Recognition of apoptotic cells by macrophages activates the peroxisome proliferator-activated receptor-gamma and attenuates the oxidative burst

It is appreciated that phagocytosis of apoptotic cells (AC) is an immunological relevant process that shapes the pro- versus anti-inflammatory macrophage phenotype. It was our intention to study the respiratory burst, a prototype marker of macrophage activation, under the impact of AC. Following incubation of RAW264.7 macrophages with AC, we noticed attenuated production of reactive oxygen species (ROS) in response to PMA treatment, and observed a correlation between attenuated ROS formation and suppression of protein kinase Calpha (PKCalpha) activation. EMSA analysis demonstrated an immediate activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) following supplementation of AC to macrophages. In macrophages carrying a dominant-negative PPARgamma mutant, recognition of AC no longer suppressed PKCalpha activation, and the initial phase of ROS formation was largely restored. Interference with actin polymerization and transwell experiments suggest that recognition of AC by macrophages suffices to attenuate the early phase of ROS formation that is attributed to PPARgamma activation.     

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.     

Roles of peroxisome proliferator-activated receptor gamma in lipid homeostasis and inflammatory responses of macrophages

Monocytes play a critical role in atherogenesis by their inflammatory signals and differentiation into macrophage foam cells through cholesterol accumulation. The seminal finding of high levels of the peroxisome proliferator-activated receptor gamma in macrophage foam cells has opened up the prospect that its ligands, most importantly the thiazolidinedione class of drugs, might directly influence the development of atheromatous lesions. The present review weighs the growing evidence on regulation of both inflammatory responses and cholesterol homeostasis in macrophages by peroxisome proliferator-activated receptor gamma ligands with regard to their overall impact as antiatherogenic agents.     

Troglitazone regulates peroxisome proliferator-activated receptors and inducible nitric oxide synthase in murine ovarian macrophages

Peroxisome proliferator-activated receptor-gamma (PPARG) and PPAR-alpha (PPARA) control metabolic processes in many cell types and act as anti-inflammatory regulators in macrophages. PPAR-activating ligands include thiazolidinediones (TZDs), such as troglitazone, once frequently used to treat insulin resistance as well as symptoms of polycystic ovary syndrome (PCOS). Since macrophages within the ovary mediate optimal follicle development, TZD actions to improve PCOS symptoms are likely to be partly mediated through these specifically localized immune cells. In mouse ovary, PPARG protein was expressed in granulosa cells and in isolated cells localized to theca, stroma, and corpora lutea, consistent with EMR1+ macrophages. Isolation of immune cells (EMR1+ or H2+) showed that Pparg and Ppara were expressed in ovarian macrophages at much higher levels than in peritoneal macrophages. Ovulatory human chorionic gonadotropin downregulated expression of Pparg and Ppara in EMR1+ ovarian macrophages, but no hormonal responsiveness was observed in H2+ cells. Downstream anti-inflammatory effects of PPARG activation were analyzed by in vitro treatment of isolated macrophages with troglitazone. Interleukin-1 beta (Il1b) expression was not altered, and tumor necrosis factor-alpha (Tnf) expression was affected in peritoneal macrophages only. In ovarian macrophages, inducible nitric oxide synthase (Nos2), an important proinflammatory enzyme that regulates ovulation, was significantly reduced by troglitazone treatment, an effect that was restricted to cells from the preovulatory ovary. Thus, expression of PPARs within ovarian macrophages is hormonally regulated, reflecting the changing roles of these cells during the ovulatory cycle. Additionally, ovarian macrophages respond directly to troglitazone to downregulate expression of proinflammatory Nos2, providing mechanistic information about ovarian effects of TZD treatment.     

Activation of peroxisome proliferator-activated receptor gamma by rosiglitazone increases sirt6 expression and ameliorates hepatic steatosis in rats

Background

Sirt6 has been implicated in the regulation of hepatic lipid metabolism and the development of hepatic steatosis. The aim of this study was to address the potential role of Sirt6 in the protective effects of rosiglitazone (RGZ) on hepatic steatosis.

Methods

To investigate the effect of RGZ on hepatic steatosis, rats were treated with RGZ (4 mg·kg⁻¹·day⁻¹) by stomach gavage for 6 weeks. The involvement of Sirt6 in the RGZ''s regulation was evaluated by Sirt6 knockdown in AML12 mouse hepatocytes.

Results

RGZ treatment ameliorated hepatic lipid accumulation and increased expression of Sirt6, peroxisome proliferator-activated receptor gamma coactivtor-1-α (Ppargc1a/PGC1-α) and Forkhead box O1 (Foxo1) in rat livers. AMP-activated protein kinase (AMPK) phosphorylation was also increased by RGZ, accompanied by alterations in phosphorylation of LKB1. Interestingly, in free fatty acid-treated cells, Sirt6 knockdown increased hepatocyte lipid accumulation measured as increased triglyceride contents (p = 0.035), suggesting that Sirt6 may be beneficial in reducing hepatic fat accumulation. In addition, Sirt6 knockdown abolished the effects of RGZ on hepatocyte fat accumulation, mRNA and protein expression of Ppargc1a/PGC1-α and Foxo1, and phosphorylation levels of LKB1 and AMPK, suggesting that Sirt6 is involved in RGZ-mediated metabolic effects.

Conclusion

Our results demonstrate that RGZ significantly decreased hepatic lipid accumulation, and that this process appeared to be mediated by the activation of the Sirt6-AMPK pathway. We propose Sirt6 as a possible therapeutic target for hepatic steatosis.     

