Toshihiro Tanioka

Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis

Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE2 biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr9), which is known to be critical for the activity of cytosolic GSHS-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE2 from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A2 in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE2 that plays a role in maintenance of tissue homeostasis.

Prostaglandin E synthase

Prostaglandin E synthase (PGES), which converts cyclooxygenase (COX)-derived prostaglandin (PG)H2 to PGE2, occurs in multiple forms with distinct enzymatic properties, modes of expression, cellular and subcellular localizations and intracellular functions. Cytosolic PGES (cPGES) is a cytosolic protein that is constitutively expressed in a wide variety of cells and tissues and is associated with heat shock protein 90 (Hsp90). Membrane-associated PGES (mPGES), the expression of which is stimulus-inducible and is downregulated by anti-inflammatory glucocorticoids, is a perinuclear protein belonging to the microsomal glutathione S-transferase (GST) family. These two PGESs display distinct functional coupling with upstream COXs in cells; cPGES is predominantly coupled with the constitutive COX-1, whereas mPGES is preferentially linked with the inducible COX-2. Several cytosolic GSTs also have the capacity to convert PGH2 to PGE2 in vitro. Accumulating evidence has suggested that mPGES participates in various pathophysiological states in which COX-2 is involved, implying that mPGES represents a potential novel target for drug development.

A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin

Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 ± 5.5 μg/ml (mean ± SD) in healthy women and 6.2 ± 3.6 μg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).

Identification of a Cellular Protein That Functionally Interacts with the C2 Domain of Cytosolic Phospholipase A2α

Cytosolic phospholipase A2(cPLA2) α plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA2α in a Ca2+-dependent manner. Peptide microsequencing revealed that purified P60 was identical to vimentin, a major component of the intermediate filament. The interaction occurred between the C2 domain of cPLA2α and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA2α and vimentin colocalized around the perinuclear area in cPLA2α-overexpressing human embryonic kidney 293 cells following A23187 stimulation. Forcible expression of vimentin in vimentin-deficient SW13 cells augmented A23187-induced arachidonate release. Moreover, overexpression of the vimentin head domain in rat fibroblastic 3Y1 cells exerted a dominant inhibitory effect on arachidonate metabolism, significantly reducing A23187-induced arachidonate release and attendant prostanoid generation. These results suggest that vimentin is an adaptor for cPLA2α to function properly during the eicosanoid-biosynthetic process.

Regulation of cytosolic prostaglandin E synthase by phosphorylation.

cPGES [cytosolic PG (prostaglandin) E synthase] is constitutively expressed in various cells and can regulate COX (cyclo-oxygenase)-1-dependent immediate PGE2 generation. In the present study, we found that cPGES underwent serine phosphorylation, which was accelerated transiently after cell activation. Several lines of evidence suggest that a cPGES-activating protein kinase is CK-II (casein kinase II). Recombinant cPGES was phosphorylated directly by and associated with CK-II in vitro, resulting in marked reduction of the K m for the substrate PGH2. In activated cells, cPGES phosphorylation occurred in parallel with increased cPGES enzymic activity and PGE2 production from exogenous and endogenous arachidonic acid, and these processes were facilitated by Hsp90 (heat-shock protein 90), a molecular chaperone that formed a tertiary complex with cPGES and CK-II. Treatment of cells with inhibitors of CK-II and Hsp90 and with a dominant-negative CK-II attenuated the formation of the cPGES-CK-II-Hsp90 complex and attendant cPGES phosphorylation and activation. Mutations of either of two predicted CK-II phosphorylation sites on cPGES (Ser113 and Ser118) abrogated its phosphorylation and activation both in vitro and in vivo. Moreover, the CK-II-Hsp90-mediated activation of cPGES was ameliorated by the p38 mitogen-activated protein kinase inhibitor SB20358 or by the anti-inflammatory glucocorticoid dexamethasone. Taken together, the results of the present study have provided the first evidence that the cellular function of this eicosanoid-biosynthetic enzyme is under the control of a molecular chaperone and its client protein kinase.

Regulation of cytosolic prostaglandin E2 synthase by 90-kDa heat shock protein.

