Fever is triggered by an elevation of prostaglandin E 2 (PGE 2 ) 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 PGE 2 biosynthesis, and examined its induction in the rat brain after intraperitoneal injection of pyrogen lipopolysaccharide (LPS). In Northern blot analysis, mPGES mRNA 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-induced mPGES mRNA signals were mainly associated with brain blood vessels, especially vein or venular-type ones, in the whole brain area. Immunohistochemical study demonstrated that mPGESlike 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 PGH 2 . Inhibition of cyclooxygenase-2 activity resulted in suppression of both PGE 2 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 PGE 2 production during fever by expressing COX-2 and mPGES.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPAR␥. Here, we report that some PPAR␥ ligands, including 15-deoxy-⌬ 12,14 -prostaglandin J 2 , covalently bind to a cysteine residue in the PPAR␥ ligand binding pocket through a Michael addition reaction by an ␣,␤-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 PPAR␥, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the ␣,␤-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the ␣,␤-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPAR␥. We propose that PPAR␥ senses oxidation of fatty acids by recognizing such an ␣,␤-unsaturated ketone as a common moiety.

No abstract

The nuclear receptor, peroxisome proliferator-activated receptor c (PPARc), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fattyacid metabolites reportedly activate PPARc through conformational changes of the X loop. Here, we report that serotonin metabolites act as endogenous agonists for PPARc to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPARc antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPARc-target gene. Collectively, these results suggest a novel agonism, in which PPARc functions as a multiple sensor in response to distinct metabolites.

Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.

No abstract

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 G 1 /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 ET B via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ET B expressed by the cells in G 1 /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 ET B -mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ET B 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.

When temperature (T) of skin decreases stepwise, cold fibers evoke transient afferent discharges, inducing cold sensation and heat-gain responses. Hence we have proposed that cold receptors at distal ends of cold fibers are thermostats to regulate skin T against cold. Here, with patch-clamp techniques, we studied the ionic basis of cold receptors in cultured dorsal root ganglion (DRG) neurons of rats, as a model of nerve endings. Cells that increased cytosolic Ca(2+) level in response to moderate cooling were identified as neurons with cold receptors. In whole-cell current-clamp recordings of these cells, in response to cooling, cold receptors evoked a dynamic receptor potential (RP), eliciting impulses briefly. In voltage-clamp recordings (-60 mV), step cooling induced an inward cold current (I(cold)) with inactivation, underlying the dynamic RP. Ca(2+) ions that entered into cells from extracellular side induced the inactivation. Analysis of the reversal potential implied that I(cold) was nonselective cation current with high Ca(2+) permeability. Threshold temperatures of cooling-induced Ca(2+) response and I(cold) were different primarily among cells. In outside-out patches, when T decreased, single nonselective cation channels became active at a critical T. This implies that a cold receptor is an ion channel and acts as the smallest thermostat. Because these thermal properties were consistent with that in cold fibers, we conclude that the same cold receptors exist at nerve endings and generate afferent impulses for cold sensation and heat-gain behaviors in response to cold.