Hiroaki Naraba

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.

Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice.

We found that intraperitoneal injection of organic acids, such as propionic and lactic acid, are able to develop writhing responses in mice similarly as that of acetic acid. These acid-induced writhing reactions were significantly attenuated by capsazepine, a VR1 receptor-specific antagonist, but the phenylbenzoquinone-induced one was not, suggesting that the acids but not phenylbenzoquinone activate the VR1 receptor, which is involved in polymodal pain perception. Hoe 140, a bradykinin B2 receptor antagonist, also suppressed the acid-induced writhing response. Furthermore, these writhing responses were significantly suppressed after neonatal treatment with capsaicin, which treatment is known to destroy peripheral sensory afferent C-fibers. Capsazepine and Hoe 140 did not further attenuate the already reduced writhing responses of capsaicin-treated mice, suggesting that the acids stimulate the VR1 and the bradykinin B2 receptor in the pathway comprising sensory afferent C-fibers. On the other hand, indomethacin further significantly suppressed the writhing number of the capsaicin-treated animals, suggesting that the acid-induced pain perception requires prostanoid receptors not only in the pathway via capsaicin-sensitive C-fibers but also in other sensory pathways. These results provide the first evidence for the involvement of the vanilloid receptor in the acid-induced inflammatory pain perception via sensory C-fibers in addition to the known mediators bradykinin, neurokinins, and prostanoids.

Transcriptional regulation of the membrane-associated prostaglandin E2 synthase gene. Essential role of the transcription factor Egr-1.

Membrane-associated prostaglandin (PG) E2 synthase (mPGES) is an inducible terminal enzyme in the biosynthetic pathway for prostaglandin E2, which participates in many biological processes. In this study, we investigated the molecular mechanism controlling the inducible expression of mPGES. The mouse mPGES gene consisted of three exons, and its 5′-proximal promoter contained consensus motifs for the binding of several transcription factors. Transgenic expression in mice of the mouse mPGES promoter flanked by a reporter gene resulted in stimulus-dependent induction of the reporter in tissues where mPGES was intrinsically induced. Deletion and site-specific mutation analyses of the 5′-flanking region demonstrated that stimulus-inducible expression of mouse and human mPGES required tandem GC boxes adjacent to the initiation site. The stimulus-induced GC box binding activity was present in nuclear extracts of cells, in which the proximal GC box was essential for binding. An 80-kDa stimulus-inducible nuclear protein that bound to this GC box was identified as the transcription factor Egr-1 (for earlygrowth response-1). These results suggest that Egr-1 is a key transcription factor in regulating the inducible expression of mPGES.

Regulation of TNFα and interleukin-10 production by prostaglandins I2 and E2: studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists

To know which receptors of prostaglandins are involved in the regulation of TNFalpha and interleukin 10 (IL-10) production, we examined the production of these cytokines in murine peritoneal macrophages stimulated with zymosan. The presence of PGE(2) or the PGI(2) analog carbacyclin in the medium reduced the TNFalpha production to one-half, whereas IL-10 production increased several fold; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have a regulatory effect on the cytokine production. Among prostaglandin E (EP) receptor-selective synthetic agonists, EP2 and EP4 agonists caused down-regulation of the zymosan-induced TNFalpha production, but up-regulation on the IL-10 production; while EP1 and EP3 agonists showed no effect. Macrophages harvested from prostaglandin I (IP) receptor-deficient mice showed the up- and down-regulatory effects on the cytokine production by the EP2 and EP4 agonists or PGE(2), but no effect was obtained by carbacyclin. On the contrary, macrophages from EP2-deficient mice showed the effect by PGE(2), carbacyclin, and the EP4 agonist, but not by the EP2 agonist; and the cells from EP4-deficient mice showed the effect by PGE(2), carbacyclin, and EP2 agonist, but not by the EP4 agonist. These functional effects of prostaglandins well accorded with the mRNA expression of TNFalpha and IL-10 when such expression was examined by the RT-PCR method. The peritoneal macrophages from normal mice expressed IP, EP2, and EP4 receptors, but not EP1 and EP3, when examined by RT-PCR. Thus the results suggest that PGI(2) and PGE(2) generated simultaneously with cytokines by macrophages treated with zymosan may influence the cytokine production through IP, EP2, and EP4 receptors.

Assessment of the MicroRNA System in Salt-Sensitive Hypertension

Most animal microRNAs are imperfectly complementary to their mRNA targets and inhibit protein synthesis through an unknown mechanism. MicroRNAs have been reported to play important roles in a number of biological processes. We assessed the microRNA system in Dahl salt-sensitive rats in order to investigate possible roles of microRNA in salt-sensitive hypertension. We constructed microRNA libraries from the kidneys of Dahl salt-sensitive and Lewis rats taking normal or high-salt diets (4 groups), and identified 91 previously reported and 12 new microRNAs expressed in the kidney. We then used Northern blotting to assess the expression levels of 118 microRNAs in the kidneys and heart ventricles. No significant differences in microRNA expression profiles were observed among the 4 groups. Thus, the microRNA system seemed to be unlikely to contribute to salt-sensitive hypertension in Dahl salt-sensitive rats.

