Biomedical subjects

Daisuke Kamei

Publications and source records attributed to Daisuke Kamei.

9 recordsLinked to original sources

PubMed2005

Coupling between cyclooxygenases and terminal prostanoid synthases.

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2, cyclooxygenase (COX), and terminal prostanoid synthase. Recent evidence suggests that lineage-specific terminal prostanoid synthases, including prostaglandin (PG) E2, PGD2, PGF2alpha, PGI2, and thromboxane synthases, show distinct functional coupling with upstream COX isozymes, COX-1 and COX-2. This can account, at least in part, for segregated utilization of the two COX isozymes in distinct phases of PG-biosynthetic responses. In terms of their localization and COX preference, terminal prostanoid synthases are classified into three categories: (i) the perinuclear enzymes that prefer COX-2, (ii) the cytosolic enzyme that prefers COX-1, and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus. Additionally, altered supply of arachidonic acid by phospholipase A2s significantly affects the efficiency of COX-terminal prostanoid synthase coupling. In this review, we summarize our recent understanding of the coupling profiles between the two COXs and various terminal prostanoid synthases.

Animals↗

PubMed2005

Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D/AUF1 functions in mRNA genesis in the nucleus and modulates mRNA decay in the cytoplasm. Although it is primarily nuclear, it shuttles between the nucleus and cytoplasm. We studied the nuclear import and export of the last exon-encoding sequence common to all its isoforms by its expression as a green fluorescent protein-fusion protein in HeLa cells and by heterokaryon assay. The C-terminal 19-residue sequence (SGYGKVSRRGGHQNSYKPY) was identified as an hnRNP D nucleocytoplasmic shuttling sequence (DNS). In vitro nuclear transport using permeabilized cells indicated that nuclear import of DNS is mediated by transportin-1 (Trn-1). DNS accumulation in the nucleus was dependent on Trn-1, Ran, and energy in multiple rounds of nuclear transport. Use of DNS with deletions, alanine scanning mutagenesis and point mutations revealed that two separate regions (the N-terminal seven residues and the C-terminal two residues) are crucial for in vivo and in vitro transport as well as for interaction with Trn-1. The N- and C-terminal motifs are conserved in the shuttling sequences of hnRNP A1 and JKTBP.

3T3 Cells↗

PubMed2005

Change in prostaglandin E synthases (PGESs) in microsomal PGES-1 knockout mice in a preterm delivery model.

Most preterm deliveries are associated with infection and inflammation. Prostaglandin E2 (PGE2) is one of the most important mediators in the processes of inflammation, and is converted from PGH2 by various kinds of PGE synthases (PGESs). Among PGESs, microsomal PGES-1 (mPGES-1) is known to be the most important subtype in the processes of inflammation. To evaluate the role of PGESs in preterm delivery, we used mPGES-1 knockout mice in a lipopolysaccharide (LPS)-induced preterm labor model. Unexpectedly, the duration of labor after LPS treatment was not statistically different between C57BL6 wild-type mice and mPGES-1 knockout mice. In wild-type mice, mPGES-1 mRNA and protein expression increased in the myometrium and fetal membrane after LPS treatment. In contrast, the expression of mPGES-2 or cytosolic PGES was not changed by LPS treatment. On mPGES-1 knockout mice, mPGES-2 increased by LPS treatment in myometrium. The present data indicate that mPGES-1 may be involved in LPS-induced preterm labor, but inhibition of mPGES-1 alone may not prevent preterm delivery, because mPGES-2 might compensate for the role of mPGES-1.

Animals↗

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↗

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

Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2.

Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease.

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↗

PubMed2002

Interactions of heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its domains with high-affinity binding sites.

JKTBP proteins consisting of two canonical RNA binding domains (RBDs) and a glycine-rich carboxyl domain are nucleocytoplasmic shuttling proteins. We studied in vivo and in vitro interactions between JKTBP and RNA. UV cross-linking experiments on HL-60 cells indicated that following RNA synthesis inhibition by actinomycin D, JKTBP1 accumulated in the cytoplasam is bound to poly(A)(+) RNAs. Recombinant JKTBP1 protein blots could bind poly(A)(+) RNAs, but not poly(A)(-) RNAs. For examination of RNA binding specificity of JKTBP, we enriched high binding sites from pools of 20 nt random sequence-containing RNAs by a selection/amplification method. After eight rounds of a selection and amplification, >20 sequences for each of JKTBPs 1 and 2 were identified. Their consensus high-affinity site was ACUAGC. Approximate K(d)s of JKTBPs 2 and 1 were estimated to be 6-12 nM for the selected sequences by filter binding assays. JKTBP deletion analysis indicated that not individual RBDs, both RBDs and the N-terminal 15 amino acids of the carboxyl domain are required for sequence-specific and high-affinity binding. These results indicate that JKTBP is a sequence-specific RNA binding protein differing from the related heterogeneous nuclear ribonucleoproteins A1 and D.

Base Sequence↗

PubMed2001

Identification of the nucleocytoplasmic shuttling sequence of heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its interaction with mRNA.

JKTBP proteins are related to a family of heterogeneous nuclear ribonucleoproteins (hnRNPs) that function in mRNA biogenesis and mRNA metabolism. JKTBP proteins constituted of isoforms 1, 2, and 1Delta6 are localized in the nucleus. We show that the dominant form JKTBP1 shuttles between the nucleus and the cytoplasm and interacts with mRNA. Immunofluorescence microscopy and immunoblotting of the subcellular fractions and overexpression of JKTBP tagged with green fluorescent protein indicated that JKTBP1 and JKTBP1Delta6, but not JKTBP2, accumulate in the cytoplasm upon polymerase II transcription inhibition. After release from inhibition, the return of accumulated cytoplasmic JKTBP to the nucleus was temperature-dependent. In heterokaryons, green fluorescent protein-tagged JKTBP1 and JKTBP1Delta6 migrated from the HeLa nucleus to the mouse nucleus, but JKTBP2 did not. Using various JKTBP deletion mutants, the 25-residue C-terminal tail was identified as a shuttling sequence like M9. It is conserved in the C-terminal tails of hnRNP D/AUF1 and type A/B hnRNP/ABBP-1. Analysis of its sequence-specific interacting protein indicated that JKTBP nuclear import is mediated by the receptor transportin 1/karyopherin beta2. UV cross-linking revealed the increased occurrence of JKTBP1 directly interacting with poly(A)(+) RNA in the cytoplasm following actinomycin D treatment. We discuss a role of JKTBP in mRNA nuclear export.

3T3 Cells↗