Endogenous prostaglandin D(2) synthesis decreases vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells

We examined the role of prostaglandin D(2) (PGD(2)) in the expression of vascular cell adhesion molecule-1 (VCAM)-1 following interleukin-1beta (IL-1) stimulation in human umbilical vein endothelial cells (HUVEC) transfected with lipocaline-type PGD(2) synthase (L-PGDS) genes. HUVEC were isolated from human umbilical vein and incubated with 20 U/ml IL-1 and various concentrations of authentic PGD(2). The isolated HUVEC were also transfected with L-PGDS genes by electroporation. The L-PGDS-transfected HUVEC were used to investigate the role of endogenous PGD(2) in IL-1-stimulated VCAM-1 biosynthesis. We also used an anti-PGD(2) antibody to examine whether an intracrine mechanism was involved in VCAM-1 production. PGD(2) and VCAM-1 levels were determined by radio- and cell surface enzyme-immunoassay, respectively. VCAM-1 mRNA was assessed by RT-PCR. IL-1-stimulated VCAM-1 expression by HUVEC was dose-dependently inhibited by authentic PGD(2). L-PGDS gene-transfected HUVEC produced more PGD(2) than HUVEC transfected with the reporter gene alone. IL-1 induced increases in VCAM-1 expression in HUVEC transfected with reporter genes alone. However, this effect was significantly attenuated in the case of IL-1 stimulation of HUVEC transfected with L-PGDS genes, and accompanied by an apparent suppression of VCAM-1 mRNA expression. Neutralization of extracellular PGD(2) by anti-PGD(2)-specific antibody influenced neither VCAM-1 mRNA expression nor VCAM-1 biosynthesis. In conclusion, HUVEC transfected with L-PGDS genes showed increased PGD(2) synthesis. This increase was associated with attenuation of both VCAM-1 expression and VCAM-1 mRNA expression. The results suggest that endogenous PGD(2) decreases VCAM-1 expression and VCAM-1 mRNA expression, probably through an intracrine mechanism.     

Prevention of paraquat-induced apoptosis in human neuronal SH-SY5Y cells by lipocalin-type prostaglandin D synthase

Paraquat is a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant 1-methyl-4-phenyl-pyridine and acts as a potential etiologic factor for the development of Parkinson's disease. In this study, we investigated the protective roles of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) against paraquat-mediated apoptosis of human neuronal SH-SY5Y cells. The treatment of SH-SY5Y cells with paraquat decreased the intracellular GSH level, and enhanced the cell death with elevation of the caspase activities. L-PGDS was expressed in SH-SY5Y cells, and its expression was enhanced with the peak at 2?h after the initiation of the treatment with paraquat. Inhibition of PGD? synthesis and exogenously added PGs showed no effects regarding the paraquat-mediated apoptosis. SiRNA-mediated suppression of L-PGDS expression in the paraquat-treated cells increased the cell death and caspase activities. Moreover, over-expression of L-PGDS suppressed the cell death and caspase activities in the paraquat-treated cells. The results of a promoter-luciferase assay demonstrated that paraquat-mediated elevation of L-PGDS gene expression occurred through the NF-κB element in the proximal promoter region of the L-PGDS gene in SH-SY5Y cells. These results indicate that L-PGDS protected against the apoptosis in the paraquat-treated SH-SY5Y cells through the up-regulation of L-PGDS expression via the NF-κB element. Thus, L-PGDS might potentially serve as an agent for prevention of human neurodegenerative diseases caused by oxidative stress and apoptosis.     

An interaction between L-prostaglandin D synthase and arrestin increases PGD2 production

L-type prostaglandin synthase (L-PGDS) produces PGD(2), a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD(2)-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2(-/-)-arr-3(-/-). Arrestin-3 promotes PGD(2) production by L-PGDS in vitro. IL-1β-induced PGD(2) production is significantly lower in MEFs-arr-2(-/-)-arr-3(-/-) than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD(2) production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD(2) synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.     

