15-deoxy-Delta12,14-prostaglandin J2 impairs phosphatidylcholine synthesis and induces nuclear accumulation of thiol-modified cytidylyltransferase

Synthesis of phosphatidylcholine, the major phospholipid of animal cell membranes, requires the key enzyme cytidylyltransferase (CCTalpha). Cysteine sulfhydryls within CCTalpha are needed for full catalytic activity. Here we show that prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) inactivates CCTalpha by inducing generation of reactive oxidant species and the appearance of a cross-linked CCTalpha dimer in cells. N-Acetyl-l-cysteine reduced oxidative stress, prevented CCTalpha cross-linking, and restored CCT function in 15d-PGJ(2)-treated cells. 15d-PGJ(2) modified critical cysteine residues within CCTalpha as determined by mutagenesis studies and by incorporation of biotin-15d-PGJ(2) into CCTalpha. These effects of 15d-PGJ(2) were associated with CCTalpha accumulation within the nucleus. The data indicate that bioactive prostanoids significantly impair membrane phospholipid production by promoting cysteine cross-bridging within CCTalpha.     

Feedback control of the arachidonate cascade in rheumatoid synoviocytes by 15-deoxy-Delta(12,14)-prostaglandin J2

Rheumatoid arthritis (RA) is a chronic polyarticular joint disease associated with massive synovial proliferation, inflammation, and angiogenesis. PPAR-gamma ligands, both 15-deoxy-Delta(12,14)-prostaglandin J2 (15d- PGJ2) and troglitazone (TRO), can inhibit the growth of RA synoviocytes in vitro, and suppress the chronic inflammation of adjuvant-induced arthritis in rats, but the potency of 15d-PGJ2 is higher than TRO. Prostaglandin (PG) E2 plays important roles in joint erosion and synovial inflammation. In the present study, 15d-PGJ2, but not TRO and other prostanoids, suppressed interleukin (IL)-1beta-induced PGE2 synthesis in rheumatoid synovial fibroblasts (RSFs) through the inhibition of cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) expression. Furthermore, the inhibition was not affected by pretreatment with anti-PPAR-gamma antibody. It means that this anti-inflammatory effect of 15d-PGJ2 for PG synthesis may be independent of PPAR-gamma and 15d-PGJ2 is a key regulator of negative feedback of the arachidonate cascade on the COX pathway. These findings provide new insight into the feedback mechanism of the arachidonate cascade.     

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells

Prostaglandin (PG) E(2) (PGE(2)) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE(2) is accomplished by conversion of the cyclooxygenase (COX) product PGH(2) by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Delta(12,14)-PGJ(2) and PGA(2) downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor gamma or PGD(2) receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE(2). Our data suggest that there is a feedback mechanism between PGE(2) and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.     

Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis

Peroxisome proliferator-activated receptors (PPAR) are members of a nuclear hormone receptor superfamily that includes receptors for steroids, retinoids, and thyroid hormone, all of which are known to affect the immune response. Previous studies dealing with PPAR-gamma expression in the immune system have been limited. Recently, PPAR-gamma was identified in monocyte/macrophage cells. In this study we examined the role of PPAR-gamma in experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. The hypothesis we are testing is whether PPAR-gamma plays an important role in EAE pathogenesis and whether PPAR-gamma ligands can inhibit the clinical expression of EAE. Initial studies have shown that the presence of the PPAR-gamma ligand 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ2) inhibits the proliferation of Ag-specific T cells from the spleen of myelin basic protein Ac(1-11) TCR-transgenic mice. 15d-PGJ2 suppressed IFN-gamma, IL-10, and IL-4 production by both Con A- and myelin basic protein Ac(1-11) peptide-stimulated lymphocytes as determined by ELISA and ELISPOT assay. Culture of encephalitogenic T cells with 15d-PGJ2 in the presence of Ag reduced the ability of these cells to adoptively transfer EAE. Examination of the target organ, the CNS, during the course of EAE revealed expression of PPAR-gamma in the spinal cord inflammatory infiltrate. Administration of 15d-PGJ2 before and at the onset of clinical signs of EAE significantly reduced the severity of disease. These results suggest that PPAR-gamma ligands may be a novel therapeutic agent for diseases such as multiple sclerosis.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.     

