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.     

Modulation of inflammatory response and parasitism by 15-Deoxy-Δ(12,14) prostaglandin J(2) in Trypanosoma cruzi-infected cardiomyocytes

Trypanosoma cruzi infection produces an intense inflammatory response in diverse tissues including the heart. The inflammatory reaction is critical for the control of the parasites’ proliferation and evolution of Chagas disease. 15-Deoxy-Δ^12,14 prostaglandin J₂ (15dPGJ2) can repress the inflammatory response in many experimental models. However, the precise role of peroxisome proliferator-activated receptor γ (PPARγ) ligands in T. cruzi infection or in Chagas disease is poorly understood. This work reports the first evidence that 15dPGJ2 treatment increases the number of intracellular parasites as shown by fluorescence microscopy and it is also able to inhibit the expression and activity of different inflammatory enzymes such as inducible nitric oxide synthase (NOS-2), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), as well as pro-inflammatory cytokine (TNF-α and IL-6) mRNA expression in neonatal mouse cardiomyocytes after T. cruzi infection. Transfection of cardiomyocytes with small interfering RNA (siRNA) induces silencing of PPARγ and impairs the effects of 15dPGJ2 on the modulation of pro-inflammatory enzymes. Moreover, transfection restores the ability of these cells to control the intracellular growth of T. cruzi. We also found that PPARγ-independent pathways are involved, since 15dPGJ2 also exerts its effect through extracellular signal-regulated kinases-mitogen-activated protein kinase (Erk-MAPK) and nuclear factor-κB (NF-κB). The use of specific pharmacological inhibitors confirmed these findings. Our data point out that 15dPGJ2 is a potent modulator of the inflammatory process and regulator of parasites growth through PPARγ-dependent and independent (Erk-MAPK- and NF-κB) pathways in T. cruzi infected neonatal cardiac cells.     

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-Delta(12,14)-prostaglandin J(2), a ligand for peroxisome proliferator-activated receptor-gamma, induces apoptosis in JEG3 choriocarcinoma cells.

Apoptosis has been described in placental (trophoblast) tissues during both normal and abnormal pregnancies. We have studied the effects of the cyclopentenone prostaglandins (PGs) on trophoblast cell death using JEG3 choriocarcinoma cells. PGJ(2), Delta(12)PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)) (10 microM) significantly reduced mitochondrial activity (MTT assay) over 16 h by 17.4 +/- 4.7%, 28 +/- 9.3%, and 62.5 +/- 2.8%, respectively (mean +/- sem), while PGA(2) and PGD(2) had no effect. The synthetic PPAR-gamma ligand ciglitizone (12.5 microM) had a potency similar to 15dPGJ(2) (69 +/- 3% reduction). Morphological examination of cultures treated with PGJ(2) and its derivatives revealed the presence of numerous cells with dense, pyknotic nuclei, a hallmark of apoptosis. FACS analysis revealed an abundance (approximately 40%) of apoptotic cells after 16-h treatment with 15dPGJ(2) (10 microM). The caspase inhibitor ZVAD-fmk (5 microM) significantly diminished the apoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2). JEG3 cells expressed PPAR-gamma mRNA by Northern analysis. These novel findings imply a role for PPAR-gamma ligands in various processes associated with pregnancy and parturition.     

Expression and function of PPARgamma in rat placental development

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor known to regulate adipogenesis. Deletion of the PPARgamma gene in the mouse results in death by embryonic day 10.0 (E10.0) due to the failure of establishment of a labyrinth layer in the placenta, which suggests that PPARgamma is involved in trophoblast differentiation. To define PPARgamma function further in placental development, the expression and localization of the PPARgamma gene in the rat placenta was investigated. RT-PCR analysis shows the presence of PPARgamma mRNA in the placenta of day 11 of pregnancy (d11). The expression level is higher at d13 and then later decreased. Immunohistochemistry detects both PPARgamma and its putative intrinsic ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2), in the trophoblast of layer I which lined the maternal sinus. Oral administration of troglitazone, an agonist of PPARgamma, to pregnant rats between d9 and d11 increases the expression level of PPARgamma in the placenta and reduces the mortality of the fetuses by half. These results suggest that PPARgamma is required not only for trophoblast differentiation but also trophoblast maturation to establish maternal-fetal transport.     

Thiol antioxidant and thiol-reducing agents attenuate 15-deoxy-delta 12,14-prostaglandin J2-induced heme oxygenase-1 expression

Heme oxygenase-1 (HO-1) is induced as a beneficial and adaptive response in cells and tissues exposed to oxidative stress. Herein we examined how various eicosanoids affect the induction of HO-1, and the possible mechanism underlying 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2))-induced HO-1 expression. PGH(2), PGD(2) and its metabolites of the PGJ(2) series, and PGA(1) markedly induced the protein expression of HO-1. Arachidonic acid (AA), docosahexaenoic acid (DHA), PGE(2), PGF(2 alpha), and thromboxane B(2) (TXB(2)) were shown to have no effect on the induction of HO-1. 15d-PGJ(2) was the most potent activator achieving significance at 5 microM. Although 15d-PGJ(2) significantly activated the MAPKs of JNK and ERK, the activation of JNK and ERK did not contribute to the induction of HO-1 as determined using transfection of dominant-negative plasmids and MAPKs inhibitors. Additional experiment indicated that 15d-PGJ(2) induced HO-1 expression through peroxisome proliferator-activated receptor (PPAR)-independent pathway. 15d-PGJ(2) significantly decreased the intracellular level of reduced glutathione; and the thiol antioxidant, N-acetyl-L-cysteine (NAC), and the thiol-reducing agent, dithiothreitol (DTT), inhibited the induction of HO-1 by 15d-PGJ(2). Finally, NAC and DTT exhibited significant inhibition of HO-1 mRNA and HO-1 promoter reporter activity induced by 15d-PGJ(2). These results suggest that thiol antioxidant and reducing agents attenuate the expression of HO-1 induced by 15d-PGJ(2), and that the cellular thiol-disulfide redox status may be linked to HO-1 activation.     

Up-regulation of heme oxygenase-1 expression through the Rac1/NADPH oxidase/ROS/p38 signaling cascade mediates the anti-inflammatory effect of 15-deoxy-delta 12,14-prostaglandin J2 in murine macrophages

We investigated the signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in murine macrophages in response to 15-deoxy-delta 12,14-prostaglandin J2 (15dPGJ2). 15dPGJ2 caused dose- and time-dependent activation of Rac1, followed by a transient increase in reactive oxygen species (ROS) via NADPH oxidase, which leads to downstream activation of p38 kinase. Inhibition of 15dPGJ2-dependent HO-1 expression significantly attenuated suppression by 15dPGJ2 of LPS-induced iNOS expression and subsequent production of nitric oxide (NO). Our findings strongly suggest that 15dPGJ2 exerts its anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1 in an in vitro inflammation model.     

Differential selectivity of protein modification by the cyclopentenone prostaglandins PGA1 and 15-deoxy-Delta12,14-PGJ2: role of glutathione

Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) and PGA(1) (PGA(1)-B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ(2)-B and PGA(1)-B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.     

HtrA3 is regulated by 15-deoxy-Δ12,14-prostaglandin J2 independently of PPARγ in clear cell renal cell carcinomas

Peroxisome proliferator-activated receptor gamma (PPARγ) ligands have been shown to possess anti-proliferative effects in many types of cancer. In clear cell renal cell carcinoma (CCRCC), the targets involved in these effects are not known. In this study, we demonstrated that, in CCRCC cell lines, the endogenous PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) induces the expression, both at the mRNA and the protein levels, of the HtrA3 gene. This gene belongs to the High-Temperature Requirement Factor A family of serine proteases that repress signaling by TGF-β family members and inhibit cell migration. Rosiglitazone or ciglitazone, synthetic PPARγ agonists, did not induce HtrA3 expression, and the PPARγ antagonist GW9662 did not prevent 15dPGJ2 induction, suggesting that the up-regulation of HtrA3 by 15dPGJ2 is independent of PPARγ. The MEK/ERK inhibitor PD98059 dramatically repressed HtrA3 induction. Altogether, these data indicate that 15dPGJ2 is able to stimulate the expression of HtrA3 through an indirect mechanism involving the MEK/ERK pathway but independent of PPARγ. Our results provide a better understanding of the mechanisms involved in the regulation of HtrA3, a potential tumor suppressor gene.     

Atomic structure of mutant PPARγ LBD complexed with 15d-PGJ2: Novel modulation mechanism of PPARγ/RXRα function by covalently bound ligands

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) activates a nuclear receptor heterodimer, peroxisome proliferators-activated receptor γ (PPARγ)/ retinoid X receptor (RXRα) through covalent binding to Cys285 in PPARγ ligand-binding domain (LBD). Here, we present the 1.9 Å crystal structure of C285S mutant LBD complexed with 15d-PGJ₂, corresponding to the non-covalently bound state. The ligand lies adjacent to a hydrogen-bond network around the helix H2 and the nearby β-sheet. Comparisons with previous structures clarified the relationships between PPARγ function and conformational alterations of LBD during the process of covalently binding ligands, such as 15d-PGJ₂, and thus suggested a mechanism, by which these ligands modulate PPARγ/RXRα function through conformational changes of the loop following helix H2′ and the β-sheet.     

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

ROS enhances CXCR4-mediated functions through inactivation of PTEN in prostate cancer cells

Inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is heavily implicated in the tumorigenesis of prostate cancer. Conversely, the upregulation of the chemokine (CXC) receptor 4 (CXCR4) is associated with prostate cancer progression and metastasis. Studies have shown that loss of PTEN permits CXCR4-mediated functions in prostate cancer cells. Loss of PTEN function is typically due to genetic and epigenetic modulations, as well as active site oxidation by reactive oxygen species (ROS); likewise ROS upregulates CXCR4 expression. Herein, we show that ROS accumulation permitted CXCR4-mediated functions through PTEN catalytic inactivation. ROS increased p-AKT and CXCR4 expression, which were abrogated by a ROS scavenger in prostate cancer cells. ROS mediated PTEN inactivation but did not affect expression, yet enhanced cell migration and invasion in a CXCR4-dependent manner. Collectively, our studies add to the body of knowledge on the regulatory role of PTEN in CXCR4-mediated cancer progression, and hopefully, will contribute to the development of therapies that target the tumor microenvironment, which have great potential for the better management of a metastatic disease.     

15-Deoxy-Δ-prostaglandin J2 enhanced the anti-tumor activity of camptothecin against renal cell carcinoma independently of topoisomerase-II and PPARγ pathways

Renal cell carcinoma (RCC) is chemoresistant cancer. Although several clinical trials were conducted to explore effective medications, the chemoresistance of RCC has not yet been conquered. An endogenous ligand for peroxisome proliferator-activated receptor-γ (PPARγ), 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), induces apoptosis in RCC. Here, we examined synergistic effects of several carcinostatics on the anti-tumor activity of 15d-PGJ₂ in Caki-2 cell line by MTT assay. A topoisomerase-I inhibitor, camptothecin (CPT), exhibited synergistically toxicity with 15d-PGJ₂, but neither 5-fluorouracil nor cisplatin did. The combination of 15d-PGJ₂ and a topoisomerase-II inhibitor, doxorubicine, did not cause synergistic cell growth inhibition. The synergistic effect of topoisomerase-I and II inhibitors was not also detected. A PPARγ antagonist, GW9662, did not prevent Caki-2 from undergoing 15d-PGJ₂-induced cytotoxicity. The treatment of CPT combined with 15d-PGJ₂ activated caspase-3 more than the separate treatment. These results suggest that 15d-PGJ₂ exhibited the anti-tumor activity synergistically with CPT independent of topoisomerase-II and PPARγ.