Lipocalin-type prostaglandin D2 synthase protein regulates glial cell migration and morphology through myristoylated alanine-rich C-kinase substrate: prostaglandin D2-independent effects

Prostaglandin D synthase (PGDS) is responsible for the conversion of PGH(2) to PGD(2). Two distinct types of PGDS have been identified: hematopoietic-type PGDS (H-PGDS) and lipocalin-type PGDS (L-PGDS). L-PGDS acts as both a PGD(2)-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. Although L-PGDS is one of the most abundant proteins in the cerebrospinal fluid, little is known about the function of L-PGDS in the central nervous system (CNS). To better understand the role of L-PGDS in the CNS, effects of L-PGDS on the migration and morphology of glial cells were investigated. The L-PGDS protein accelerated the migration of cultured glial cells. Expression of the L-pgds gene was detected in glial cells and neurons. L-PGDS protein also induced morphological changes in glia similar to the characteristic phenotypic changes in reactive gliosis. L-PGDS-induced cell migration was associated with augmented formation of actin filaments and focal adhesion, which was accompanied by activation of AKT, RhoA, and JNK pathways. L-PGDS protein injected into the mouse brain promoted migration and accumulation of astrocytes in vivo. Furthermore, the cell migration-promoting effect of L-PGDS on glial cells was independent of the PGD(2) products. The L-PGDS protein interacted with myristoylated alanine-rich protein kinase C substrate (MARCKS) to promote cell migration. These results demonstrate the critical role of L-PGDS as a secreted lipocalin in the regulation of glial cell migration and morphology. The results also indicate that L-PGDS may participate in reactive gliosis in an autocrine or paracrine manner, and may have pathological implications in neuroinflammatory diseases.     

Induction and function of lipocalin prostaglandin D synthase in host immunity

Although mainly expressed in neuronal cells, lipocalin-type PGD synthase (L-PGDS) is detected in the macrophages infiltrated to atherosclerotic plaques. However, the regulation and significance of L-PGDS expression in macrophages are unknown. Here, we found that treatment of macrophages with bacterial endotoxin (LPS) or Pseudomonas induced L-PGDS expression. Epigenetic suppression of L-PGDS expression in macrophages blunted a majority of PGD(2) produced after LPS treatment. Chromatin immunoprecipitation assays show that L-PGDS induction was regulated positively by AP-1, but negatively by p53. L-PGDS expression was detected in whole lung and alveolar macrophages treated with LPS or Pseudomonas. L-PGDS overexpressing transgenic mice improved clearance of Pseudomonas from the lung compared with nontransgenic mice. Similarly, intratracheal instillation of PGD(2) enhanced removal of Pseudomonas from the lung in mice. In contrast, L-PGDS knockout mice were impaired in their ability to remove Pseudomonas from the lung. Together, our results identify induction of L-PGDS expression by inflammatory stimuli or bacterial infection, the regulatory mechanism of L-PGDS induction, and the protective role of L-PGDS expression in host immune response. Our study suggests a potential therapeutic usage of L-PGDS or PGD(2) against Pseudomonas pneumonia.     

Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells

The promoter function of the rat lipocalin-type prostaglandin D synthase (L-PGDS) gene was characterized in primary cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of the promoter region (-1250 to +77) showed that an AP-2 element at -109 was required for activation and an E-box at +57, for repression. Binding of nuclear factors to each of these cis-elements was demonstrated by an electrophoretic mobility shift assay. Several components of the Notch-Hes signaling pathway, Jagged, Notch1, Notch3, and Hes-1, were expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and repressed the promoter activity of the rat L-PGDS gene in a dose-dependent manner. The L-PGDS gene expression was up-regulated slowly by interleukin-1 beta to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two NF-kappa B elements at -1106 and -291 were essential for this up-regulation. Binding of two NF-kappa B subunits, p65 and c-Rel, to these two NF-kappa B elements occurred after the interleukin-1 beta treatment. Therefore, the L-PGDS gene is the first gene identified as the target for the Notch-Hes signal through the E-box among a variety of genes involved in the prostanoid biosynthesis, classified to the lipocalin family, and expressed in the leptomeninges. Moreover, the L-PGDS gene is a unique gene that is activated slowly by the NF-kappa B system.     

