Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to J(2) series of PGs including 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2), which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ(12)-PGJ(2) has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ(12)-PGJ(2). According to the standard curve, the amount of Δ(12)-PGJ(2) can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ(2), while it only showed the cross-reaction of 28% with unstable PGJ(2). This immunological assay was applied to the determination of the endogenous formation of Δ(12)-PGJ(2) in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ(12)-PGJ(2) increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ(2). Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ(12)-PGJ(2) in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ(2) series at various doses on adipogenesis during the maturation phase. Although Δ(12)-PGJ(2) was slightly less potent than 15d-PGJ(2), each of these PGJ(2) series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ(12)-PGJ(2) contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ(2) during the maturation phase of cultured adipocytes.     

Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE₂ upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) and troglitazone as peroxisome proliferator-activated receptor γ (PPARγ) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARγ and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.     

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-Δ(12,14)-prostaglandin J(2) interferes inducible synthesis of prostaglandins E(2) and F(2α) that suppress subsequent adipogenesis program in cultured preadipocytes

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E(2) and PGF(2α) involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ(2) interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ(12)-PGJ(2) and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ(2). Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ(2) or PDTC although 15d-PGJ(2) alone has no stimulatory effect. Moreover, 15d-PGJ(2) did not block the inhibitory effects of exogenous PGE(2) and PGF(2α) on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ(2) can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.     

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 J(2) suppresses IL-6-induced STAT3 phosphorylation via electrophilic reactivity in endothelial cells

In this study, the effects of 15d-PGJ(2) were investigated in IL-6-activated endothelial cells (ECs). 15d-PGJ(2) was found to abrogate phosphorylation on tyr705 of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner, but did not inhibit serine phosphorylation of STAT3 and the upperstream JAK2 phosphorylation. Other PPAR activators, such as WY1643 or ciglitazone, had no effect upon IL-6-induced STAT3 phosphorylation. Additionally, neither orthovanadate nor l-NAME treatment reverses the inhibition of STAT3 phosphorylation by 15d-PGJ(2). Otherwise, the effect of 15d-PGJ(2) requires the alpha,beta-unsaturated carbonyl group in the cyclopentane ring. A 15d-PGJ(2) analog, 9,10-Dihydro-15d-PGJ(2), which lack alpha,beta-unsaturated carbonyl group showed no increase in ROS production and no effect in inhibition of IL-6-induced STAT3 phosphorylation. The electrophilic compound, acrolein, mimics the inhibition effect of 15d-PGJ(2). Among the antioxidants, only NAC and glutathione reversed the effects of 15d-PGJ(2). NAC, glutathione and DTT all reversed the inhibition of STAT3 phosphorylation when preincubated with 15d-PGJ(2). The inhibition of ICAM-1 gene expression by 15d-PGJ(2) was abrogated by NAC and glutathione in IL-6-treated ECs. Taken together, these results suggest that 15d-PGJ(2) inhibits IL-6-stimulated phosphorylation on tyr705 of STAT3 dependent on its own electrophilic reactivity in ECs.     

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.     

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.     

Stimulation of NGF expression and secretion in 3T3-L1 adipocytes by prostaglandins PGD2, PGJ2, and Delta12-PGJ2

Nerve growth factor (NGF) has recently been shown to be secreted from white adipocytes, its production being strongly stimulated by the proinflammatory cytokine tumor necrosis factor-alpha. In this study, we have examined whether a series of prostaglandins and other inflammation-related factors also stimulate NGF expression and secretion by adipocytes, using 3T3-L1 cells. Although interleukin (IL)-1beta, IL-10, and IL-18 each induced a small decrease in NGF mRNA level in 3T3-L1 adipocytes, there was no significant effect of these cytokines on NGF secretion. A small reduction in NGF expression and/or secretion was also observed with adiponectin and prostaglandins PGE(2), PGF(2alpha), and PGI(2). In marked contrast, prostaglandin PGD(2) induced a major, dose-dependent increase (up to 20- to 40-fold) in NGF expression and secretion. The PGD(2) metabolites, PGJ(2) and Delta(12)-PGJ(2), also induced major increases (up to 30-fold) in NGF production. A further metabolite of PGJ(2), 15-deoxy-Delta(12,14)-PGJ(2), a peroxisome proliferator-activated receptor-gamma agonist, led paradoxically to a small increase in NGF mRNA level but a fall in NGF secretion. Both PGD(2) and PGJ(2) induced significant increases in NGF gene expression by 4 h after their addition. It is concluded that PGD(2) and the J series prostaglandins, PGJ(2) and Delta(12)-PGJ(2), can play a significant role in the regulation of NGF production by white adipocytes. These results provide support for the view that NGF is an important inflammatory response protein, as well as a target-derived neurotrophin, in white adipose tissue.     

