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-Δ12,14-prostaglandin J2 induces p53 expression through Nrf2-mediated upregulation of heme oxygenase-1 in human breast cancer cells

Abstract

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that has antioxidant and cytoprotective functions. However, HO-1 has oncogenic functions in cancerous or transformed cells. In the present work, we investigated the effects of HO-1 on the expression of p53 induced by 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) in human breast cancer (MCF-7) cells. Treatment of MCF-7 cells with 15d-PGJ₂ led to time-dependent increases in the expression of p53 as well as HO-1. Upregulation of p53 expression by 15d-PGJ₂ was abrogated by si-RNA knock-down of HO-1. In MCF-7 cells transfected with HO-1 si-RNA, 15d-PGJ₂ failed to induce expression of p53 as well as HO-1. In addition, HO-1 inducers enhanced the p53 expression. We speculated that iron, a by-product of HO-1-catalyzed reactions, could mediate 15d-PGJ₂–induced p53 expression. Upregulation of p53 expression by 15d-PGJ₂ was abrogated by the iron chelator desferrioxamine in MCF-7 cells. Iron released from heme by HO-1 activity is mostly in the Fe²⁺ form. When MCF-7 cells were treated with the Fe²⁺-specific chelator phenanthroline, 15d-PGJ₂–induced p53 expression was attenuated. In addition, levels of the Fe-sequestering protein H-ferritin were elevated in 15d-PGJ₂-treated MCF-7 cells. In conclusion, upregulation of p53 and p21 via HO-1 induction and subsequent release of iron with accumulation of H-ferritin may confer resistance to oxidative damage in cancer cells frequently challenged by redox-cycling anticancer drugs.     

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.     

15-deoxy-delta12,14-prostaglandin J2-induced apoptosis in amnion-like WISH cells.

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.     

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.     

Differential regulation of chemokine gene expression by 15-deoxy-delta 12,14 prostaglandin J2

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-Delta(12,14)PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 x 10(-6) M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARgamma-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.     

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.     

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.     

Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2

15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.     

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.     

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.     

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-D12,14-prostaglandin J2 inhibits CX3CL1/fractalkine expression in human endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma)is a member of nuclear hormone receptor superfamily, and is knownto play a role in various biological processes including inflammatoryresponses and adipocyte differentiation. CX3CL1/fractalkineis a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes.Endothelial cells produce fractalkine when stimulated with cytokinessuch as interleukin-1 (IL-1), tumour necrosis factor-alpha andinterferon-gamma (IFN-gamma). We herein report that 15-deoxy-n12,14 -prostaglandinJ2 (15d-PGJ2), a PPAR-gamma agonist,inhibits the expression of fractalkine induced by IFN-gamma orIL-1beta in human endothelial cells. Agonist for PPAR-alpha (WY14643)or PPAR-gamma (ciglitazone) did not inhibit the cytokine-inducedfractalkine expression, and the effect of 15d-PGJ2 maybe independent of PPAR. 15-Deoxy-D12,14 prostaglandinJ2 also inhibited the adhesion of blood mononuclear cellsto endothelial monolayers treated with IFN-gamma or IL-1beta.The data suggest that 15d-PGJ2 regulates inflammatoryreactions, at least in part, through the inhibition of fractalkineexpression and leucocyte traffic through the endothelium.     

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.     

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.