A negative regulator of delayed prostaglandin D2 production in mouse mast cells

We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D(2) production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcepsilonRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD(2) generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD(2) generation and cytokine/chemokine expression. The suppression of delayed PGD(2) generation by NLRP3 was preceded by a transient decrease of NF-kappaB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), COX-1 and hematopoietic PGD(2) synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD(2) generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD(2), a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.     

Activated human dendritic cells express inducible cyclo-oxygenase and synthesize prostaglandin E2 but not prostaglandin D2

Prostaglandins (PG) are well known lipid mediators with important immunoregulatory properties. While exogenous PGE2 has the ability to modulate the function and maturation of antigen presenting cells, such as dendritic cells (DC), it is not clear whether human DC have the capacity to synthesize PGE2 and other prostaglandins themselves. We therefore examined the expression of inducible cyclo-oxygenase (COX-2) by monocyte derived DC and the production of PGE2 and PGD2. Both monocyte derived DC and freshly isolated blood myeloid DC expressed little COX-2 constitutively, though COX-2 expression was rapidly but transiently upregulated in response to lipopolysaccharide stimulation. COX-2 mRNA was detectable within 1 h of LPS exposure, peaked at 4-6 h, and rapidly declined thereafter. COX-2 expression was accompanied by DC synthesis of PGE2, with peak levels present at 6-18 h post-stimulation. In contrast, PGD2 synthesis was not detected at any time point. When DC were activated with LPS in the presence of nimesulide, a COX-2 selective inhibitor, IL-10 synthesis was inhibited, indicating that endogenous prostaglandins regulate DC cytokine production. PGE2 production by DC may therefore modulate DC and T-cell function, thereby shaping the character of the immune response.     

Effect of endotoxin treatment on the expression and localization of spinal cyclooxygenase, prostaglandin synthases, and PGD2 receptors

Systemic inflammation leads to increased expression of spinal cyclooxygenase (COX)-2 and to a subsequent increase of prostaglandin (PG) biosynthesis, which contribute to the development of hyperalgesia and allodynia. In this study, endotoxin caused a sequential induction of membrane bound prostaglandin E synthase-1 and lipocalin-type PGD synthase (L-PGDS) in the mouse spinal cord. L-PGDS expression was detected in the leptomeninges, oligodendrocytes, and interestingly, in discrete perivascular cells. Endotoxin-caused increase was most prominent in oligodendrocytes. Endotoxin-induced COX-2 and membrane bound prostaglandin E synthase-1 were restricted to the leptomeninges and perivascular cells. COX-1 was not influenced by endotoxin. We found COX-1 expressed in microglia, some of them in close proximity to L-PGDS-positive oligodendrocytes and co-localization of COX-1 with L-PGDS in perivascular and leptomeningeal cells under control conditions. It can be assumed, that PGD₂ biosynthesis under control conditions is mediated via COX-1 and that during inflammation, increased PGD₂ is dependent on COX-2. We found the PGD₂ receptors DP1 and chemoattractant receptor homologous molecule expressed on T helper type 2 cells (CRTH2) localized in neurons of the dorsal, and motoneurons in the ventral horn. The localization of the PGD₂ receptors DP1 and CRTH in spinal cord neurons, particularly in neurons of lamina I and II involved in the processing of nociceptive stimuli, supports a role of PGD₂ under inflammatory conditions.     