15-deoxy-Delta12,14-prostaglandin J2-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction

A natural ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), decreases endothelial nitric oxide synthase (eNOS) expression by an unknown mechanism. Here we found that 15d-PGJ(2)-induced eNOS reduction is inversely associated with heat shock protein 70 (HSP70) induction in endothelial cells. Treatment of cells with 15d-PGJ(2) decreased eNOS protein expression in a concentration- and time-dependent manner, but independently of PPARgamma with no effect on mRNA levels. Although 15d-PGJ(2) elicited endothelial apoptosis, inhibition of both pan-caspases and cathepsins failed to reverse reduction of eNOS protein. Interestingly, we observed that 15d-PGJ(2) induced HSP70 in a dose-dependent manner. Immunoprecipitation and heat shock treatment demonstrated that eNOS reduction was strongly related to HSP70 induction. Cellular fractionation revealed that treatment with 15d-PGJ(2) increased eNOS distribution 2.5-fold from soluble to insoluble fractions. These findings provide new insights into mechanisms whereby eNOS regulation by 15d-PGJ(2) is related to HSP70 induction.     

15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone modulate Staphylococcus aureus-dependent astrocyte activation primarily through a PPAR-gamma-independent pathway

Brain abscesses arise from a focal parenchymal infection by various pathogens, particularly Staphylococcus aureus. We have shown that astrocytes are activated upon exposure to S. aureus and may contribute to the excessive tissue damage characteristic of brain abscess. Therefore, modulating astrocyte activation may facilitate a reduction in brain abscess severity. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists are potent inhibitors of microglial activation; however, the effects of these compounds on S. aureus-dependent astrocyte activation have not yet been examined. Here, we demonstrate that two chemically distinct PPAR-gamma agonists, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, suppress the production of several pro-inflammatory molecules in S. aureus-stimulated astrocytes including interleukin-1beta and nitric oxide (NO). Interestingly, 15d-PGJ2 attenuated Toll-like receptor 2 (TLR2) and inducible nitric oxide synthase expression, but failed to modulate macrophage inflammatory protein-2 (MIP-2/CXCL2) production, suggesting that 15d-PGJ2 is not a global inhibitor of astrocyte activation. Another novel finding of this study was the fact that both 15d-PGJ2 and ciglitazone were capable of attenuating pre-existing astrocyte activation, indicating their potential benefit in a therapeutic setting. Importantly, 15d-PGJ2 and ciglitazone were still capable of inhibiting S. aureus-induced pro-inflammatory mediator release in PPAR-gamma-deficient astrocytes, supporting PPAR-gamma-independent effects of these compounds. Collectively, these results suggest that 15d-PGJ2 and ciglitazone exert their anti-inflammatory actions on astrocytes primarily independent of the PPAR-gamma pathway.     

Covalent binding of 15-deoxy-delta12,14-prostaglandin J2 to PPARgamma

Since 15-deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2)) has been identified as an endogenous ligand of PPARgamma thus inducing adipogenesis, it has been reported to play active parts in numerous cellular regulatory mechanisms. As 15dPGJ(2) has been shown to covalently bind several peptides and proteins, we investigated whether it also covalently binds PPARgamma. We first observed that after incubation of 15dPGJ(2) with recombinant PPARgamma, the quantity of free 15dPGJ(2) measured was always lower than the initial amount. We then measured the ability of the labeled agonist rosiglitazone to displace the complex PPARgamma(2)/15dPGJ(2) obtained after pre-incubation. We observed that the binding of rosiglitazone was dependent on the initial concentration of 15dPGJ(2). Finally using MALDI-TOF mass spectrometry analysis, after trypsinolysis of an incubate of the PPARgamma(2) ligand binding domain (GST-LBD) with 15dPGJ2, we found a fragment (m/z = 1314.699) corresponding to the addition of 15dPGJ(2) (m/z = 316.203) to the GST-LBD peptide (m/z = 998.481). All these observations demonstrate the existence of a covalent binding of 15dPGJ(2) to PPARgamma, which opens up new perspectives to study the molecular basis for selective activities of PPARs.     

Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J2, reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ2-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ2 administration decreases leukocyte rolling and adhesion to the inflamed mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS-/- mice were not susceptible to 15d-PGJ2-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ2-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-1 expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ2, whereas 15d-PGJ2 inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ2 suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.     

