Takuma Shiraki

Coexpression of Microsomal-Type Prostaglandin E Synthase with Cyclooxygenase-2 in Brain Endothelial Cells of Rats during Endotoxin-Induced Fever

Fever is triggered by an elevation of prostaglandin E2(PGE2) in the brain. However, the mechanism of its elevation remains unanswered. We herein cloned the rat glutathione-dependent microsomal prostaglandin E synthase (mPGES), the terminal enzyme for PGE2 biosynthesis, and examined its induction in the rat brain after intraperitoneal injection of pyrogen lipopolysaccharide (LPS). In Northern blot analysis, mPGESmRNA was weakly expressed in the brain under the normal conditions but was markedly induced between 2 and 4 hr after the LPS injection.In situ hybridization study revealed that LPS-inducedmPGES mRNA signals were mainly associated with brain blood vessels, especially vein or venular-type ones, in the whole brain area. Immunohistochemical study demonstrated that mPGES-like immunoreactivity was expressed in the perinuclear region of brain endothelial cells, which were identified as von Willebrand factor-positive cells. Furthermore, in the perinuclear region of the endothelial cells, mPGES was colocalized with cyclooxygenase-2 (COX-2), which is the enzyme essential for the production of the mPGES substrate PGH2. Inhibition of cyclooxygenase-2 activity resulted in suppression of both PGE2 level in the CSF and fever (Cao et al., 1997), suggesting that the two enzymes were functionally linked and that this link is essential for fever. These results demonstrate that brain endothelial cells play an essential role in the PGE2 production during fever by expressing COX-2 and mPGES.

α,β-Unsaturated Ketone Is a Core Moiety of Natural Ligands for Covalent Binding to Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptor γ (PPARγ) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPARγ. Here, we report that some PPARγ ligands, including 15-deoxy-Δ12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPARγ ligand binding pocket through a Michael addition reaction by an α,β-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPARγ, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the α,β-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the α,β-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPARγ. We propose that PPARγ senses oxidation of fatty acids by recognizing such an α,β-unsaturated ketone as a common moiety.

Endothelin-induced Apoptosis of A375 Human Melanoma Cells

Endothelin-1 (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nm ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G1/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ETB expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides.

Activation of Orphan Nuclear Constitutive Androstane Receptor Requires Subnuclear Targeting by Peroxisome Proliferator-activated Receptor γ Coactivator-1α A POSSIBLE LINK BETWEEN XENOBIOTIC RESPONSE AND NUTRITIONAL STATE

In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1β, a PGC-1α homologue, also activated CAR to less of an extent than PGC-1α. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor α, enhanced the PGC-1α-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1α. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1α were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1α was required for CAR localization at nuclear speckles. These results indicate that PGC-1α mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1α.

Brain-specific endothelial induction of prostaglandin E2 synthesis enzymes and its temporal relation to fever

Brain endothelial cells are hypothesized to be the major source of prostaglandin E(2) (PGE(2)) responsible for fever because they express 2 PGE(2)-synthesizing enzymes (cyclooxygenase-2 and microsomal-type PGE synthase) in response to pyrogens. To further validate this hypothesis, we examined in rats whether endothelial expression of these enzymes occurs only in the brain, and whether the time course of enzyme expression in brain endothelial cells can explain the time courses of brain PGE(2) level and fever. Intraperitoneal injection of lipopolysaccharide induced these enzymes only in brain endothelial cells, but not in those of peripheral organs including the neck, heart, lung, liver and kidney. Induction of these enzymes in brain endothelial cells was first noticed at 1.5 h after lipopolysaccharide injection, at which time elevation of PGE(2) was also first detected. Fever started just after this time point. These results demonstrate the significance of brain endothelial cells in the PGE(2) production during fever. Unexpectedly, PGE(2) level markedly dropped at 5 h in spite of high levels of these enzymes, implicating the existence of an unknown mechanism that suppresses PGE(2) level during the recovery phase of fever.