Increased expression of lipocalin-type-prostaglandin D synthase in ulcerative colitis and exacerbating role in murine colitis

The pathogenesis of ulcerative colitis (UC) is unclear, but enhancement of disease activity by usage of nonsteroidal anti-inflammatory drugs suggests involvement of prostanoid in its pathophysiology. However, biological effect of prostaglandin (PG) D(2) on intestinal inflammation remains unknown. We investigated the expression of enzymes for PGD(2) synthesis, prostaglandin D synthase (PGDS), and its relation to the activity of colitis in UC patients. The role of lipocalin-type PGDS (L-PGDS) using a murine colitis model was also assessed. Tissue samples were obtained by colonic biopsies from patients with UC. Expression levels of mRNAs for L-PGDS and hematopoietic-type PGDS were investigated by quantitative RT-PCR. COX-2 and L-PGDS expression was investigated by immunohistochemistry. Localization of L-PGDS expression was also determined by in situ hybridization. In experimental study, mice were treated with dextran sodium sulfate in the drinking water to induce colitis. The degree of colonic inflammation was compared with L-PGDS(-/-) mice and control mice. The level of L-PGDS mRNA expression was increased in UC patients in parallel with disease activity. Colocalization of L-PGDS and cyclooxygenase (COX) 2 was observed in lamina proprial infiltrating cells and muscularis mucosa in UC patients. The level of hematopoietic PGDS mRNA expression did not differ from control mucosa. Dextran sodium sulfate treatment to L-PGDS(-/-) mice showed lower disease activity than control mice. We reported for the first time the presence of L-PGDS in the COX-2-expressing cells in the mucosa of active UC patients and that only L-PGDS increased with disease activity. An animal model study suggests that PGD(2) derived from L-PGDS-expressing cells plays proinflammatory roles in colitis.     

7beta-Hydroxy-epiandrosterone-mediated regulation of the prostaglandin synthesis pathway in human peripheral blood monocytes

7alpha-Hydroxy-DHEA, 7beta-hydroxy-DHEA and 7beta-hydroxy-EpiA are native metabolites of dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA). Since numerous steroids are reported to interfere with inflammatory and immune processes, our objective was to test the effects of these hydroxysteroids on prostaglandin (PG) production and related enzyme gene expression. Human peripheral blood monocytes were cultured for 4 and 24 h in the presence of each of the steroids (1-100 nM), with and without addition of TNF-alpha (10 ng/mL). Levels of PGE(2), PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were measured in the incubation medium, and cell content of cyclooxygenase (COX-2), and PGE and PGD synthases (m-PGES1, H-PGDS, L-PGDS), and peroxisome proliferator activated receptor (PPAR-gamma) was assessed by quantitative RT-PCR and Western blots. Addition of TNF-alpha resulted in elevated PG production and increased COX-2 and m-PGES1 levels. Among the three steroids tested, only 7beta-hydroxy-EpiA decreased COX-2, m-PGES1 and PPAR-gamma expression while markedly decreasing PGE(2) and increasing 15d-PGJ(2) production. These results suggest that 7beta-hydroxy-EpiA is a native trigger of cellular protection through simultaneous activation of 15d-PGJ(2) and depression of PGE(2) synthesis, and that these effects may be mediated by activation of a putative receptor, specific for 7beta-hydroxy-EpiA.     

A novel pathway to enhance adipocyte differentiation of 3T3-L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol regulatory element-binding protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.     

Lipocalin-type prostaglandin D synthase protects against oxidative stress-induced neuronal cell death

L-PGDS [lipocalin-type PGD (prostaglandin D) synthase] is a dual-functional protein, acting as a PGD2-producing enzyme and a lipid transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. In the present study we demonstrate the protective effect of L-PGDS on H2O2-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H2O2-treated neuronal cells, and the L-PGDS level was highly associated with H2O2-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H2O2-mediated cell death. A cell viability assay revealed that L-PGDS protected against H2O2-induced cell death in a concentration-dependent manner. Furthermore, the titration of free thiols in H2O2-treated L-PGDS revealed that H2O2 reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight)-MS spectrum of H2O2-treated L-PGDS showed a 32 Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable with those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases.