Differential effects of serum constituents on apoptosis induced by the cyclopentenone prostaglandin 15-deoxy-delta12,14-prostaglandin J2 in WISH epithelial cells

Cyclopentenone prostaglandins, delta12-PGJ2 and 15d-PGJ2, have potent anti-tumour and anti-inflammatory activities, and have been shown to induce apoptosis in amnion-derived WISH cells. In this study, we have investigated the protective effects of serum and its constituents (growth factors and albumin) on delta12-PGJ2 and 15d-PGJ2-induced apoptosis in WISH cells. Serum (0.5% w/v) was protective against both delta12-PGJ2 and 15d-PGJ2-induced apoptosis. This was not due to the presence of serum-derived growth factors (EGF, IGF-1 and IGF-2), since they had no significant effect on 15d-PGJ2-induced cell death. In contrast, IGF-1 partially inhibited etoposide-induced apoptosis, confirming the presence of a functional IGF-1 receptor signalling system. Albumin was identified as the key survival factor in serum, since albumin and delipidated albumin exhibited the same level of protection from 15d-PGJ2-induced apoptosis as serum itself. The potential for serum albumin to regulate the bioactivity of cyclopentenone PGs may be of considerable importance in pathological conditions where roles for cyclopentenone PGs have been identified.     

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.     

Involvement of clusterin in 15-deoxy-delta12,14-prostaglandin J2-induced vascular smooth muscle cell differentiation

To establish an in vitro model of vascular smooth muscle cell (VSMC) differentiation, we examined the effect of 15-deoxy-delta12,14-prostaglandin J(2) (15d-PGJ(2)) on the expression of VSMC differentiation markers. After the addition of 15d-PGJ(2) to confluent human umbilical artery smooth muscle cells synchronized in the G(0) phase, cells showed a "hill and valley" appearance and thereafter aggregated and formed macroscopic nodules. Cells forming nodules expressed high levels of SM2, the most specific VSMC differentiation marker, comparable to medial VSMCs in vivo. 15d-PGJ(2) significantly increased the mRNA and protein expression levels of clusterin, a secreted glycoprotein reported to induce nodule formation and differentiation of VSMCs. Moreover, addition of an anti-clusterin antibody completely inhibited the nodule formation induced by 15d-PGJ(2) and induced apoptosis. Our results suggested that clusterin is involved in 15d-PGJ(2)-induced nodule formation and cell differentiation in VSMCs.     

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.     

15-deoxy-delta 12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes.

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J(2) series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), which have been suggested to exert anti-inflammatory effects in vivo. Meanwhile, the mechanism of these effects is not well understood and the natural site and the extent of its production in vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ(2) and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ(2)-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ(2) specifically. The presence of 15d-PGJ(2) in vivo was immunohistochemically verified in the cytoplasm of most of the foamy macrophages in human atherosclerotic plaques. In addition, the immunostaining of lipopolysaccharide-stimulated RAW264.7 macrophages with mAb11G2 demonstrated an enhanced intracellular accumulation of 15d-PGJ(2), suggesting that the PGD(2) metabolic pathway, generating the anti-inflammatory PGs, is indeed utilized in the cells during inflammation. The activation of macrophages also resulted in the extracellular production of PGD(2), which was associated with a significant increase in the extracellular 15d-PGJ(2) levels, and the extracellular 15d-PGJ(2) production was reproduced by incubating PGD(2) in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD(2) metabolites, we established a novel metabolic pathway, in which PGD(2) is converted to 15d-PGJ(2) via an albumin-independent mechanism.     

15-deoxy-delta 12,14-prostaglandin J2 and laminar fluid shear stress stabilize c-IAP1 in vascular endothelial cells

Laminar shear stress strongly inhibits vascular endothelial cell apoptosis by unknown mechanisms. We reported that shear stress stimulates endothelial cells to produce 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) by elevating the expression level of lipocalin-type prostaglandin D synthase. To investigate the role of 15d-PGJ2 produced in the vascular wall, we examined the effect of 15d-PGJ2 on endothelial cell apoptosis. We induced apoptosis in human umbilical vein endothelial cells (HUVECs) by growth factor deprivation. 15d-PGJ2 strongly inhibited DNA ladder formation, nuclear fragmentation, and caspase-3-like activity in HUVECs. To elucidate the mechanism by which 15d-PGJ2 inhibits endothelial cell apoptosis, we examined expression of the inhibitor of apoptosis proteins (IAP) cellular-IAP1 (c-IAP1), c-IAP2, x-linked IAP, and survivin in HUVECs. In parallel with the inhibition of apoptosis, 15d-PGJ2 elevated the expression level of c-IAP1 protein in a dose- and time-dependent manner without changing the mRNA level. Laminar shear stress also induced c-IAP1 expression. Chase experiments with the use of cycloheximide revealed that 15d-PGJ2 and shear stress both inhibited the proteolytic degradation of c-IAP1 protein. These results suggested that 15d-PGJ2 inhibits endothelial cell apoptosis through, at least in part, c-IAP1 protein stabilization. This mechanism might be involved in the antiapoptotic effect of laminar shear stress.     