Cytosolic prostaglandin (PG) E(2) synthase (cPGES) is constitutively expressed in a wide variety of cells and converts cyclooxygenase (COX)-1-derived PGH(2) to PGE(2). Given the fact that cPGES is identical to p23, a heat shock protein 90 (Hsp90)-binding protein, we herein examined the effect of Hsp90 on PGE(2) generation by cPGES. Incubation of cPGES with Hsp90 resulted in a significant increase in PGES activity in vitro. Association of cPGES with Hsp90 was increased in cells stimulated with A23187 or bradykinin, accompanied by concomitant increases in cPGES activity and PGE(2) production. Moreover, treatment of cells with Hsp90 inhibitors, which destabilized the cPGES/Hsp90 complex, reduced cPGES activity and PGE(2) production to basal levels. These results suggest that the regulation of cPGES activity in cells depends on its association with Hsp90 and provide the first line of evidence that eicosanoid biosynthesis is under the control of the molecular chaperone.

Influence of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children

Although many single nucleotide polymorphism (SNP) studies have reported an association of atopy, allergic diseases and total serum immunoglobulin E (IgE) levels, almost all of these studies sought risk factors for the onset of these allergic diseases. Furthermore, many studies have analyzed a single gene and hardly any have analyzed environmental factors. In these analyses, the results could be masked and the effects of other genes and environmental factors may be decreased. Here, we described the correlation between four genes [interleukin (IL)‐4 (C‐590T), IL‐4 receptor (A1652G), FCER1B (G6842A) and STAT6 (G2964A)] in connection with IgE production; the role of IL‐10 (C‐627A) as a regulatory cytokine of allergy; and the severity of food allergy (FA) and atopic eczema (AE) in 220 Japanese allergic children. In addition to these SNPs, environmental factors, i.e., patients attitude, indoor envirmonment, and so on, were also investigated in this study.

Regulation of the human leukocyte-derived arginine aminopeptidase endoplasmic reticulum-aminopeptidase 2 gene by interferon-γ

The leukocyte‐derived arginine aminopeptidase (L‐RAP) is the second aminopeptidase localized in the endoplasmic reticulum (ER) processing antigenic peptides presented to major histocompatibility complex (MHC) class I molecules. In this study, the genomic organization of the gene encoding human L‐RAP was determined and the regulatory mechanism of its expression was elucidated. The entire genomic structure of the L‐RAP gene is similar to both placental leucine aminopeptidase (P‐LAP) and adipocyte‐derived leucine aminopeptidase (A‐LAP) genes, confirming the close relationship of these three enzymes. Interferon (IFN)‐γ up‐regulates the expression of the L‐RAP gene. Deletion and site‐directed mutagenic analyses of the 5′‐flanking region of the L‐RAP gene and electrophoretic mobility shift assay indicated that while interferon regulatory factor (IRF)‐2 is important in the basal condition, IRF‐1 is the primary regulator of IFN‐γ‐mediated augmentation of the gene expression. In addition, PU.1, a member of the E26 transformation‐specific family of transcription factors, also plays a role in the regulation of gene expression. The maximum expression of the gene was achieved by coexpression of IRF‐1 and PU.1 in HEK293 cells and IRF‐2 suppressed the IRF‐1‐mediated enhancement of gene expression, suggesting that IFN‐γ‐induced L‐RAP gene expression is cooperatively regulated by IRFs and PU.1 transcription factors.

Inducible nitric-oxide synthase and nitric oxide donor decrease insulin receptor substrate-2 protein expression by promoting proteasome-dependent degradation in pancreatic beta-cells: involvement of glycogen synthase kinase-3beta.

Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic β-cells. Gene disruption of IRS-2 results in failure of the β-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in β-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in β-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in β-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1β (IL-1β), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK/SAPK) in β-cells. Inhibition of GSK-3β by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in β-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated β-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3β-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expresssion may contribute to the progression and/or exacerbation of β-cell failure in diabetes.

Coupling between cyclooxygenases and prostaglandin F2α synthase: Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F2α-synthetic activity

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.