Fumiaki Kojima

Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2.

Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE2 biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathioneS-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE2 generation. Arg110, a residue well conserved in the microsomal GSHS-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A2 allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE2 biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer.

Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis

Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H2 to PGE2 downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription–polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH2 to PGE2 was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1β or tumor necrosis factor (TNF)-α, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-γ, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1β or TNF-α. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF1α and thromboxane B2, the production of PGE2 was greater after chondrocytes were stimulated by IL-1β or TNF-α. Conversion of PGH2 to PGE2 (PGES activity) was significantly increased in the lysate from IL-1β-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE2 synthesis in patients with OA.

Prostaglandin E synthase in the pathophysiology of arthritis

Prostaglandin E synthase (PGES) is a recently identified terminal enzyme that acts downstream of cyclooxygenase and catalyzes the conversion of prostaglandin (PG) H2 to PGE2. At least three isozymes have been cloned so far, which are called membrane‐associated PGES (mPGES)‐1, mPGES‐2, and cytosolic PGES. Among them, mPGES‐1 is induced by various inflammatory stimuli in some cells and tissues. Induction of mPGES‐1 in the component of articular tissues of patients with rheumatoid arthritis and osteoarthritis has been demonstrated in vitro. Recent studies using adjuvant induced arthritis model have shown the increase of mPGES‐1 expression resulted in the increase of PGE2 production at the sites of inflammation. In addition, reports of mPGES‐1‐deficient mice clearly suggest the role of mPGES‐1 in the process of chronic inflammation such as collagen‐induced arthritis and collagen antibody induced arthritis in vivo. Thus, recent in vitro and in vivo findings suggest that mPGES‐1 may be a novel therapeutic target for arthritis. This paper introduces recent advances in research about the role of PGES in the pathophysiology of arthritis.

Prostaglandin I2 Plays a Key Role in Zymosan-Induced Mouse Pleurisy

Zymosan, the cell wall of Saccharomyces cerevisiae, induces innate immune responses involving prostanoid production and complement activation. However, the roles of prostanoids in zymosan-induced inflammation and their interaction with the complement system remain to be determined. To clarify these issues, we examined zymosan-induced pleurisy in mice lacking receptors for prostaglandin (PG) E2 (EP–/– mice) or PGI2 (IP–/– mice). Zymosan-induced exudate formation was significantly reduced in IP–/– mice compared with wild-type (WT) mice, whereas none of the EP–/– mice (EP1–/–, EP2–/–, EP3–/–, and EP1–/–4 mice) showed any significant difference from WT mice. Furthermore, indomethacin, an inhibitor of prostanoid biosynthesis, suppressed exudate formation in WT mice to almost the same level as that of IP–/– mice. Accordingly, significant production of PGI2 in the pleural cavity, suggested to be cyclooxygenase-2-dependent, was observed after zymosan injection. Complement activation in the pleural cavity after zymosan injection was confirmed, and preinjection of cobra venom factor (CVF), to deplete blood complement C3, was significantly suppressed after zymosan-induced exudate formation in WT mice. Simultaneous treatment with indomethacin and CVF further suppressed exudate formation in WT mice compared with each treatment alone. Because, some degree of exudate formation was still observed, other factor(s) seem to be involved. However, platelet-activating factor, a promising candidate as one such factor, was not involved in zymosan-induced exudate formation. These results clearly indicate that the PGI2-IP system together with the complement system plays a key role in exudate formation in zymosan-induced pleurisy.

FR190997, a novel bradykinin B2 agonist, expresses longer action than bradykinin in paw edema formation and hypotensive response.

Biological actions of a novel non-peptide B2 receptor agonist, FR190997, were examined by comparing them with those of bradykinin. The paw edema was induced by subcutaneous injection of 30 microl of solution of bradykinin (0.3, 0.6, and 1.2 nmol) or FR190997 (0.1, 0.3, and 0.9 nmol) into the right hind paw of ICR male mice. Bradykinin caused a dose-dependent edema formation, which peaked at 15 min and ceased after 150 min. FR190997 also formed a dose-dependent edema, peaking at 15-30 min with a slight delay compared to bradykinin and this response continued over 200 min. The edema formed by bradykinin or FR190997 was inhibited by pretreatment with HOE140 (1 mg/kg) injected intraperitoneally 30 min before the injection of each agonist. A novel non-peptide B2 antagonist, FR173657 (30 mg/kg, i.p. 30 min before the agonist), also diminished these responses by bradykinin and FR190997 dose-dependently. Indomethacin (10 mg/kg, i.p. 30 min before) inhibited the response to FR190997, suggesting that release of prostaglandins induced by the B2 agonistic action might be involved in this inflammatory process induced by FR190997. The hypotensive action of FR190997 was also examined. Intravenously injected FR190997 caused the systemic hypotensive response in Sprague-Dawley male rats anesthetized with pentobarbital. The potency of FR190997 was weaker than that of bradykinin, when compared with the maximal hypotension. Duration of the hypotensive response of FR190997 was significantly longer than that of bradykinin. These results indicate that FR190997 has the B2 agonistic action similar to bradykinin and is also a good tool for in vivo examination of the B2 receptor.