A potential role of 15-deoxy-delta(12,14)-prostaglandin J2 for induction of human articular chondrocyte apoptosis in arthritis

The cyclopentenone prostaglandin (PG) J2 is formed within the cyclopentenone ring of the endogenous prostaglandin PG D2 by a nonenzymatic reaction. The PG J family is involved in mediating various biological effects including the regulation of cell cycle progression and inflammatory responses. Here we demonstrate the potential role of 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PG J2) in human articular chondrocyte apoptosis. 15d-PG J2 was released by human articular chondrocytes and found in joint synovial fluids taken from osteoarthritis or rheumatoid arthritis patients. Proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) up-regulated chondrocyte release of 15d-PG J2. PG D2 synthase mRNA expression was up-regulated by IL-1beta, TNF-alpha, or nitric oxide. 15d-PG J2 induced apoptosis of chondrocytes from osteoarthritis or rheumatoid arthritis patients as well as control nonarthritic subjects in a time- and dose-dependent manner and in a peroxisome proliferator-activated receptor gamma-dependent manner. Peroxisome proliferator-activated receptor gamma expression was up-regulated by IL-1beta and TNF-alpha. Inhibition of NF-kappaB, and the activation of p38 MAPK were also found to be involved in 15d-PG J2-induced chondrocyte apoptosis. Such signal pathways led to the activation of the downstream pro-apoptotic molecule p53 and caspase cascades. Together, these results suggest that 15d-PGJ2 may play an important role in the pathogenesis of arthritic joint destruction via a regulation of chondrocyte apoptosis.     

15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.     

15-deoxy-delta12,14-prostaglandin J2 regulates the functional state and the survival of microglial cells through multiple molecular mechanisms

We have previously reported that rat primary microglial cultures express the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and that several functions associated with the activation of these cells, including nitric oxide (NO) and tumor necrosis factor-alpha synthesis, are down-regulated by 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, two specific PPAR-gamma agonists. Here we demonstrate that microglial cells not only express a functionally active PPAR-gamma, but also synthesize large amounts of 15d-PGJ2 upon stimulation with lipopolysaccharide (LPS). In addition, we show that, although 15d-PGJ2 and ciglitazone were equally effective in reducing microglial activation when used at 1-5 microm concentrations, 15d-PGJ2, but not of ciglitazone, reduced PGE2 production at low concentration (0.1 microm) and induced a time-dependent microglial impairment and apoptosis at high concentration (10 microm). Interestingly, the inhibition of PGE2 production was achieved mainly through the inhibition of cycloxygenase-2 enzymatic activity, as the expression of this enzyme and that of the microsomal isoform of PGE synthase remained unaltered. These findings suggest that 15d-PGJ2 affects the functional state and the survival of activated microglia through mechanisms only in part dependent on PPAR-gamma and that the concentration of 15d-PGJ2 is crucial in determining the particular microglial function affected.     

Translational regulation of cyclin D1 by 15-deoxy-delta(12,14)-prostaglandin J(2).

