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.     

Prostaglandin J2 and its metabolites promote neurite outgrowth induced by nerve growth factor in PC12 cells

Although A- and J-type prostaglandins (PG's) arrest the cell cycle at the G1 phase in vitro and suppress tumor growth in vivo, their effects on neuronal cells have not so far been clarified. Here, we found promotion of neurite outgrowth as a novel biological function of PGJ's. In PC12h cells, PGJ's (PGJ2, Delta12-PGJ2 and 15-deoxy-Delta12,14-PGJ2) promoted neurite outgrowth in the presence of nerve growth factor (NGF), whereas they themselves did not show such a promotion. The potency of promoting neurite outgrowth was PGJ2 < Delta12-PGJ2 < 15-deoxy-Delta12,14-PGJ2. However, troglitazone, an activator of peroxisome proliferator-activated receptorgamma (PPARgamma), and other PG's including PGA1, PGA2 and PGD2 did not promote neurite outgrowth. These results suggest that PGJ's promote neurite outgrowth independently of PPARgamma activation.     

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.     

Delta 12-prostaglandin J2, a plasma metabolite of prostaglandin D2, causes eosinophil mobilization from the bone marrow and primes eosinophils for chemotaxis

PGD(2), a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD(2) is rapidly transformed into its major metabolite delta(12)-PGJ(2), the effect of which on eosinophil migration has not yet been characterized. In this study we found that delta(12)-PGJ(2) was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD(2), PGJ(2), or 15-deoxy-delta(12,14)-PGJ(2). Moreover, pretreatment of eosinophils with delta(12)-PGJ(2) markedly enhanced the chemotactic response to eotaxin, and in this respect delta(12)-PGJ(2) was more effective than PGD(2). delta(12)-PGJ(2)-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that delta(12)-PGJ(2) signaled through the same receptor, CRTH2, as PGD(2). Finally, delta(12)-PGJ(2) was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that delta(12)-PGJ(2) is present in the systemic circulation at relevant levels, a role for this PGD(2) metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.     

Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine

We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.     

Prostaglandin D(2) and J(2) induce apoptosis in human leukemia cells via activation of the caspase 3 cascade and production of reactive oxygen species

The presence of prostaglandins (PGs) has been demonstrated in the processes of carcinogenesis and inflammation. In the present study, we found that 12-o-tetradecanoylphorbol 13-acetate (TPA) induced cyclooxygenase 2 (COX-2), but not COX-1, protein expression in HL-60 cells, and the addition of arachidonic acid (AA) in the presence or absence of TPA significantly reduced the viability of HL-60 cells, an effect that was blocked by adding the COX inhibitors, NS398 and aspirin. The AA metabolites, PGD(2) and PGJ(2), but not PGE(2) or PGF(2alpha), reduced the viability of the human HL60 and Jurkat leukemia cells according to the MTT assay and LDH release assay. Apoptotic characteristics including DNA fragmentation, apoptotic bodies, and hypodiploid cells were observed in PGD(2)- and PGJ(2)-treated leukemia cells. A dose- and time-dependent induction of caspase 3 protein procession, and PARP and D4-GDI protein cleavage with activation of caspase 3, but not caspase 1, enzyme activity was detected in HL-60 cells treated with PGD(2) or PGJ(2). Additionally, DNA ladders induced by PGD(2) and PGJ(2) were significantly inhibited by the caspase 3 peptidyl inhibitor, Ac-DEVD-FMK, but not by the caspase 1 peptidyl inhibitor, Ac-YVAD-FMK, in accordance with the blocking of caspase 3, PARP, and D4-GDI protein procession. An increase in intracellular peroxide levels by PGD(2) and PGJ(2) was identified by the DCHF-DA assay, and anti-oxidant N-acetyl cysteine (NAC), mannitol (MAN), and tiron significantly inhibited cell death induced by PGD(2) and PGJ(2) by reducing reactive oxygen species (ROS) production. The PGJ(2) metabolites, 15-deoxy-Delta(12,14)-PGJ(2) and Delta(12)-PGJ(2), exhibited effective apoptosis-inducing activity in HL-60 cells through ROS production via activation of the caspase 3 cascade. The proliferator-activated receptor-gamma (PPAR-gamma) agonists, rosiglitazone (RO), troglitazone (TR), and ciglitazone (CI), induced apoptosis in cells which was blocked by the addition of the PPAR-gamma antagonists, GW9662 and BADGE, via blocking of caspase 3 and PARP cleavage. However, neither GW9662 nor BADGE showed any protective effect on PGD(2)- and PGJ(2)-induced apoptosis. A differential apoptotic effect of PGs through ROS production, followed by activation of the caspase 3 cascade, was demonstrated.     