Nrf2-mediated induction of cytoprotective enzymes by 15-deoxy-Delta12,14-prostaglandin J2 is attenuated by alkenal/one oxidoreductase

NADPH-dependent alkenal/one oxidoreductase (Aor) was discovered to be highly inducible in rat liver following treatment with the cancer chemopreventive agent 3H-1, 2-dithiole-3-thione. Aor was further characterized as an Nrf2-regulated antioxidative enzyme that reduces carbon-carbon double bonds in a variety of alpha, beta-unsaturated aldehydes and ketones. 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is a reactive membrane lipid metabolite that activates multiple pathways, including Nrf2-mediated induction of cytoprotective enzymes. Physiologically, it is postulated that 15d-PGJ2 alkylates key regulatory proteins via the electrophilic carbon centers found in two alpha, beta-unsaturated ketone moieties. This current study addresses the metabolism of 15d-PGJ2 by rat Aor (rAor) and subsequent deactivation of the Nrf2 signaling pathway by both rat and human AOR. We demonstrate that induction of NADPH-dependent quinone oxidoreductase activity by 15d-PGJ2 is markedly attenuated in mouse embryonic fibroblasts that overexpress rAor. Luciferase reporter assay and quantitative real-time PCR confirmed these findings. Concentrations required for doubling the NADPH-dependent quinone oxidoreductase response are increased from 1.8 microm in wild-type to >10 microm in rat Aor transgenic fibroblasts. 15d-PGJ2 is metabolized by recombinant rAor with a Km of 9.6 microm and k(cat) of 18.5 min(-1). The major product is 12,13-dihydro-15-deoxy-Delta12,14-prostaglandin J2 (dihydro-15d-PGJ2). The reduction of C=C by Aor yielding dihydro-15d-PGJ2 abolishes the inducibility in an antioxidant response element-driven luciferase assay. Collectively, these results demonstrate that 15d-PGJ2 can be catabolized by Aor, thereby attenuating subsequent Nrf2 signaling and possibly inflammatory and apoptotic processes also influenced by 15d-PGJ2.     

15-deoxy-Delta(12,14)-prostaglandin J2 induces vascular endothelial cell apoptosis through the sequential activation of MAPKS and p53

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a potent anti-angiogenic factor and induces endothelial cell apoptosis, although the mechanism remains unclear. In this study, 15d-PGJ(2) was found to increase p53 levels of the human umbilical vein endothelial cells by stabilizing p53. Both 15d-PGJ(2)-induced apoptosis and the induction of p21(Waf1) and Bax can be abolished by p53 small interfering RNA but not by peroxisome proliferator-activated receptor gamma inhibitors. Moreover, 15d-PGJ(2) activated JNK and p38 MAPK while inducing p53 phosphorylation at sites responsible for p53 activity. JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) pretreatment attenuated 15d-PGJ(2)-mediated apoptosis and suppressed the p21(Waf1) and Bax expressions without affecting p53 protein accumulation. Pretreatment with SP600125 partially prevented the phosphorylation of p53 at serines 33 and 392 induced by 15d-PGJ(2). 15d-PGJ(2) was also found to induce reactive oxygen species generation and partially blocked nuclear factor-kappaB activity. Pretreatment with antioxidant N-acetylcysteine prevented the p53 accumulation, the phosphorylations of JNK and p38 MAPK, the inhibition of NF-kappaB activity, as well as the apoptosis induced by 15d-PGJ(2). Using a mouse model of corneal neovascularization, it was demonstrated in vivo that 15d-PGJ(2) induced reactive oxygen species generation, activated JNK and p38 MAPK, induced p53 accumulation/phosphorylation, and induced vascular endothelial cell apoptosis, which could be abolished by N-acetylcysteine, SP600125, SB203580, or a virus-derived amphipathic peptides-based p53 small interfering RNA. This is the first study that 15d-PGJ(2) induces vascular endothelial cell apoptosis through the signaling of JNK and p38 MAPK-mediated p53 activation both in vitro and in vivo, further establishing the potential of 15d-PGJ(2) as an anti-angiogenesis agent.     

A PPAR-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J(2), inhibited gastric mucosal injury induced by ischemia-reperfusion in rats

