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Yoshihito Nakatani

Publications and source records attributed to Yoshihito Nakatani.

13 recordsLinked to original sources
PubMed2005

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2.

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.

Adenocarcinoma↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin e synthase-1.

We examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice. Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice. LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained. Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice. This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice. Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice. Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice. Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation.

Acetic Acid↗
PubMed2004

Subcellular localization and regulation of hypoxia-inducible factor-2alpha in vascular endothelial cells.

The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as transcription factors that mediate hypoxia-inducible gene expression through hypoxia-responsive element (HRE). They play critical roles not only in normal development, but also in tumor progression. Endothelial cells (EC) express both HIF-1alpha and -2alpha. In this study, we examined the subcellular localization of HIF-1alpha and -2alpha in bovine arterial EC (BAEC) by immunoblotting and immunocytostaining analysis and found that even under normoxic conditions, as with its heterodimeric partner ARNT, HIF-2alpha was stable, and was localized in the nucleus of BAEC differently than HIF-1alpha. HIF-2alpha might be regulated by a different mechanism than HIF-1alpha and might mediate the expression of some EC-specific genes under normoxic conditions. We further found that cardiovascular helix-loop-helix factor (CHF) 2, which had been identified as an ARNT-interacting protein, was expressed in BAEC and suppressed HRE-dependent gene expression both under normoxia and hypoxia. CHF2 might be one of the key regulators of HIF-2alpha-mediated gene expression in normoxic EC.

Amino Acid Sequence↗
PubMed2003

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F(2alpha)-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.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

Potential role of microsomal prostaglandin E synthase-1 in tumorigenesis.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is a stimulus-inducible enzyme that functions downstream of cyclooxygenase (COX)-2 in the PGE2-biosynthetic pathway. Given the accumulating evidence that COX-2-derived PGE2 participates in the development of various tumors, including colorectal cancer, we herein examined the potential involvement of mPGES-1 in tumorigenesis. Immunohistochemical analyses demonstrated the expression of both COX-2 and mPGES-1 in human colon cancer tissues. HCA-7, a human colorectal adenocarcinoma cell line that displays COX-2- and PGE2-dependent proliferation, expressed both COX-2 and mPGES-1 constitutively. Treatment of HCA-7 cells with an mPGES-1 inhibitor or antisense oligonucleotide attenuated, whereas overexpression of mPGES-1 accelerated, PGE2 production and cell proliferation. Moreover, cotransfection of COX-2 and mPGES-1 into HEK293 cells resulted in cellular transformation manifested by colony formation in soft agar culture and tumor formation when implanted subcutaneously into nude mice. cDNA array analyses revealed that this mPGES-1-directed cellular transformation was accompanied by changes in the expression of a variety of genes related to proliferation, morphology, adhesion, and the cell cycle. These results collectively suggest that aberrant expression of mPGES-1 in combination with COX-2 can contribute to tumorigenesis.

Adenocarcinoma↗
PubMed2003

Contribution of membrane-associated prostaglandin E2 synthase to bone resorption.

This study initially confirmed that, among prostaglandins (PGs) produced in bone, only PGE(2) has the potency to stimulate osteoclastogenesis and bone resorption in the mouse coculture system of osteoblasts and bone marrow cells. For the PGE(2) biosynthesis two isoforms of the terminal and specific enzymes, membrane-associated PGE(2) synthase (mPGES) and cytosolic PGES (cPGES) have recently been identified. In cultured mouse primary osteoblasts, both mPGES and cyclooxygenase-2 were induced by the bone resorptive cytokines interleukin-1, tumor necrosis factor-alpha, and fibroblast growth factor-2. Induction of mPGES was also seen in the mouse long bone and bone marrow in vivo by intraperitoneal injection of lipopolysaccharide. In contrast, cPGES was expressed constitutively both in vitro and in vivo without being affected by these stimuli. An antisense oligonucleotide blocking mPGES expression inhibited not only PGE(2) production, but also osteoclastogenesis and bone resorption stimulated by the cytokines, which was reversed by addition of exogenous PGE(2). We therefore conclude that mPGES, which is induced by and mediates the effects of bone resorptive stimuli, may make a target molecule for the treatment of bone resorptive disorders.

Animals↗
PubMed2002

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.

Alzheimer Disease↗
PubMed2002

[Prostaglandin E2 synthases].

Prostaglandin E2 (PGE2) is widely distributed in various tissues, and exhibits various biologically important activities. PGE2 synthase (PGES) catalyzes conversion of COX-derived PGH2 to PGE2. It now appears that there are at least three distinct types of PGES in mammals. We identified two distinct glutathione-dependent PGESs. Cytosolic PGES (cPGES), known as p23, is constitutively and ubiquitously expressed and predominantly converts COX-1-derived PGH2 to PGE2. We find that the regulation of cPGES/p23 activity in cells depends on its association with hsp90. Microsomal PGES-1 (mPGES-1), identical to MGST1-L1, is an inducible perinuclear enzyme that is functionally linked with COX-2 in marked preference to COX-1. COX-2 and mPGES-1 are essential components for delayed PGE2 synthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer. Most recently, glutathione-nonspecific mPGES-2, homologous to glutaredoxin and thioredoxin, was identified. These PGESs seem to be a potential novel target for drug development.

Animals↗