Regulation of prostaglandin endoperoxide synthase-2 and IL-6 expression in mouse bone marrow-derived mast cells by exogenous but not endogenous prostanoids.

Mouse bone marrow-derived mast cells (BMMC), stimulated with stem cell factor, IL-1beta, and IL-10, secrete IL-6 and demonstrate a delayed phase of PGD(2) generation that is dependent upon the induced expression of PG endoperoxide synthase (PGHS)-2. We have examined the potential for exogenous prostanoids, acting in a paracrine fashion, and endogenous prostanoids, acting in an autocrine fashion, to regulate PGHS-2 induction and IL-6 secretion in mouse BMMC. Exogenous PGE(2), which acts through G protein-coupled receptors, and 15-deoxy-Delta(12,14)-PGJ(2), which is a ligand for peroxisome proliferator-activated receptor (PPAR)gamma, elicited a 2- to 3-fold amplification of PGHS-2 induction, delayed-phase PGD(2) generation, and IL-6 secretion in response to stem cell factor, IL-1beta, and IL-10. The effect of PGE(2) was reproduced by the E prostanoid (EP)1 receptor agonist 17-trinor-PGE(2), and the EP1/EP3 agonist, sulprostone, but not the EP2 receptor agonist, butaprost. Although BMMC express PPARgamma, the effects of 15-deoxy-Delta(12,14)-PGJ(2) were not reproduced by the PPARgamma agonists, troglitazone and ciglitazone. PGHS-2 induction, but not IL-6 secretion, was impaired in cPLA(2)-deficient BMMC. However, there was no impairment of PGHS-2 induction in BMMC deficient in hematopoietic PGD synthase or PGHS-1 in the presence or absence of the PGHS-2 inhibitor, NS-398. Thus, although exogenous prostanoids may contribute to amplification of the inflammatory response by augmenting PGD(2) generation and IL-6 secretion from mast cells, endogenous prostanoids do not play a role.     

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

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

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.     

Novel prostaglandin D(2)-derived activators of peroxisome proliferator-activated receptor-gamma are formed in macrophage cell cultures

Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9alpha,13E,15(S))-Prosta-5,13-dien-1-oic acid [prostaglandin D(2) (PGD(2))] induced formation of considerable peroxisome proliferator-activated receptor-gamma (PPARgamma) activity [Nature 391 (1998) 79]. Because PGD(2) itself is a poor PPARgamma ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D(2) for 24 h and studied the ability of the metabolites formed to activate PPARgamma. PGD(2) products were extracted and fractionated by reverse phase high-performance liquid chromatography. Chemical identification was achieved by UV spectroscopy, gas-liquid chromatography/mass spectrometry and chemical syntheses of reference compounds. PGD(2) was converted to eight products, six of which were identified. Ligand-induced interaction of PPARgamma with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARgamma activation was investigated by transient transfection of RAW 264.7 macrophages. In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-delta(12,14)-PGJ(2)), a novel PPARgamma ligand and activator viz. 9-hydroxy-11-oxo-, (5Z,9alpha,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-delta(12,14)-PGD(2)) was identified. The biological significance of these results is currently under investigation.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.     

15-deoxy-delta 12,14-prostaglandin J(2) inhibits the synthesis of the acute phase protein SIP24 in cartilage: Involvement of COX-2 in resolution of inflammation

We previously demonstrated that, in the MC615 cartilage cell line, the p38/NF-kB pathway is activated both during differentiation and in response to an inflammatory stimulus. In both cases, the p38/NF-kB pathway activation leads to the expression of the lipocalin SIP24 and of COX-2. Given the fact that, in the same cells, the COX-2 expression is sustained during the inflammation resolution, at the same time that the SIP24 expression is suppressed, in the present study we tested the hypothesis that COX-2 products play a role in SIP24 repression. Taken together, our results suggest that, during the resolution of inflammation, COX-2 represses the acute phase protein SIP24 and restores physiological conditions, possibly through a pathway involving PPARgamma. Experimental evidences being the following: (1) 15-deoxy-delta 12,14-prostaglandin J(2), but not PGE(2): (i) inhibits the expression of SIP24 in the inflammatory phase and induces COX-2 synthesis; (ii) represses NF-kB activation induced by LPS; (iii) represses the synthesis of microsomal PGE Synthase-1 induced by LPS. (2) PPARgamma and PPARalpha are present in MC615 cells in both proliferating and hyperconfluent cultures. (3) PPARgamma ligand GW7845, but not PPARalpha ligand GW7647: (i) represses the expression of SIP24 induced by LPS; (ii) induces COX-2 expression. (4) p38 is involved in the PPARgamma mediated induction of COX-2. In fact 15-deoxy-delta 12,14-prostaglandin J(2) activates p38 and the cell pretreatment with the p38 specific inhibitor SB203580 represses the expression of COX-2 induced by both the 15-deoxy-delta12,14-prostaglandin J(2) and the PPARgamma ligand GW7845.     

