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.     

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-Delta(12,14)-prostaglandin J2 inhibits the expression of proinflammatory genes in human blood monocytes via a PPAR-gamma-independent mechanism

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in inhibition of the expression of proinflammatory cytokines and inducible enzymes such as cyclooxygenase-2 (COX-2). Using real-time RT-PCR the present study investigates the impact of two PPAR-gamma agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and ciglitazone, on the expression of several proinflammatory genes in lipopolysaccharide (LPS)-stimulated human blood monocytes. Stimulation of cells with LPS resulted in a profound induction of the expression of COX-2, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of cells with 15d-PGJ(2) (10 microM) was associated with a nearly complete inhibition of the expression of all genes that remained unaltered in the presence of the PPAR-gamma antagonist bisphenol A diglycidyl ether (BADGE; 100 microM). By contrast, treatment of cells with another potent PPAR-gamma agonist, ciglitazone (50 microM), and the PPAR-alpha agonist WY-14,643 (100 microM) did not suppress LPS-induced expression of the investigated genes. Stimulation of monocytes with LPS resulted in an 88% inhibition of PPAR-gamma mRNA expression that was fully restored by 15d-PGJ(2) but only to a partial extent by ciglitazone and WY-14,643. Again, BADGE did not alter the effect of 15d-PGJ(2). Collectively, our results show that alterations of gene expression by 15d-PGJ(2) in LPS-stimulated human blood monocytes are mediated by PPAR-gamma-independent mechanisms. Moreover, it is concluded that both inhibition of proinflammatory gene expression and restoration of LPS-induced decrease of PPAR-gamma expression may contribute to the biological action of 15d-PGJ(2).     

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-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.     

Induction of heme oxygenase-1 mediates the anti-inflammatory effects of the ethanol extract of Rubus coreanus in murine macrophages

Foods of plant origin, especially fruits and vegetables, draw increased attention because of their potential benefits to human health. The aim of the present study was to determine in vitro anti-inflammatory activity of four different extracts obtained from the fruits of Rubus coreanus (aqueous and ethanol extracts of unripe and ripe fruits). Among the four extracts, the ethanol extract of unripe fruits of R. coreanus (URCE) suppressed nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. We also demonstrated that URCE by itself is a potent inducer of heme oxygenase-1 (HO-1). Inhibition of HO-1 activity by tin protoporphyrin, a specific HO-1 inhibitor, suppressed the URCE-induced reductions in the production of NO and PGE(2) as well as the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Our data suggest that URCE exerts anti-inflammatory effects in macrophages via activation of the HO-1 pathway and helps to elucidate the mechanism underlying the potential therapeutic value of R. coreanus extracts.     

15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-alpha by decreasing levels of acetylated histone H3 and H4 at its promoter

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch-clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K(1/2)) for cGMP in the homomeric CNGA3-T565M channels (160microM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0microM). Conversely, the K(1/2) for cGMP in the homomeric CNGA3-E593K channels (3.0microM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.     

15-Deoxy-Δ-prostaglandin J2 impairs the functions of histone acetyltransferases through their insolubilization in cells

The cyclopentenonic prostaglandin 15-deoxy-Δ^12,14-PG J₂ (15d-PGJ₂) is a metabolite derived from PGD₂. Although 15d-PGJ₂ has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor γ (PPARγ), the functions are not fully understood. In order to examine the effect of 15d-PGJ₂ on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ₂. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-κB-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ₂ attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ₂ on the function of HATs was independent of PPARγ, because PPARγ agonists could not mimick 15d-PGJ₂ and PPARγ antagonists did not inhibit 15d-PGJ₂.     

15-Deoxy-delta(12,14)-prostaglandin J(2) down-regulates CXCR4 on carcinoma cells through PPARgamma- and NFkappaB-mediated pathways

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE(2), PGA(2), PGD(2), PGJ(2) and 15dPGJ(2) each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD(2) and its metabolites PGJ(2) and 15dPGJ(2). Down-regulation was most rapid with the end-product 15dPGJ(2) and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ(2) is known to be a ligand for the nuclear receptor PPARgamma. Down-regulation of CXCR4 was also observed with the PPARgamma agonist rosiglitazone, while 15dPGJ(2)-induced CXCR4 down-regulation was substantially diminished by the PPARgamma antagonists GW9662 and T0070907. These data support the involvement of PPARgamma. However, the 15dPGJ(2) analogue CAY10410, which can act on PPARgamma but which lacks the intrinsic cyclopentenone structure found in 15dPGJ(2), down-regulated CXCR4 substantially less potently than 15dPGJ(2). The cyclopentenone grouping is known to inhibit the activity of NFkappaB. Consistent with an additional role for NFkappaB, we found that the cyclopentenone prostaglandin PGA(2) and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFkappaB p50 and that 15dPGJ(2) interfered with this p50 nuclear localization. These data suggest that 15dPGJ(2) can down-regulate CXCR4 on cancer cells through both PPARgamma and NFkappaB. 15dPGJ(2), present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.     

Suppression of chondrosarcoma cells by 15-deoxy-Delta 12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21

We previously reported that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent agonist for peroxisome proliferator-activated receptor gamma (PPAR gamma), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ(2)-induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ(2)-induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ(2), but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ(2) was partly, but not completely, blocked by PPAR gamma antagonist (GW9662) suggesting the 15d-PGJ(2) exerted its effect by PPAR gamma-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ(2) in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma.     

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.     

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.     

Nrf2 regulates the alternative first exons of CD36 in macrophages through specific antioxidant response elements

We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARγ specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.     

PPARγ stabilizes HO-1 mRNA in monocytes/macrophages which affects IFN-β expression

NADPH oxidase activation in either RAW264.7 cells or peritoneal macrophages (PM) derived from PPARγ wild-type mice increased reactive oxygen species (ROS) formation, caused PPARγ activation, heme oxygenase-1 (HO-1) induction, and concomitant IFN-β expression. In macrophages transduced with a dominant negative (d/n) mutant of PPARγ (RAW264.7 AF2) as well as PPARγ negative PM derived from Mac-PPARγ-KO mice, NADPH oxidase-dependent IFN-β expression was attenuated. As the underlying mechanism, we noted decreased HO-1 mRNA stability in RAW264.7 AF2 cells as well as PPARγ negative PM, compared to either parent RAW264.7 cells or wild-type PM. Assuming mRNA stabilization of HO-1 by PPARγ we transfected macrophages with a HO-1 3′-UTR reporter construct. The PPARγ agonist rosiglitazone significantly up-regulated luciferase expression in RAW264.7 cells, while it remained unaltered in RAW264.7 AF2 macrophages. Deletion of each of two AU-rich elements in the 3′-UTR HO-1 decreased luciferase activity in RAW264.7 cells. Using LPS as a NADPH oxidase activator, PM from Mac-PPARγ-KO mice showed a decreased HO-1 mRNA half-life in vitro and in vivo compared to PPARγ wild-type mice. These data identified a so far unappreciated role of PPARγ in stabilizing HO-1 mRNA, thus, contributing to the expression of the HO-1 target gene IFN-β.     

Assessing bioenergetic function in response to oxidative stress by metabolic profiling

It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.