The D-group cyclins play a key role in the progression of cells through the G(1) phase of the cell cycle. Treatment of MCF-7 breast cancer cells with the cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) results in rapid down-regulation of cyclin D1 protein expression and growth arrest in the G(0)/G(1) phase of the cell cycle. 15d-PGJ(2) also down-regulates the expression of cyclin D1 mRNA; however, this effect is delayed relative to the effect on cyclin D1 protein levels, suggesting that the regulation of cyclin D1 occurs at least partly at the level of translation or protein turnover. Treatment of MCF-7 cells with 15d-PGJ(2) leads to a rapid increase in the phosphorylation of protein synthesis initiation factor eukaryotic initiation factor 2alpha (eIF-2alpha) and a shift of cyclin D1 mRNA from the polysome-associated to free mRNA fraction, indicating that 15d-PGJ(2) inhibits the initiation of cyclin D1 mRNA translation. The selective rapid decrease in cyclin D1 protein accumulation is facilitated by its rapid turnover (t(1/2) = 34 min) after inhibition of cyclin D1 protein synthesis. The half-life of cyclin D1 protein is not significantly altered in cells treated with 15d-PGJ(2). Treatment of cells with 15d-PGJ(2) results in strong induction of heat shock protein 70 (HSP70) gene expression, suggesting that 15d-PGJ(2) might activate protein kinase R (PKR), an eIF-2alpha kinase shown previously to be responsive to agents that induce stress. 15d-PGJ(2) strongly stimulates eIF-2alpha phosphorylation and down-regulates cyclin D1 expression in a cell line derived from wild-type mouse embryo fibroblasts but has an attenuated effect in PKR-null cells, providing evidence that PKR is involved in mediating the effect of 15d-PGJ(2) on eIF-2alpha phosphorylation and cyclin D1 expression. In summary, treatment of MCF-7 cells with 15d-PGJ(2) results in increased phosphorylation of eIF-2alpha and inhibition of cyclin D1 mRNA translation initiation. At later time points, repression of cyclin D1 mRNA expression may also contribute to the decrease in cyclin D1 protein.     

Inhibition of IFN-gamma -induced STAT1 activation by 15- deoxy-Delta 12,14-prostaglandin J2

The inhibitory actions of 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) on inflammatory gene expression have been attributed to the ability of this prostaglandin to inhibit the activation of NF-kappaB. In this study, we have identified an additional signaling pathway sensitive to inhibition by PGJ(2). We show that PGJ(2) inhibits interferon (IFN)-gamma-stimulated phosphorylation and DNA-binding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1- to 2-h incubation and are maximal after a 6-h incubation with PGJ(2), and they correlate with the expression of heat shock protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ(2) on inducible nitric oxide synthase (iNOS) expression and NF-kappaB activation in response to IL-1 with the increased expression of HSP70. Using overexpression and antisense depletion, we provide evidence that HSP70 does not mediate the inhibitory actions of PGJ(2) on IL-1-induced NF-kappaB or IFN-gamma-induced STAT1 activation or cytokine-stimulated iNOS expression by beta-cells. Last, we show that the inhibitory actions of a short 6-h pulse with PGJ(2) on IL-1 plus IFN-gamma-stimulated iNOS expression and NO production by beta-cells are persistent for extended periods (< or =48 h). These findings suggest that PGJ(2) inhibits multiple cytokine-signaling pathways (IL-1 and IFN-gamma), that the inhibitory actions are persistent for extended periods, and that increased HSP70 expression correlates with, but does not appear to mediate, the inhibitory actions of PGJ(2) on IL-1 and IFN-gamma signaling in beta-cells.     

Effect of 15-deoxy-delta12,14-prostaglandin J2 on IL-1beta-induced expression of epithelial neutrophil-activating protein-78 in human endothelial cells

Epithelial neutrophil-activating peptide-78 (ENA-78) is a member of CXC chemokines. It is produced by endothelial cells stimulated with interleukin-1 (IL-1), along with other CXC chemokines such as IL-8 and growth-related oncogene protein-alpha (GRO-alpha). IL-1-induced ENA-78 production by endothelial cells may be important for the regulation of neutrophil activation. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a natural ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and affects the expression of various genes. We examined the effect of 15d-PGJ(2) on the expression of ENA-78 in cultured endothelial cells stimulated with IL-1beta. 15d-PGJ(2) inhibited the IL-1beta-induced expression of ENA-78, but not the expression of IL-8 or GRO-alpha in response to IL-1. Ciglitazone, another agonist for PPAR-gamma, had no effect on the expression of ENA-78, suggesting that 15d-PGJ(2) may inhibit the expression of ENA-78 in a PPAR-gamma-independent manner. 15d-PGJ(2) may modulate inflammatory reactions by regulating the balance of CXC chemokines in endothelial cells.     

