Physiology and pharmacology of the prostaglandin J2 family

The prostaglandin (PG) J(2) family including PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are metabolites of PGD(2). They had been known as powerful inhibitors of cell proliferation and viral replication until 15d-PGJ(2) was found to be a natural ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). Since then, several new pharmacological actions of the PGJ(2) family have been found, such as pro- and anti-apoptotic effects, cell differentiation-inducing effects, and inhibitory effects on inflammatory processes, whether they depend on PPAR gamma or not. We reported that the PGJ(2) family, particularly 15d-PGJ(2), inhibits cell proliferation by reducing the expression of G(1) cyclins and inducing the expression of cyclin-dependent kinase inhibitor p21 and moreover, induces cell differentiation in vascular smooth muscle cells. In vascular endothelial cells, we found that 15d-PGJ(2) inhibits apoptotic cell death at least in part by the induction of the inhibitor of apoptosis protein c-IAP1. More importantly, physiological levels of laminar fluid shear stress loaded on endothelial cells upregulate the expression of lipocalin-type PGD(2) synthase, which converts PGH(2) to PGD(2), the precursor of the PGJ(2) family. Based on these results, we have hypothesized that the PGJ(2) family synthesized in vascular wall plays an important physiological role to protect vascular cells from atherogenic stimuli.     

Modulation of peroxisome proliferator-activated receptors (PPARs) by PPAR(alpha)- and PPAR(gamma)-specific ligands and by 17beta-estradiol in isolated zebrafish hepatocytes.

Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPAR(alpha) include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPAR(beta) activators include fatty acids, prostaglandin A2 (PGA2) and prostacyclin (PGI2). PPAR(gamma) is the most selective receptor and, among others, 15-deoxy-Delta(12,14) prostaglandin J2 (PGJ2) has been described to be a PPAR(gamma)-specific ligand. The aim of the present study was to determine if known PPAR(alpha) and PPAR(gamma) ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPAR(alpha) specific ligand (8S-HETE), a PPARgamma specific ligand (PGJ) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17beta-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPAR(alpha) and PPAR(gamma) by clofibrate (at 0.5 mM for PPAR(alpha) and at 1 and 2 mM for PPAR(gamma)), by HETE (1 microM), and by PGJ2 (0.3 and 1 microM for PPAR(alpha) and 0.3 microM for PPAR(gamma)). Expression of PPARgamma was also induced at 10 microM by 17beta-estradiol. The percentage of PPAR(alpha) positive nuclei increased significantly at 1 microM HETE and the percentage of PPAR(gamma) positive cells decreased at 10 microM 17beta-estradiol. As a conclusion, clofibrate, HETE and PGJ2 are able to induce expression of both PPAR(alpha) and PPAR(gamma) in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions.     

The biphasic effects of cyclopentenone prostaglandins, prostaglandin J(2) and 15-deoxy-Delta(12,14)-prostaglandin J(2) on proliferation and apoptosis in rat basophilic leukemia (RBL-2H3) cells

Mast cells produce chemical mediators, including histamine and arachidonate metabolites such as prostaglandin D(2) (PGD(2)) after antigen stimulation. Cyclopentenone prostaglandins of the J series, prostaglandin J(2) (PGJ(2)) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), are thought to be derivatives of PGD(2). In this study, the biphasic effects of the PGJ(2) and 15d-PGJ(2) on proliferation and apoptosis in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mast cells, were examined. At low concentrations, 1 or 3 microM PGJ(2) and 15d-PGJ(2) induced cell proliferation, respectively. At high concentrations (10-30 microM) both the inhibition of viability and decrease in histamine content in RBL-2H3 cells were dose dependent. These effects were independent of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), since troglitazone, an agonist of PPARgamma did not cause any effects in RBL-2H3 cells. Cell death induced by PGJ(2) and 15d-PGJ(2) was the result of apoptotic processes, since RBL-2H3 cells treated with 30 microM of the prostaglandins had condensed nuclei, DNA fragmentation and increase in activities of caspase-3 and -9. Moreover, PGJ(2) or 15d-PGJ(2)-induced apoptotic effects were prevented by the caspase inhibitor, z-VAD-fmk. In conclusion, the PGJ(2) or 15d-PGJ(2)-induced apoptosis in RBL-2H3 cells occurs mainly via mitochondrial pathways instead of by PPARgamma-dependent mechanisms.     

Peroxisome proliferator-activated receptor gamma augments tumor necrosis factor family-induced apoptosis in hepatocellular carcinoma

Proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor, which mainly associates with adipogenesis, but also appears to facilitate cell differentiation or apoptosis in certain malignant cells. This apoptosis induction by PPARgamma is increased by co-stimulation with tumor necrosis factor (TNF)-alpha-related apoptosis-inducing ligand (TRAIL), a member of the TNF family. In this study, we investigated the effect of PPARgamma on Fas-mediated apoptosis in hepatocellular carcinoma (HCC) cell lines. PPARgamma was expressed on all seven HCC cell lines and located in their nuclei. 15-Deoxy-Delta-12,14-prostaglandin J2 (15d- PGJ2), a PPARgamma ligand, inhibited cellular proliferation in HepG2, SK-Hep1 or HLE cells, unlike pioglitazone, another PPARgamma ligand, which did not have a significant influence on proliferation of these cells. However, 15d-PGJ2 facilitated Fas-mediated HCC apoptosis that could not be induced by Fas alone. These results suggest that PPARgamma can augment TNF-family-induced apoptosis.     

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.     

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.     

