Activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) induces cell death through MAPK-dependent mechanism in osteoblastic cells

The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPARgamma agonists in osteoblastic cells. Ciglitazone and troglitazone, PPARgamma agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPARalpha agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPARgamma antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.     

Resveratrol inhibits macrophage expression of EMMPRIN by activating PPARgamma

The effect of resveratrol on macrophage EMMPRIN expression and its potential mechanism was investigated. Both EMMPRIN expression and MMP-9 activity, respectively assayed by Western blot and zymography, were greatly up-regulated during PMA-induced macrophage differentiation from THP-1 monocytes. Both resveratrol and a PPARgamma agonist, pioglitazone, significantly inhibited EMMPRIN expression and MMP-9 activity in a concentration-dependent manner. The effects of pioglitazone and resveratrol were reversed by pretreatment of THP-1 cells with a PPARgamma antagonist, GW9662, prior to PMA induction. Thus, data suggest that resveratrol may down-regulate EMMPRIN and MMP-9 through PPARgamma activation. This possibility was further examined in resveratrol-or pioglitazone-treated U937 cells, which had been co-transfected with a PPARgamma expression vector and a luciferase reporter vector containing three tandem repeats of PPRE in cis. Results of the agonist-activated luciferase assay showed that resveratrol activated PPARgamma in a concentration-dependent manner. Since EMMPRIN and MMP-9 up-regulation is associated with activation of the NF-kappaB pathway, we investigated the effect of pioglitazone and resveratrol on TNF-alpha-induced NF-kappaB activation. Western blot results indicated that both pioglitazone and resveratrol markedly inhibited the NF-kappaB pathway through suppressing IkappaB protein phosphorylation in macrophages, although this effect of resveratrol was not reversed by GW9662. In conclusion, resveratrol can down-regulate EMMPRIN expression by macrophages via activating PPARgamma. This may be a primary mechanism of its inhibitory effect on MMP-9.     

Role of mitogen-activated protein kinase (MAPK) in troglitazone-induced osteoblastic cell death

Troglitazone, a PPARgamma agonist, has been reported to induce cell death on different cell types. However, its mechanism of action remains unclear. The present study was undertaken to investigate the effect of troglitazone on cell death and to determine its underlying mechanism in MC3T3-E1 cells, an established osteoblast cell line. Troglitazone induced loss of cell viability in a dose- and time-dependent manner, which was accompanied by apoptosis. Troglitazone increased reactive oxygen species (ROS), but troglitazone-induced cell death was not affected by the antioxidant N-acetylcysteine, suggesting that the ROS generation is not involved in the cytotoxicity of troglitazone. Troglitazone-induced cell death was prevented by the PPARgamma antagonist GW9662. Troglitazone treatment inhibited activation of extracellular signal-regulated protein kinase (ERK) and stimulated p38 activation. Troglitazone-induced cell death was increased by the ERK inhibitor U0126 and prevented by transfection with constitutively active MEK1 and the p38 inhibitor SB203580. Troglitazone induced depolarization of mitochondrial membrane potential and its effect was blocked by SB203580 and GW9662. Caspase-3 was activated by troglitazone treatment and pharmacological inhibition of caspase blocked troglitazone-induced cell death. Taken together, these data suggest that troglitazone induces apoptosis via a caspase-dependent mechanism associated with down-regulation of ERK and up-regulation of p38.     

An L-tyrosine derivative and PPARgamma agonist, GW7845, activates a multifaceted caspase cascade in bone marrow B cells.