7beta-hydroxy-epiandrosterone modulation of 15-deoxy-delta12,14-prostaglandin J2, prostaglandin D2 and prostaglandin E2 production from human mononuclear cells

7β-hydroxy-epiandrosterone (7β-OH-EPIA) has been shown to be cytoprotective in various organs including the brain. It has also been shown that prostaglandin D₂ (PGD₂) and its spontaneous metabolite 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) are also cytoprotective. It is possible that these prostaglandins derived from circulating mononuclear cells may mediate the actions of 7β-OH-EPIA. The aim of this study, therefore, was to ascertain the effect of 7β-OH-EPIA (in the absence or presence of tumour necrosis factor-α (TNF-α)), a pro-inflammatory stimulus, on the biosynthesis of PGD₂, PGE₂ and 15d-PGJ₂ from human mononuclear cells. Prostaglandins were measured by enzyme immunoassay (EIA). 7β-OH-EPIA alone induced a concentration-dependant increase in the production of PGD₂. TNF-α increased PGD₂ levels which were enhanced by 7β-OH-EPIA. 7β-OH-EPIA increased 15d-PGJ₂ levels both in the absence and presence of TNF-α. 7β-OH-EPIA alone had no effect on PGE₂ biosynthesis but suppressed TNF-α-induced PGE₂ circa 50%. 7β-OH-EPIA also increased the level of free arachidonic acid and radiolabelled prostaglandins in cells pre-incubated with radiolabelled arachidonic acid, indicating that the increase may occur via the enhanced release of substrate arachidonic acid. 7β-OH-EPIA did not affect levels of the anti-inflammatory cytokine IL-10 indicating that this is an unlikely mechanism by which 7β-OH-EPIA induces its actions but more likely exerts its effects via the production of cytoprotective prostaglandins.     

Molecular recognition of 15-deoxy-delta(12,14)-prostaglandin J2 by nuclear factor-kappa B and other cellular proteins

15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), a dehydration product of prostaglandin D2, is an important pharmacological molecule, which with the virtue of its electrophilicity, has been reported to covalently modify some cellular proteins (such as nuclear factor-kappa B (NF-kappaB), AP-1, p53, and thioredoxin) and elicit its physiological effects. The aim of the present computational study is to understand the role molecular recognition plays in the association of 15d-PGJ2 with NF-kappaB and other proteins. Another aim is to characterize whether p53 is a direct target for covalent modification by 15d-PGJ2. A docking strategy is applied along with calculation of ab initio electrostatic potential maps to analyze the mode of binding of prostaglandin molecule with critical cysteine-containing sites in each protein. The results provide identification of important sites in the target proteins, which provide recognition and stability to the prostaglandin molecule. Fit of shape and complementarity of electrostatic interactions are derived as significant determinants of molecular recognition of 15d-PGJ2. Further, comparative results indicate that p53 protein may also be a target for direct modification by 15d-PGJ2. The molecular models obtained should allow the rational design of more specific analogs of 15d-PGJ2.     

Quantitative characterization of 15-deoxy-delta(12,14)-prostaglandin J2 in regulating EGFPSmad2 translocation in CHO cells through PPARgamma/TGFbeta/Smad2 pathway.

Smad2 is an important factor in TGFbeta/Smad2 signal transduction pathway with ability for signal propagation, it could translocate from cytoplasm to nucleus after the TGFbeta receptor-mediated phosphorylation. 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), a natural agonist of the peroxisome proliferator-activated receptor gamma (PPARgamma), is found recently to be able to function in the regulation of Smad2 activity. However, no quantification data have been yet reported, and it still keeps suspenseful whether or not 15d-PGJ2 could regulate Smad2 activity by depending on PPARgamma through PPAR gamma/TGFbeta/ Smad2 pathway. In this work, by analyzing the EGFP-Smad2 location in CHO cells according to the Nucleus Trafficking Analysis Module based on IN Cell Analyzer 1000 platform, TGFbeta stimulated EGFP-Smad2 translocation regulated by 15d-PGJ2 was quantitatively investigated. The results showed that TGFbeta could induce EGFP-Smad2 translocation from cytoplasm to nucleus by EC50 of 8.83 pM, and 15d-PGJ2 could impede the TGFbeta-stimulated Smad2 translocation by IC50 of 0.68 microM. Moreover, GW9662, a PPARgamma antagonist, could attenuate such a 15d-PGJ2 inhibitory activity by almost one order of magnitude. This result thereby implies that 15d-PGJ2 might inhibit Smad2 translocation through PPARgamma/TGFbeta/Smad2 pathway. Further investigation discovered that different from the case for 15d-PGJ2, rosiglitazone, another PPARgamma agonist, could enhance Smad2 translocation to nucleus, suggesting that rosiglitazone and 15d-PGJ(2) might take different modes in the activation of PPARgamma within the signaling pathway.