Agonist stimulation of B1 and B2 kinin receptors causes activation of the MAP kinase signaling pathway, resulting in the translocation of AP-1 in HEK 293 cells

In response to bradykinin, phosphorylated MAP kinases (ERK‐1 and ERK‐2) were abundantly increased in HEK 293 cells, which overexpress the rat B2 kinin receptor. In a similar way des‐Arg9‐bradykinin stimulation of B1 kinin receptor‐overexpressing HEK 293 cells caused activation of the same species of MAP kinase. Furthermore, nuclear translocation of transcription factor AP‐1 was also found in the cells after stimulation with either agonist. PD98059, a MAP kinase kinase (MEK‐1) inhibitor, blocked the agonist‐induced AP‐1 translocation as well as the phosphorylation of the MAP kinases. This communication provides the first evidence for both B1 and B2 kinin receptors mediating the MAP kinase signaling pathway to activate AP‐1.

Klk1 as One of the Genes Contributing to Hypertension in Dahl Salt-Sensitive Rat

A genome-wide quantitative trait loci analysis for blood pressure was performed using 107 male F2 rats derived from Dahl salt-sensitive and Lewis rats. Blood pressure was assessed by telemetry, and >400 microsatellite markers were used for genotyping. Two major quantitative trait loci for blood pressure were identified at chromosome 1 and chromosome 10. The expression levels of 366 transcripts around the chromosome 1 quantitative trait loci were assessed by RT-PCR, and we found that the Klk1 (kallikrein 1) and Ngfg (nerve growth factor gamma) mRNA levels were significantly reduced in the kidneys of Dahl salt-sensitive rats compared with those in Lewis rats. The expression levels of kallikrein 1 protein were also suppressed in Dahl salt-sensitive rats compared with those in Lewis rats. Because the kallikrein–kinin system has been shown to be involved in renal function, including salt homeostasis, it is likely that the reduced expression of Klk1 contributes to salt-sensitive hypertension in Dahl salt-sensitive rats.

Enhanced production of platelet-activating factor in stimulated rat leukocytes pretreated with triacsin C, a novel acyl-CoA synthetase inhibitor

Triacsin C, a product of Streptmyces sp. SK-1894, was previously reported as an inhibitor of long chain acyl-CoA synthetase. Pretreatment with triacsin C (500 nM) for 1h enhanced production of platelet-activating factor in rat neutrophils, followed by stimulation with A23187 or fMLP. Amount of lyso-PAF was also augumented. Triacsin C alone did not increase PAF content and did not modulate enzymatic activities of acytransferase, cholinephosphotransferase, acetylhydrolase, acetyltransferase or phospholipase A2. These results suggest that triacsin C might enhance supply of substrate for PAF synthesis, i.e. accumulation of lyso-PAF by interfering reacylation pathway.

Examination of signal transduction pathway of stimulated B1 and B2 kinin receptors; MAP kinase pathway to AP-1 translocation in HEK 293 cells.

B1 or B2 kinin receptor-overexpressing HEK293 cells were stimulated with des-Arg9-BK or BK, respectively. Each agonist induced translocation of AP-1 into the nuclear fraction as well as activation of MAP kinases in each cells. MAP kinase inhibitor PD98059 suppressed translocation of AP-1 and agonist-induced MAP kinase activation in both cells. These results indicate that stimulation of B1 or B2 receptor expresses a feature of the signal transduction pathway of MAP kinase activation to translocation of AP-1. This signal transduction pathway of HEK cells through B1 and B2 receptors may be similar in response to respective agonists.

Involvement of bradykinin in endotoxin-induced vascular permeability increase in the skin of rats

The aim of the present study was to investigate the role of bradykinin as well as that of platelet-activating factor in the endotoxin-induced acute vascular permeability increase in the dorsal skin of rats by use of kininogen-deficient and normal Brown-Norway rats. In the kininogen-deficient rats, the dose-dependent dye exudation induced by endotoxin was about one half of that in the normal rats at any doses of endotoxin tested (0.1-1.0 mg per site), whereas the dose-response curves obtained by bradykinin (1-100 nmol per site), platelet-activating factor (0.1-1 nmol per site) or histamine (50-500 nmol per site) were the same in both rats. This effect induced by endotoxin in the kininogen-deficient rats was not changed by pretreatment with a bradykinin B2 receptor antagonist, HOE140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, 1 mg kg-1 i.v.), whereas the endotoxin-induced response in the normal rats was attenuated by the receptor antagonist. These responses in both kininogen-deficient and normal rats were significantly inhibited by a selective platelet-activating factor antagonist, TCV309 (3-bromo-5-[N-phenyl-N-[2-[[2-(1,2,3,4,-tetrahydro-2- isoquinolylcarbonyl-oxy)-ethyl]-carbamoyl]-ethyl]carbamoyl]-1-prop yl- pyridinium nitrate, 0.1 mg kg-1 i.v.). These results suggest that bradykinin could be one of the major mediators in the endotoxin-induced vascular permeability increase in rat skin in addition to platelet-activating factor.