Role of peroxisome proliferator-activated receptor gamma in glucose-induced insulin secretion

Peroxisome proliferator-activated receptor (PPAR) isoforms (α and γ) are known to beexpressed in pancreatic islets as well as in insulin-producing cell lines.Ligands of PPAR have been shoWn toenhance glucose-induced insulin secretion in rat pancreatic islets.However,their effect on insulin secretionis still unclear.To understand the molecular mechanism by which PPAR7 exerts its effect on glucose-induced insulin secretion,we examined the endogenous activity of PPAR isoforms,and studied the PPARyfunction and its target gene expression in INS-1 cells.We found that:(1)endogenous PPARγ was activatedin a ligand-dependent manner in INS-1 cells;(2)overexpression of PPARy in the absence of PPARγ ligandsenhanced glucose-induced insulin secretion,which indicates that the increased glucose-induced insulin secretionis a PPARγ-mediated event;(3)the addition of both PPARγ and retinoid X receptor (RXR) ligands showed asynergistic effect on the augmentation of reporter activity,suggesting that the hetero-dimerization of PPAR7and RXR is required for the regulation of the target genes;(4)PPARs upregulated both the glucose transporter2 (GLUT2) and Cbl-associated protein (CAP) genes in INS-1 cells.Our findings suggest an importantmechanistic pathway in which PPARγ enhances glucose-induced insulin secretion by activating the expressionof GLUT2 and CAP genes in a ligand-dependent manner.     

Activation of peroxisome proliferator-activated receptor gamma inhibits endothelin-1-induced cardiac hypertrophy via the calcineurin/NFAT signaling pathway

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) has been described as a negative regulator of cardiac hypertrophy. A better understanding of PPAR-gamma and cardiac hypertrophy may facilitate the development of novel therapeutic strategies to treat heart diseases related to cardiac hypertrophy by mimicking the naturally preferred mechanisms. In the present study, we investigated the interaction between PPAR-gamma and calcineurin/nuclear factor of activated T-cells (NFAT) in endothelin-1 (ET-1)-induced hypertrophy of neonatal rat cardiac myocytes. The results suggest that the treatment of cultured cardiac myocytes with a PPAR-gamma ligand, rosiglitazone, inhibited the ET-1-induced increase in protein synthesis, surface area, calcineurin enzymatic activity, and protein expression. Both the application of rosiglitazone and overexpression of the PPAR-gamma inhibited the nuclear translocation of NFATc4. Moreover, co-immunoprecipitation studies showed that rosiglitazone enhanced the association between PPAR-gamma and calcineurin/NFAT. These results suggest that ET-1-induced cardiac hypertrophy is inhibited by activation of PPAR-gamma, which is at least partly due to cross-talk between PPAR-gamma and calcineurin/NFAT.     

Activation of peroxisome proliferator-activated receptor gamma inhibits osteoprotegerin gene expression in human aortic smooth muscle cells

Increasing evidence indicates an important role of PPAR gamma activation in modulating the development and progression of atherosclerosis, however, the mechanisms involved in these effects are not well understood since the PPAR gamma-regulated genes in vascular smooth muscle cells (VSMC) are poorly defined. Here we reported that PPAR gamma ligands, GW7845, ciglitazone and troglitazone had the effect of inhibiting osteoprotegerin (OPG) expression in human aortic smooth muscle cells (HASMC). The effect of GW7845 and ciglitazone on OPG expression was completely abolished by GW9662, a PPAR gamma antagonist. Overexpression of PPAR gamma in HASMC by the infection of a PPAR gamma adenovirus dramatically decreased OPG expression. In addition, PPAR gamma activation inhibited OPG promoter activity. Taken together, our data suggest that OPG expression is a novel PPAR gamma target gene in VSMC and downregulation of OPG expression by PPAR gamma activation provides a new insight into the understanding of the role of PPAR gamma in atheroscelrosis and hypertension.     

Activation of peroxisome proliferator-activated receptor gamma does not inhibit IL-6 or TNF-alpha responses of macrophages to lipopolysaccharide in vitro or in vivo

We have investigated the potential use of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARgamma agonists. Although 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-alpha and IL-6, four other high affinity PPARgamma ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARgamma or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARgamma ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARgamma does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARgamma-independent mechanism. To examine the anti-inflammatory activity of PPARgamma agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-alpha and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARgamma in leukocytes will not be useful for the treatment of acute inflammation.     

Activation of human peroxisome proliferator-activated receptor (PPAR) subtypes by pioglitazone

Pioglitazone, a thiazolidinedione (TZD) derivative, is an antidiabetic agent that improves hyperglycaemia and hyperlipidaemia in obese and diabetic animals via a reduction in hepatic and peripheral insulin resistance. The TZDs including pioglitazone have been identified as high affinity ligands for peroxisome proliferator-activated receptor (PPAR) gamma. The selectivity of pioglitazone for the human PPAR subtypes has not been reported, thus, we investigated the effect of pioglitazone on the human PPAR subtypes. Transient transactivation assay showed that pioglitazone is a selective hPPARgamma1 activator and a weak hPPARalpha activator. Binding assay indicated that the transactivation of hPPARgamma1 or hPPARalpha by pioglitazone is due to direct binding of pioglitazone to each subtype. Furthermore, pioglitazone significantly increased the apoA-I secretion from the human hepatoma cell line HepG2.     

Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.     

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.