Effects of dexamethasone and protein kinase C inhibitors on the induction of bradykinin B1 mRNA and the bradykinin B1 receptor-mediated contractile response in isolated rat ileum

We detected the expression of inducible bradykinin (BK) B1 receptor mRNA in the rat ileum by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, when the isolated ileum was suspended for at least 1 hr in an aerated Tyrodes solution at 37 degrees. The induction of this mRNA was both time- and temperature-dependent, and was followed by a contractile response to des-Arg9-BK at around 3 hr of incubation; this response increased in magnitude with time and was maximal at 6 hr. In contrast, the contraction in response to BK and the expression of B2 receptor mRNA were constant throughout this 6-hr incubation period. The contraction due to des-Arg9-BK was selectively suppressed by B1 receptor antagonists, i.e. des-Arg9[Leu8]-BK and des-Arg10-HOE140, but not by the B2 antagonists D-Arg-[Hyp3,Thi5,8,D-Phe7]-BK and HOE140. The inducible des-Arg9-BK contractile response was suppressed by continuous in vitro exposure of the ileum to cycloheximide or actinomycin D, but neither inhibitor affected the contraction induced by BK, suggesting that the B1 receptor could be induced de novo. In vitro and ex vivo treatment of the ileum with dexamethasone suppressed the induction of the contractile response to des-Arg9-BK, but had no significant effect on the expression of B1 receptor mRNA. Some protein kinase C inhibitors, i.e. H7 and calphostin C, suppressed the expression of B1 receptor mRNA and diminished the contractile response to des-Arg9-BK. These results suggest that the de novo synthesis of the B1 receptor in the ileum preparation can be up-regulated at the transcriptional level (a process in which a specific isoform of protein kinase C may be involved). Additionally, these data suggest that the contractile response to des-Arg9-BK involves a process sensitive to some post-transcriptional action of dexamethasone.

Suppression of lymphangiogenesis by soluble vascular endothelial growth factor receptor-2 in a mouse lung cancer model

The vascular endothelial growth factor (VEGF) family has a key role in the formation of blood vessels and lymphatics. Among the members of this family, VEGF-C is one of the most important factors involved in lymphangiogenesis via binding with two receptors (vascular endothelial growth factor receptor-2 and -3: VEGFR-2 and VEGFR-3). Soluble VEGFR-2 (sVEGFR-2) has a role in maintaining the alymphatic state of the cornea associated with binding to VEGF-C, and selectively inhibits lymphangiogenesis but not angiogenesis. In this study, we introduced sVEGFR-2 into lung cancer cells and evaluated the influence on tumor progression and on genes regulating lymphatic formation and metastasis in vivo. A retroviral vector was used to introduce the sVEGFR-2 gene into Lewis lung carcinoma cells (LLC), which were designated as LLC-sVEGFR-2 cells. Proteins secreted into the culture supernatant by these cells were detected by western blotting using specific antibodies. To examine lymphangiogenesis by primary lung cancer in vivo, LLC-sVEGFR-2 cells were subcutaneously injected into C57BL/6 mice. At 14days after injection, immunohistochemistry was performed using an antibody directed against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a marker of lymphatics. Expression of mRNA for VEGFR-2, VEGFR-3 and matrix metalloproteinases (MMPs) was also determined by real-time PCR. Furthermore, LLC-sVEGFR-2 cells were directly inoculated into the left lung in C57BL/6 mice and the number of micro-metastases in pulmonary lymph nodes was determined. Introduction of sVEGFR-2 into LLC cells resulted in secretion of sVEGFR-2 protein into the culture supernatant. There were fewer LYVE-1 positive lymphatics after inoculation of LLC-sVEGFR-2 into mice compared with the control group. In addition, VEGFR-2, VEGFR-3, and MMPs gene expression was suppressed in the primary tumors of the LLC-sVEGFR-2 group compared with the control group. Furthermore, there were fewer micro-metastases in the pulmonary lymph nodes of the LLC-sVEGFR-2 group compared with the control group after cells were directly inoculated into the lung. These findings indicate that introduction of sVEGFR-2 suppressed lymphangiogenesis in primary lung cancer and also suppressed lymphogenic metastasis by inhibiting VEGF-C, followed by down-regulation of VEGFR-2, VEGFR-3 and MMPs. Accordingly, sVEGFR-2 might be a promising target for treatment of cancer by regulating lymphangiogenesis and lymphogenic metastasis.

MPGES1-Dependent Prostaglandin E2 (PGE2) Controls Antigen-Specific Th17 and Th1 Responses by Regulating T Autocrine and Paracrine PGE2 Production

The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly PGE2, are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal PGE synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell (Treg) development. During a type II collagen–CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Ag-experienced mPGES1−/− CD4+ cells showed impaired IL-17A, IFN-γ, and IL-6 production when rechallenged ex vivo with their cognate Ag compared with their wild-type counterparts. Additionally, production of PGE2 by cocultured APCs synergized with that of Ag-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFN-γ responses. However, in contrast with CD4+ cells that were Ag primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFN-γ production under Th17 polarization of naive T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an Ag-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFN-γ responses.

Cigarette Smoke Extract Inhibits Platelet Aggregation by Suppressing Cyclooxygenase Activity

The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A 2 receptor agonist, and that induced by collagen with respective IC 50 values of 1.05u2009±u20090.14% and 1.34u2009±u20090.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid–induced TXA 2 production in murine platelets with an IC 50 value of 7.32u2009±u20092.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA 2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I 2 receptor and PGE 2 receptor subtypes EP 2 and EP 4 , were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA 2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC 50 value of 1.07u2009±u20090.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA 2 production in platelets, leading to inhibition of platelet aggregation.