Background

A Presentation at Ernst Schering Prize ceremony (Sep. 27, 2000, Berlin)

• Lipid Mediator

Lipids were long thought of solely as sources of energy or as components of cellular membranes. We know now, however, that lipids function in concert with cytokines, hormones and/or neurotransmitters to play important roles in cellular signal transduction. For example, a polyunsaturated fatty acid, arachidonic acid, is converted to the biologically active compounds, prostaglandins and leukotrienes (collectively termed "eicosanoids", "eicosa-" comes from 20 in Greek). Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) are phospholipid mediators. Eicosanoids, and phospholipid mediators are not only proinflammatory compounds but are also involved in respiratory, cardiovascular, reproductive and central nervous system functions. Furthermore, these lipids have been shown to function in cellular proliferation and differentiation. Thus, in addition to constitutive roles, lipids play essential roles in intercellular and transcellular signaling pathways. Lipid molecules possessing various biological activities are termed "lipid mediators".

During the last few years, we have isolated the enzymes arachidonate 5-lipoxygenase, leukotriene A4 hydrolase and leukotriene B4 dehydrogenase, which are responsible for the synthesis and metabolism of the lipid mediator leukotriene B4. We have also succeeded in cloning receptors for PAF and leukotriene B4, both of which have potent inflammatory and chemotactic properties. On the basis of these and other findings, we are now examining the metabolic regulation and mechanism of actions of various lipid mediators. We are also directing our attention toward the role of bacterial lipopolysaccharide (LPS, endotoxin) in self-defense systems, and role of LPA in neuronal development and cell migration.

• Recent developments in research on the arachidonate cascade system

The structure of most eicosanoids (prostaglandin, leukotriene etc.) was identified during 1960-70's, and their metabolic pathways largely elucidated during 1980's. A significant breakthrough was made in 1991, with the cloning of the gene for a novel, inducible type of cyclooxygenase (COX-2), and the discovery of an arachidonate-specific phospholipase (cPLA2, cytosolic phospholipase A2). In 1995, several important papers appeared in Cell and Nature showing that the targeted disruption of COX-2 causes multiple abnormalities, including massive peritonitis and defective development of the glomerulus. These results suggest that COX-2 is involved not only in inflammation/cell defense mechanisms, but also in organogenesis. COX-2 itself or its metabolites may play different roles in the early stages of development. Inhibition of COX-2 is a potential method for preventing colon cancer. Another interesting observation came from two groups that showed that arachidonate metabolites are ligands for PPARs (peroxisome proliferator activated receptors, a member of an orphan nuclear receptor superfamily); 15-deoxy-delta11,12-PGJ2 for PPAR gamma, and LTB4 for PPAR alpha. Although it has not been confirmed that these compounds are indeed the endogenous ligands, the results obtained suggest that some nonpolar icosanoids may be interacting with nuclear receptors, rather than with seven-transmembrane receptors on the cell surface. In this respect, it is quite interesting that LTB4 has recentlybeen shown to bind to both cell-surface (Nature 387, 620-624, 97) and nuclear receptors (Nature 384, 39-43, 96), the first initiating and the second terminating inflammatory reactions. The observations may be relevant to the enzymologic evidence obtained invarious laboratories showing that COX-1, COX-2, LTC4 synthase, and FLAP (five-lipoxygenase activation protein) are localized in the nuclear membrane, and that cytosolic enzymes such as cPLA2 and 5-lipoxygenase can translocate to the nuclear membrane. Icosanoids and PAF may also have as yet unknown functions. Indeed, we have recently shown that the transgenic mice overexpressing PAF receptor produce melanocytic tumors.