Effects of 1400W, a potent selective inducible NOS inhibitor, on histamine- and leukotriene D4-induced relaxation of isolated guinea pig nasal mucosa.

To clarify the role of inducible nitric oxide synthase (iNOS) in the relaxation of nasal vasculature, the effects of a potent selective iNOS inhibitor, N-[(3-aminomethyl)benzyl]acetamidine (1400W), on histamine- and leukotriene D4 (LTD4)-induced relaxations of isolated nasal septal mucosae were examined in naive guinea pigs. In addition to eNOS and nNOS, Western blots demonstrated a distinct expression of iNOS in nasal mucosal tissues of naive guinea pigs. In isolated nasal septal mucosae precontracted with norepinephrine (3 x 10(-5)M), both histamine (10(-7)-10(-3)M) and LTD4 (10(-10)-10(-7)M) exhibited relaxations, which were inhibited by a NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 10(-4)M). The inhibitory effect of L-NMMA was reversed by L-arginine (10(-3)M), indicating that the relaxations induced by histamine and LTD4 are mediated by NO. Furthermore, both the histamine- and LTD4-induced relaxations were also significantly attenuated by 1400W (10(-5)M). These findings suggest an involvement of NO generated by iNOS in agonist-induced relaxation of nasal mucosal vasculature in naive guinea pigs.     

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.     

Feedback control of cyclooxygenase-2 expression through PPARgamma

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandins (PG), plays a key role in inflammation, tumorigenesis, development, and circulatory homeostasis. The PGD(2) metabolite 15-deoxy-Delta(12, 14) PGJ(2) (15d-PGJ(2)) was identified as a potent natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma expressed in macrophages has been postulated as a negative regulator of inflammation and a positive regulator of differentiation into foam cell associated with atherogenesis. Here, we show that 15d-PGJ(2) suppresses the lipopolysaccharide (LPS)-induced expression of COX-2 in the macrophage-like differentiated U937 cells but not in vascular endothelial cells. PPARgamma mRNA abundantly expressed in the U937 cells, not in the endothelial cells, is down-regulated by LPS. In contrast, LPS up-regulates mRNA for the glucocorticoid receptor which ligand anti-inflammatory steroid dexamethasone (DEX) strongly suppresses the LPS-induced expression of COX-2, although both 15d-PGJ(2) and DEX suppressed COX-2 promoter activity by interfering with the NF-kappaB signaling pathway. Transfection of a PPARgamma expression vector into the endothelial cells acquires this suppressive regulation of COX-2 gene by 15d-PGJ(2) but not by DEX. A selective COX-2 inhibitor, NS-398, inhibits production of PGD(2) in the U937 cells. Taking these findings together, we propose that expression of COX-2 is regulated by a negative feedback loop mediated through PPARgamma, which makes possible a dynamic production of PG, especially in macrophages, and may be attributed to various expression patterns and physiological functions of COX-2.     

Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE(2), PGA(2), PGA(1), PGF(2 alpha)) either delayed apoptosis or had no effect, whereas PGD(2) and its metabolite PGJ(2) selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca(2+). Intriguingly, the sequential metabolites Delta(12)PGJ(2) and 15-deoxy-Delta(12,) Delta(14)-PGJ(2) (15dPGJ(2)) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Delta(12)PGJ(2) and 15dPGJ(2) are peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators, apoptosis was not mimicked by synthetic PPAR-gamma and PPAR-alpha ligands or blocked by an irreversible PPAR-gamma antagonist. Furthermore, Delta(12)PGJ(2) and 15dPGJ(2) inhibited LPS-induced I kappa B alpha degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-gamma-independent inhibition of NF-kappa B activation. In addition, we show that TNF-alpha-mediated loss of cytoplasmic I kappa B alpha in eosinophils is inhibited by 15dPGJ(2) in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD(2) and PGJ(2) may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2) in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.