The nuclear bile acid receptor FXR is activated by PGC-1α in a ligand-dependent manner

The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1alpha [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR. We found that the transcriptional activation of a reporter plasmid by a GAL4-FXR chimaera was strongly enhanced by PGC-1alpha, in a ligand-dependent manner. Transcriptional activation of the SHP (small heterodimer partner) gene by the FXR-RXRalpha (retinoid X receptor alpha) heterodimer was also enhanced by PGC-1alpha in the presence of CDCA (chenodeoxycholic acid). Co-immunoprecipitation and pull-down studies using glutathione S-transferase-PGC-1alpha fusion proteins revealed that the ligand-binding domain of FXR binds PGC-1alpha in a ligand-influenced manner both in vivo and in vitro. Furthermore, our studies revealed that SHP represses its own transcription, and the addition of excess amounts of PGC-1alpha can overcome the inhibitory effect of SHP. These observations indicate that PGC-1alpha mediates the ligand-dependent activation of FXR and transcription of SHP gene.

l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons.

We investigated the effects of l-menthol on cultured dorsal root ganglion (DRG) cells, instead of free nerve endings of sensory fibers. Using Fura-2 microfluorimetry, we identified a few DRG neurons that showed an increase in intracellular free Ca2+ concentration ([Ca2+]i in response to l-menthol. They made up only 10% of the neurons activated by a high K+ solution. l-Menthol induced the [Ca2+]i increase in a dose-dependent manner, with an EC50 of 37.9 μM and a Hill coefficient of 0.97. A related compound, cyclohexanol, had no effect. When extracellular Ca2+ was removed, l-menthol did not induce the [Ca2+]i increase. Whole-cell current-clamp recordings revealed that l-menthol induced depolarization (13.2 mV, receptor potential) leading to impulses. We conclude that l-menthol induced the impulses through activation of menthol receptors in a small subset of the cultured sensory neurons.

Proline cis/trans-Isomerase Pin1 Regulates Peroxisome Proliferator-activated Receptor γ Activity through the Direct Binding to the Activation Function-1 Domain

The important roles of a nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARγ activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARγ activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARγ. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARγ-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARγ.

Cyclooxygenase in the vagal afferents: is it involved in the brain prostaglandin response evoked by lipopolysaccharide?

The vagal afferents are proposed to transmit abdominal immune signals to the brain. In this immune-brain communication, prostaglandins might play a mediator role. In fact, prostaglandin receptors are abundant in the vagal afferents. We examined here the presence of cyclooxygenase, an enzyme necessary for prostaglandin biosynthesis, in the vagal afferents of rats. We also tested whether the vagal afferents contribute to the elevation of prostaglandin E2 in the brain after intraperitoneal injection of lipopolysaccharide. Under normal conditions, cyclooxygenase-1-like immunoreactivity was constitutively expressed in the vagal afferents at their central terminals and in their cell bodies. Cyclooxygenase-2-like immunoreactivity was absent in the vagal afferents under normal as well as lipopolysaccharide-challenged conditions. Instead, cyclooxygenase-2-like immunoreactivity was induced in brain endothelial cells by the lipopolysaccharide challenge. The elevation of prostaglandin E2 in the cerebrospinal fluid after lipopolysaccharide challenge was not inhibited, but was rather enhanced, by the bilateral vagotomy. These results suggest that the vagal afferents potentially generate prostaglandins, which may locally modulate the vagal signal transmission, but that the vagal afferents are not essential to the elevation of prostaglandin E2 in the brain after intraperitoneal challenge with LPS.

Spectroscopic analyses of the binding kinetics of 15d-PGJ2 to the PPARγ ligand-binding domain by multi-wavelength global fitting

PPARgamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor that is activated by natural lipid metabolites, including 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2). We previously reported that several oxidized lipid metabolites covalently bind to PPARgamma through a Michael-addition to activate transcription. To separate the ligand-entering (dock) and covalent-binding (lock) steps in PPARgamma activation, we investigated the binding kinetics of 15d-PGJ2 to the PPARgamma LBD (ligand-binding domain) by stopped-flow spectroscopy. We analysed the spectral changes of 15d-PGJ2 by multi-wavelength global fitting based on a two-step chemical reaction model, in which an intermediate state represents the 15d-PGJ2-PPARgamma complex without covalent binding. The extracted spectrum of the intermediate state in wild-type PPARgamma was quite similar to the observed spectrum of 15d-PGJ2 in the C285S mutant, which cannot be activated by 15d-PGJ2, indicating that the complex remains in the inactive, intermediate state in the mutant. Thus lock rather than dock is one of the critical steps in PPARgamma activation by 15d-PGJ2.