INTRODUCTION: Recent studies have demonstrated the anti-inflammatory action of 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), a derivative of the PGD(2) metabolic pathway. Acute inflammation, including neutrophil activation, plays a critical role in the pathogenesis of ischemia-reperfusion (I/R). The aim of the present study was to determine the effect of 15d-PGJ(2) on I/R-induced gastric mucosal injury in rats.METHODS: Gastric mucosal damage was induced in male Wistar rats by clamping the celiac artery for 30 min followed by reperfusion. 15d-PGJ(2) (0.01-1.0 mg/kg) was given to the rats intraperitoneally 1 h before the vascular clamping. The area of gastric mucosal erosions (erosion index) was measured. Thiobarbituric acid reactive substances (TBARS) and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expression of tumor necrosis factor-alpha (TNF-alpha) in gastric mucosa was measured by ELISA. In addition, to elucidate whether the protective effects of 15d-PGJ(2) are related to the activation of the PPAR-gamma receptor, we also investigated the effects of a PPAR-gamma antagonist, GW9662.RESULTS: After 60 min of reperfusion, the area of gastric erosion index had significantly increased from the mean basal levels. The increase in the erosion index was significantly inhibited by pretreatment with 15d-PGJ(2) in a dose-dependent manner. On the other hand, GW9662 reversed the protective effect of 15d-PGJ(2). The concentration of TBARS and MPO activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with 15d-PGJ(2) significantly reduced these increases. The TNF-alpha content was significantly higher in the I/R group than in the sham-operated group. However, the increase in TNF-alpha was significantly inhibited by pretreatment with 15d-PGJ(2).CONCLUSIONS: 15d-PGJ(2) significantly inhibited the severity of acute gastric mucosal injury induced by I/R in rats through PPAR-gamma-dependent mechanisms. This effect may be due, in part, to a reduction in the infiltration of neutrophils into the gastric mucosa, possibly via the inhibition of inflammatory cytokine.     

15-Deoxy-Delta12,14-prostaglandin J2 regulates endogenous Cot MAPK kinase kinase 1 activity induced by lipopolysaccharide

Cot is a MAPK kinase kinase that has been implicated in cellular activation and proliferation. Here, we show that the addition of lipopolysaccharide (LPS) to RAW264 macrophages induces a 10-fold increase of endogenous Cot activity, measured as MAPK kinase kinase 1 activity. Taxol, but not phorbol 12-myristate 13-acetate (PMA), induces a similar activation of Cot. A tyrosine kinase activity is involved in Cot activation by LPS. 15-Deoxy-Delta12,14-prostaglandin J2, but not rosiglitazone, blocks Cot activation by LPS. Furthermore, 15-deoxy-Delta12,14-prostaglandin J2 also inhibited the LPS-induced Cot in vitro. However, 15-deoxy-Delta12,14-prostaglandin J2 does not inhibit MAPK kinase 1 or ERK1/ERK2 activation/phosphorylation induced by PMA and mediated by c-Raf. Considering these data, we propose that the inhibition of LPS-induced Cot activation is one mechanism by which 15-deoxy-Delta12,14-prostaglandin J2 acts as an anti-inflammatory.     

15-deoxy-Delta 12,14-prostaglandin J2 induces apoptosis of human hepatic myofibroblasts. A pathway involving oxidative stress independently of peroxisome-proliferator-activated receptors

Hepatic myofibroblasts (hMFs) play a key role in the development of liver fibrosis associated with chronic liver diseases. Apoptosis of these cells is emerging as a key process in the resolution of liver fibrosis. Here, we examined the effects of cyclopentenone prostaglandins on apoptosis of human hMFs. Cyclopentenone prostaglandins of the J series markedly reduced hMF viability, with 15-deoxy-Delta(12,14)-prostaglandin J2 (15-d-PGJ2) being the most potent. This effect was independent of peroxisome-proliferator-activated receptors (PPARs), because PPARgamma and PPARalpha agonists did not affect hMF cell viability, and PPARgamma, the nuclear receptor for 15-d-PGJ2, was not expressed in hMFs. Moreover, 15-d-PGJ2 did not act via a cell surface G protein-coupled receptor, as shown in guanosine-5'-O-(3-thiotriphosphate) binding assays. Cell death resulted from an apoptotic process, because 15-d-PGJ2-treated hMFs exhibited condensed nuclei, fragmented DNA, and elevated caspase-3 activity. Moreover, the caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethyl ketone blocked the cytotoxic effect of 15-d-PGJ2. The apoptotic effects of 15-d-PGJ2 were reproduced by H2O2 and blocked by the antioxidants N-acetylcysteine (NAC), N-(2-mercapto-propionyl)-glycine (NMPG) and pyrrolidine dithiocarbamate (PDTC). Accordingly, 15-d-PGJ2 generated rapid production of reactive oxygen species in hMFs, via a NAC/NMPG/PDTC-sensitive pathway. In conclusion, 15-d-PGJ2 induces apoptosis of human hMFs via a novel mechanism involving oxidative stress and unrelated to activation of its nuclear receptor PPARgamma. These data underline the antifibrogenic potential of 15-d-PGJ2.