Apoptosis is a critical event in the deletion of B lymphocytes prior to their migration to the periphery. Synthetic peroxisome proliferator activated receptor gamma (PPARgamma) agonists, including the drug GW7845 and the environmental contaminant mono-(2-ethylhexyl) phthalate, as well as an endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), induce clonally unrestricted apoptosis in pro/pre-B cells. Considering that PPARgamma agonists are used clinically for the treatment of diabetes and postulated to be useful as chemotherapeutics, we used GW7845 as a model PPARgamma agonist to examine the mechanism of cell death that may contribute to tumor killing as well as normal bone marrow B lymphocyte toxicity. GW7845 induced rapid mitochondrial membrane depolarization and release of cytochrome c, along with nearly concurrent activation of capases-2, -3, -8, and -9 in primary pro-B cells and BU-11 cells, a nontransformed pro/pre-B cell line. GW7845-induced apoptosis was reduced significantly in Bax-deficient and Apaf-1 mutant primary pro-B cells, supporting the conclusion that GW7845-induced apoptosis is mitochondria- and apoptosome-dependent. Using benzyloxycarbonyl-VAD-fluoromethyl ketone (VAD-FMK) as a pan-caspase inhibitor, we demonstrated that an initial cytochrome c release occurred independently of caspase activation and that only caspase-9 activation was partially caspase independent. The attenuation of GW7845-induced apoptosis by multiple FMK-labeled peptide sequences suggests that multiple caspase pathways are responsible for initiating and executing apoptosis. The strong activation of Bid provides a mechanism by which caspases-2, -3, and -8 may amplify the apoptotic signal. These data support the hypothesis that pharmacologic concentrations of PPARgamma agonists induce an intrinsic apoptotic pathway that is driven in normal bone marrow B cells by multiple amplification loops.     

Induction of apoptosis by mono(2-ethylhexyl)phthalate (MEHP) in U937 cells

Treatment of U937 cells with mono(2-ethylhexyl)phthalate (MEHP) for 20 h led to a dose-dependent loss of cell viability, assessed by propidium iodide (PI) staining with fluorescent activated cell sorting (FACS) analysis. The cytotoxic behavior of MEHP is attributed to the induction of apoptosis. MEHP induced activation of caspase-3, internucleosomal DNA fragmentation and the morphological features of nuclear apoptosis. Analysis with LightCycler quantitative RT-PCR demonstrated the decrease of bcl-2 and increase of bax mRNA levels. Peroxisome proliferator-activated receptor (PPAR) gamma antagonists, bisphenol A diglycidyl ether (BADGE) and GW9662, significantly inhibited the MEHP-induced caspase-3 activity and apoptotic nuclear morphological changes. Furthermore, a PPARgamma ligand, rosiglitazone synergized the MEHP-induced caspase-3 activity. These results suggest that MEHP can induce apoptosis in U937 cells through modulation of the balance of bcl-2/bax in part by PPARgamma activation.     

Pleiotropic vascular effects of PPARgamma ligands

Peroxisome proliferator-activated receptor-gamma (PPARgamma) ligands have been used for several years as modulators of insulin sensitivity and glucose metabolism. Recent data from numerous studies have shown that PPARgamma ligands have numerous beneficial effects in the vasculature. They have been shown to regulate proliferation and migration of vascular smooth muscle cells as well as improving endothelial cell function. They improve blood pressure by actions at the resistance arteries and kidneys. Clinical trials have indicated that PPARgamma ligands can minimize the development of atherosclerosis as well as regulating other vascular inflammation. PPARgamma has been detected in all the cells found in the vessel wall as well as those cells associated with vascular pathophysiologies. In the monocyte/macrophage, PPARgamma ligands downregulate production of inflammatory cytokines and migration. Also, PPARgamma ligands regulate the expression of SR-A and CD36 receptors that take up lipids in the macrophage. PPARgamma has also been demonstrated to act through the liver X receptor alpha to increase the activity of reverse cholesterol transport in these cells. In dendritic cells and T-cells, PPARgamma ligands have been shown to inhibit activation and the initiation of inflammation. Inflammatory cytokines are downregulated in animal models administered PPARgamma ligands, leading to decreased atherosclerosis. While PPARgamma ligands have been useful in the treatment of type 2 diabetes, the important vasculoprotection elicited by these compounds could prove to be of greater benefit in the future.     

Role of PPARgamma in the development of the central nervous system

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that plays a central role in adipocyte differentiation and insulin sensitivity. Recently, a diversity of the action of PPARgamma on many other cell types or organs is indicated. We summarize here the possible role of PPARgamma in the development of the murine central nervous system. Expressions of PPARgamma in newborn or adult mouse brain are extremely low, but high in embryo or fetal mouse brain. Furthermore, we investigated the role of PPARgamma in proliferation or differentiation of neural stem cells (NSCs) isolated from murine embryonic brains, because NSCs are considered to be a major source of neurons in developmental brains. Administrations of PPARgamma-specific ligands on the NSCs from wild-type mice resulted in the stimulation of cell growth. On the other hand, administration of PPARgamma-antagonist showed the cell death and apoptosis of NSCs. These results may indicate that PPARgamma plays an important role during the early stage of the development of the central nervous system.     

