesis. Representative compound 3a exhibits PPARδ-preferential antagonistic activity.     

Pioglitazone and dexamethasone induce adipogenesis in D1 bone marrow stromal cell line, but not through the peroxisome proliferator-activated receptor-gamma pathway

Osteoblasts and adipocytes share a common progenitor in bone marrow. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in adipogenesis. Using a mouse pluripotent mesenchymal cell, D1, as a model, several reports have demonstrated that dexamethasone, a glucocorticoid, can induce adipogenesis. We first examined whether adipogenesis induction in D1 cells is initiated by activation of PPAR-gamma. The results revealed that pioglitazone induces adipogenesis in D1 cells in a dose-dependent manner and decreases alkaline phosphatase activity in D1 cells. Interestingly, this adipogenesis was not blocked by bisphenol A diglycidyl ether, a PPAR-gamma antagonist. A PPAR-gamma-mediated reporter gene assay showed no response to pioglitazone. We then asked whether dexamethasone-induced adipogenesis can be repressed by mifepristone (RU486), an antagonist of glucocorticoid receptor. The results disclosed that mifepristone cannot counteract dexamethasone-induced adipogenesis, and mifepristone itself induced adipogenesis in D1 cells. Moreover, glucocorticoid receptor-mediated reporter gene assay was not responsive to dexamethasone or mifepristone. We concluded that the adipogenesis induced by pioglitazone and dexamethasone in D1 cells may not occur via a PPAR-gamma and glucocorticoid receptor pathway. Finally, we analyzed the gene expression profile of D1 by cDNA microarray after treatment with dexamethasone. We found that the expression of several adipogenesis-related genes is highly provoked by this agent.     

Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPARγ). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-κB activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression via PPARγ activation.     

Beta-arrestin-1 protein represses adipogenesis and inflammatory responses through its interaction with peroxisome proliferator-activated receptor-gamma (PPARgamma)

One of the master regulators of adipogenesis and macrophage function is peroxisome proliferator-activated receptor-γ (PPARγ). Here, we report that a deficiency of β-arrestin-1 expression affects PPARγ-mediated expression of lipid metabolic genes and inflammatory genes. Further mechanistic studies revealed that β-arrestin-1 interacts with PPARγ. β-Arrestin-1 suppressed the formation of a complex between PPARγ and 9-cis-retinoic acid receptor-α through its direct interaction with PPARγ. The interaction of β-arrestin-1 with PPARγ repressed PPARγ/9-cis-retinoic acid receptor-α function but promoted PPARγ/nuclear receptor corepressor function in PPARγ-mediated adipogenesis and inflammatory gene expression. Consistent with these results, a deficiency of β-arrestin-1 binding to PPARγ abolished its suppression of PPARγ-dependent adipogenesis and inflammatory responses. These results indicate that the regulation of PPARγ by β-arrestin-1 is critical. Furthermore, in vivo expression of β-arrestin-1 (but not the binding-deficient mutant) significantly repressed adipogenesis, macrophage infiltration, and diet-induced obesity and improved glucose tolerance and systemic insulin sensitivity. Therefore, our findings not only reveal a molecular mechanism for the modulation of obesity by β-arrestin-1 but also suggest a potential tactical approach against obesity and its associated metabolic disorders.     

IL-13 attenuates gastrointestinal candidiasis in normal and immunodeficient RAG-2(-/-) mice via peroxisome proliferator-activated receptor-gamma activation

We recently demonstrated that in vitro peroxisome proliferator-activated receptor-gamma (PPARgamma) activation of mouse peritoneal macrophages by IL-13 or PPARgamma ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2(-/-)) mice with natural and synthetic PPARgamma-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARgamma antagonist, and are reduced in PPARgamma(+/-) mice. Overall, these data demonstrate that IL-13 or PPARgamma ligands attenuate C. albicans infection of the GI tract through PPARgamma activation and hence suggest that PPARgamma ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.     

Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep

Peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that is activated by fatty acids and derivatives and the antidiabetic glitazones, which plays a role in the control of lipid and glucose homeostasis. In the present work, we tested the hypothesis that PPARgamma plays a role in reproductive tissues by studying its expression and function in the hypothalamo-pituitary-ovary axis in the sheep. PPARgamma 1 and PPARgamma 2 proteins and mRNAs were detected in whole ovine pituitary and ovary but not in hypothalamic extracts. In situ hybridization on ovarian section localized PPARgamma mRNA in the granulosa layer of follicles. Interestingly, PPARgamma expression was higher in small antral (1-3 mm diameter) than in preovulatory follicles (>5 mm diameter) (P < 0.001) and was not correlated with healthy status. To assess the biological activity of ovarian PPARgamma, ovine granulosa cells were transfected with a reporter construct driven by PPARgamma-responsive elements. Addition of rosiglitazone, a PPARgamma ligand, stimulated reporter gene expression, showing that endogenous PPARgamma is functional in ovine granulosa cells in vitro. Moreover, rosiglitazone inhibited granulosa cell proliferation (P < 0.05) and increased the secretion of progesterone in vitro (P < 0.05). This stimulation effect was stronger in granulosa cells from small than from large follicles. In contrast, rosiglitazone had no effect on LH, FSH, prolactin and growth hormone secretion by ovine pituitary cells in vitro. Overall, these data suggest that PPARgamma ligands might stimulate follicular differentiation in vivo likely through a direct action on granulosa cells rather than by modulating pituitary hormone secretion.