Anti-inflammatory effects and changes in prostaglandin patterns induced by 7beta-hydroxy-epiandrosterone in rats with colitis

High dose levels of dehydroepiandrosterone and its 7-hydroxylated derivatives have been shown to reduce oxidative stress and inflammatory responses in dextran sodium sulfate (DSS)-induced colitis in rats. Another endogenous steroid, 7beta-hydroxy-epiandrosterone (7beta-hydroxy-EpiA) has been shown to exert neuroprotective effects at much smaller doses. Our aims were to evaluate whether 7beta-hydroxy-EpiA pre-treatment prevents DSS-induced colitis and to determine whether the effects involve changes in anti-inflammatory prostaglandin (PG) D(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) levels. Rats were administered 0.01, 0.1 and 1mg/kg 7beta-hydroxy-EpiA i.p. once a day for 7 days. Thereafter, colitis was induced by administration of 5% DSS in drinking water for 7 days. Levels of the PGs and the expression of cyclooxygenase (COX-2) and PG synthases were assessed during the course of the experiment. Administration of 7beta-hydroxy-EpiA caused a transient increase in COX-2 and PGE synthase expression within 6-15h and augmented colonic tissue levels of 15d-PGJ(2) levels starting at day 2. Treatment with DSS resulted in shortened colon length, depleted mucus in goblet cells and induced oxidative stress. COX-2 and mPGES-1 synthase expression were enhanced and accompanied by increased PGE(2), D(2) and 15d-PGJ(2) production. Although all dose levels of 7beta-hydroxy-EpiA reduced PGE(2) production, only the lowest dose (0.01mg/kg) of the steroid completely prevented colitis damage and tissue inflammation. 7beta-Hydroxy-EpiA pre-treatment prevents the occurrence of DSS-induced colitis through a shift from PGE(2) to PGD(2) production, associated with an early but transient increase in COX-2 expression and a sustained increase in the production of the anti-inflammatory prostaglandin 15d-PGJ(2).     

Expression of adipogenesis markers in a murine stromal cell line treated with 15-deoxy Delta(12,14)-prostaglandin J2, interleukin-11, 9-cis retinoic acid and vitamin K2.

Recent studies have demonstrated that bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vivo, and in vitro, and that peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a central role in the control of adipocyte differentiation. In the present study, we treated a murine stromal cell line (TMS-14) with a cocktail of dexamethasone, insulin and glucose (DIG cocktail), which caused the cells to convert to fat-laden cells with adipocyte-like morphology. We also exposed TMS-14 cells to DIG cocktail followed by 15-deoxy Delta(12,14)-prostaglandin J2 (15d-PGJ2), a ligand of PPAR gamma, interleukin- 11 (IL-11), 9-cis retinoic acid (9-cis RA) and vitamin K2. 15d-PGJ2 enhanced DIG cocktail-induced adipogenesis, whereas IL-11, 9-cis RA and vitamin K2 each inhibited adipogenesis induced by DIG cocktail. The gene expressions of four adipogenesis markers, PPAR gamma 2, adipocyte P2 (aP2), adipocyte determination and differentiation factor 1 (ADD1), and fatty acid synthase (FAS) were enhanced by DIG cocktail and these expressions were more enhanced by 15d-PGJ2, in contrast they were attenuated by 9-cis RA. IL-11 also attenuated the adipogenesis markers except ADD1. Western blotting showed that 15d-PGJ2 enhanced the levels of PPAR gamma, C/EBP alpha and RXR alpha proteins, while IL-11 and 9-cis RA decreased the level of PPAR gamma protein, but not C/EBP alpha protein and vitamin K2 decreased the level of C/EBP alpha protein. We also tested the effect of 15d-PGJ2 on osteoblastogenesis, using TMS-12 cells, another stromal cell clone from the same mouse, which differentiate into osteoblasts spontaneously. 15d-PGJ2 did not affect osteoblastogenesis, as detected by von Kossa staining and Cbfa-1 gene expression. These data indicate that 15d-PGJ2 enhances the expression of both PPAR gamma and C/EBP alpha and as a result it stimulates adipogenesis in murine bone marrow cells.     

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.     

Mast cells function as an alternative modulator of adipogenesis through 15-deoxy-delta-12, 14-prostaglandin J2

Mast cells are one of the major producers of prostaglandins (PGs). The final metabolite of PGs 15-deoxy-delta-12,14-PGJ(2) (15-deoxy-delta PGJ(2)) is the endogenous ligand of the peroxisome proliferator-activated receptor (PPAR) γ. PPARγ modulates adipocyte differentiation; therefore, we attempted to investigate whether PGs derived from mast cells influenced on adipogenesis. We found the increase of mast cell numbers in fat tissue of obese mice fed with a high-fat diet allowed us to speculate contributions of mast cells to adipogenesis. Mast cell-mediated induction of adipogenesis was evaluated by using 3T3 L1 cells. Supernatants obtained from mast cells stimulated with calcium ionophore or the high-glucose condition contained 15-deoxy-delta PGJ(2) and induced adipogenesis of 3T3 L1 cells. Agonistic activity of PGJ(2) from the supernatants on PPARγ was confirmed by a reporter gene assay. Culture medium collected from calcium ionophore-stimulated bone marrow-derived cultured mast cells (BMCMC) activated PPAR-responsive element in NIH3T3 fibroblasts, and the specific inhibitor of PPARγ canceled the activation. Contribution of mast cells to obesity was evaluated by using mast cell-deficient mice fed with a Western diet. Weight gain of mast cell-deficient mice during high-fat feeding was impaired compared with their littermate wild-type mice; on the other hand, transplantation of bone marrow-derived cultured mast cells to mast cell-deficient mice restored the weight gain by intake of a high-fat diet. In this study, we clearly demonstrated that mast cells produced PGs in response to the high-glucose condition and induced adipocyte differentiation and possibly obesity. This is the first study that provides evidence for a novel role of mast cells in adipogenesis via PPARγ activation.     

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).     

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.