PPAR gamma ligands, 15-deoxy-delta12,14-prostaglandin J2 and rosiglitazone regulate human cultured airway smooth muscle proliferation through different mechanisms

The influence of two peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, a thiazolidinedione, rosiglitazone (RG) and the prostaglandin D2 metabolite 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) on the proliferation of human cultured airway smooth muscle (HASM) was examined. The increases in HASM cell number in response to basic fibroblast growth factor (bFGF, 300 pm) or thrombin (0.3 U ml-1) were significantly inhibited by either RG (1-10 microM) or 15d-PGJ2 (1-10 microM). The effects of RG, but not 15d-PGJ2, were reversed by the selective PPARgamma antagonist GW9662 (1 microM). Neither RG nor 15d-PGJ2 (10 microM) decreased cell viability, or induced apoptosis, suggesting that the regulation of cell number was due to inhibition of proliferation, rather than increased cell death. Flow-cytometric analysis of HASM cell cycle distribution 24 h after bFGF addition showed that RG prevented the progression of cells from G1 to S phase. In contrast, 15d-PGJ2 caused an increase in the proportion of cells in S phase, and a decrease in G2/M, compared to bFGF alone. Neither RG nor 15d-PGJ2 inhibited ERK phosphorylation measured 6 h post mitogen addition. The bFGF-mediated increase in cyclin D1 protein levels after 8 h was reduced in the presence of 15d-PGJ2, but not RG. Although both RG and 15d-PGJ2 can inhibit proliferation of HASM irrespective of the mitogen used, only the antiproliferative effects of RG appear to be PPARgamma-dependent. The different antimitogenic mechanisms of 15d-PGJ2 and synthetic ligands for PPARgamma may be exploited to optimise the potential for these compounds to inhibit airway remodelling in asthma.British Journal of Pharmacology (2004) 141, 517-525. doi:10.1038/sj.bjp.0705630     

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands as bifunctional regulators of cell proliferation

Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the ligand-activated nuclear receptor superfamily, plays a key role in mediating differentiation of adipocytes and regulating fat metabolism. PPARgamma has been implicated in the pathophysiology of atherosclerosis, inflammation, obesity, diabetes, immune response, and ageing. Recently, it has been shown that activation of PPARgamma by J(2) series cyclopentenone prostaglandins (cyPGs), especially 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) or synthetic agents, such as antidiabetic thiazolidinediones, causes anti-proliferation, apoptosis, differentiation, and anti-inflammation of certain types of cancer cells. The anti-proliferative effects of PPARgamma activators are associated with de novo synthesis of proteins involved in regulating the cell cycle and cell survival/death. Anti-inflammatory effects of 15d-PGJ(2) are associated with interruption of nuclear factor-kappaB and subsequent blockade of inflammatory gene expression. Furthermore, 15d-PGJ(2) at nontoxic doses induce expression of phase II detoxification or stress-responding enzymes, which may confer cellular resistance or adaptation to oxidative stress. The presence of a reactive alpha,beta-unsaturated carbonyl moiety in the cyclopentenone ring of 15d-PGJ(2) is important for part of biological functions this cyPG has. Recently, attention has been focused on the anti-proliferative activity of nonsteroidal anti-inflammatory drugs (NSAIDs) in cancerous or transformed cells, which is mediated through interaction with PPARgamma irrespective of their ability to inhibit COX-2. Despite the fact that abnormally elevated COX-2 is associated with resistance to cell death, induction of apoptosis by certain NSAIDs is accompanied by up-regulation of COX-2 expression. This commentary focuses on dual effects of the typical PPARgamma agonist 15d-PGJ(2) on cell proliferation and growth, and its possible involvement in the NSAID-induced COX-2 expression and apoptosis.