Induction of proliferation by 15-deoxy-delta12,14-prostaglandin J2 and the precursors in monocytic leukemia U937

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in several human tumors including gastric, lung, colon, prostate and breast. However, the role of PPARgamma signals in leukemia is still unclear. The aim of this study is to evaluate the ability of 15-deoxy-Delta12,14-prostaglandin J2 (15dPGJ2), that is a ligand for PPARgamma, on proliferation of human leukemia cell line U937. 15dPGJ2 at 5 micromol/l stimulated the proliferation. In contrast, 15dPGJ2 at concentrations of >10 micromol/l inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2 and Delta12-PGJ2 (DeltaPGJ2), those are precursors of 15dPGJ2, had similarly proliferative effects, whereas they showed antiproliferative effects at high concentrations. FACScan analysis revealed that PGD2 at 5 micromol/l, PGJ2 at 1 micromol/l, DeltaPGJ2 at 1 micromol/l and 15dPGJ2 at 5 micromol/l, all accelerated cell cycle progression. Immunoblotting analysis revealed that PGD2 at 5 micromol/l and 15dPGJ2 at 5 micromol/l inhibited the expression of phospho-p38, phospho-MKK3/MKK6 and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p27. In contrast, PGJ2 at 1 micromol/l and DeltaPGJ2 at 1 micromol/l did not affect the expression of them. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 MAPK pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of proliferation.     

Effects of 15-deoxy-Δ^12,14-prostaglandin J2 on cell proliferation and apoptosis in ECV304 endothelial cells

AIM: To investigate the effects of 15-deoxy-Δ^12,14-prostaglandin J2 (15d-PGJ2) on cell proliferation and apoptosis in ECV304 endothelial cells and related molecular mechanism. METHODS: MTT, Hoechst33258, TUNEL, Flow cytometry, DNA ladder, RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA) analysis were employed. RESULTS: The 15d-PGJ2 induced apoptosis in ECV304 endothelial cells in a dose-dependent manner(the percentage of apoptosis was enhanced from 10.0 % 1.3 % to 32.8 % 1.6 %), which was accompanied by inhibition of NF-κB and AP-1 DNA binding activity, down-regulation of c-myc, upregulation of Gadd45 and p53,and activation of p38 kinase. However, the expression of p21 was found no significant change. CONCLUSION:peroxisome proliferator-activated receptor gamma ligand, 15d-PGJ2, can inhibit proliferation and induce apoptosisin ECV304 endothelial cells through different mechanisms.     

Cyclooxygenase-independent inhibition of H2O2-induced cell death by S-ketoprofen in renal cells.

The stress response of the distal tubule to oxidative attack may be relevant to recovery from acute renal failure. In distal tubular Madin-Darby cells (MDCK), H(2)O(2) induced up-regulation of cyclooxygenases (COX-1 and COX-2), prostaglandin-E(2) production and caspase-independent cell death. Cell death was inhibited by S-ketoprofen, but not by the much weaker COX inhibitor R-ketoprofen. Interestingly, we identified 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome-proliferator activated receptor-gamma agonist, as a lethal prostaglandin whose effect was reproduced by the PPAR-gamma agonist ciglitazone. Nevertheless, H(2)O(2)-induced cell death was unaffected by other non-steroidal anti-inflammatory drugs (NSAIDs) or all-trans-retinoic acid. Moreover, c-Jun-N-terminal kinase inhibitor SP600125 prevented 15-deoxy-Delta(12,14)-PGJ(2)-induced cell death, but not H(2)O(2)-induced cell death. PPAR-gamma antagonist GW9662 showed no affect on the cell death. These results indicated that protection by S-ketoprofen was COX-independent and PPARgamma independent. Moreover, the IC(50) value of the action of S-ketoprofen for the inhibition of H(2)O(2)-induced MDCK cell death ( approximately equal 140microM) was much higher than the IC(50) value for the inhibition of COX-1 and COX-2 activities ( approximately equal 1microM). Further design of S-ketoprofen derivatives devoid of COX inhibitory activity will give opportunity to protect the kidney against oxidative attack while avoiding unwanted effects of NSAID.     

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.     

Possible involvement of p38 in mechanisms underlying acceleration of proliferation by 15-deoxy-Delta(12,14)-prostaglandin J2 and the precursors in leukemia cell line THP-1

15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ2), which is a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), induced apoptosis of several human tumors including gastric, lung, colon, prostate, and breast. However, the role of PPARgamma signals in other types of cancer cells (e.g., leukemia) except solid cancer cells is still unclear. The aim of this study is to evaluate the ability of 15dPGJ2 to modify the proliferation of the human leukemia cell line THP-1. 15dPGJ2 at 5 microM stimulated the proliferation in THP-1 at 24 to 72 h after incubation. In contrast, 15dPGJ2 at concentrations above 10 microM inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2, and Delta12-PGJ2 (DeltaPGJ2), precursors of 15dPGJ2, had similar proliferative effects at lower concentrations, whereas they induced apoptosis at high concentrations. 15dPGJ2 and three precursors failed to induce the differentiation in THP-1 as assessed by using the differentiation marker CD11b. FACScan analysis revealed that PGD2 at 5 microM, PGJ2 at 1 microM, DeltaPGJ2 at 1 microM and 15dPGJ2 at 5 microM all accelerated cell cycle progression in THP-1. Immunoblotting analysis revealed that PGD2 at 5 microM and 15dPGJ2 at 5 microM inhibited the expression of phospho-p38, phospho-MKK3/MKK6, and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p21, and p27 in THP-1. In contrast, PGJ2 at 1 microM and DeltaPGJ2 at 1 microM did not affect their expressions. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 mitogen-activated protein kinase pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of the THP-1 proliferation.