Research Projects in our laboratory

• Metabolic regulation of biosynthesis and metabolism of lipid mediators

We have isolated the enzymes, 5-lipoxygenase, leukotriene A4 hydrolase, 12-lipoxygenase, and 15-lipoxygenase. A metabolic pathway for LTB4 was recently elucidated, and the key enzyme (12-hydroxy-LTB4 dehydrogenase) isolated (J. Biol. Chem., 268, 18128-18135, 1993; J. Biol.Chem. 271,2844-2850,1996). Isolation and cloning of other enzymes related to the synthesis and degradation of lipid mediators, and the study of the mechanisms by which these enzymes are regulated (including transcriptional and post transcriptional regulation), intracellular localization and translocation of key enzymes are ongoing projects in our laboratory (see a Figure for metabolic pathways of prostaglandins and leukotrienes).

• Receptors and signal transduction functions of lipid mediators

We are pursuing the isolation of receptors for various lipid mediators using techniques of protein purification, monoclonal-antibody-affinity chromatography, and expression cloning. Our goal is to elucidate intracellular signaling pathways mediated by lipid mediators in both CHO cells and native cells (blood cells, and neuronal cells etc.). We havecloned the genes for several 7-transmembrane receptors, including the gene for the PAF receptor, and we are using these clones to study intracellular signaling (see a Figure) Special attention is being directed to the mechanism of signal transduction from cell-surface 7-transmembrane receptors to the nucleus. Just recently, after many years of efforts, we succeeded in cloning the LTB4 receptor. CHO cells expressing LTB4 receptor show marked chemotactic responses toward nM concentrations of LTB4.

• Phospholipase A2 and lysophospholipid acyltransferases

In 1997, we have established mice deficient in cPLA2alpha (other name, groupIVA enzyme) (Uozumi, Nature, 1997). The mice are healthy but abnormalities in reproduction and synaptic plasticity. Furthermore, we found that cPLA2alpha-null mice are resistant to various disease models (collagen-induced arthritis, intestinal polyposis, allergic encephalomyelitis, bleomycin-induced lung fibrosis, ARDS and anaphylactic symptoms, see reviews by Shimizu et al. (IUBMB Life, 2006) and Kita et al. (BBA, 2006). We also discovered three new types of cPLA2 (cPLA2delta, epsilon, and zeta, Ohto et al., JBC, 2005). The analyses of biochemical properties and in vivo functions of cPLA2 enzymes are one of the major research projects in our laboratory.

In 2006, we made a significant breakthrough in isolating enzymes involved in membrane remodeling and surfactant production. Two lysophosphatidylcholine acyltransferase (LPCAT1 and LPCAT2) (Nakanishi, Shindou, JBC 2006). By genomic database screening, we discovered about 20 different genes possessing putative acyltransferase motifs. It is intriguing to determine how many enzymes are present, and how these enzyme recognize fatty acyl-CoAs, and polar head groups of lysophospholipids. See Lands' cycle for clarification (picture of Lands' cycle).

• Profiling of lipid mediators by LC-MS/MS

Dr. Kita in collaboration with Dr. Takahashi made a significant progress in quantitative determination of arachidonate derivatives and PAF (Kita et al., Anal. Biochem and BBRC). Using these methods, profiling of lipid mediators in various disease models are ongoing (Yoshikawa et al).

• Maintenance and guidance in the use of shared analytical instruments

Assistant Professor Toshie Takahashi is in charge of the maintenance and guidance in the use of various shared instruments in the Faculty of Medicine. These instruments include several mass spectrometers (JEOL HX110, Hitachi M-80, Thermo Electron TSQ-7000), a Beckman capillary zone electrophoresis apparatus (CZE, P/ACE system 2000), a Fuji Bas imageanalyzer (2000), a PDI densitometer, and a Dicton FACscan. The structures of lipid molecules and quantitation, and a primary structure determination of peptides and protein by LC-MS on-line systems are being examined. The methods are sufficiently powerful to identify post-translationally modified proteins, and will be used in the coming proteomics projects.

Experimental methods and protocols

Although our research uses primarily the techniques of biochemistry and molecular biology, we also make use of physiological assays, including Ca imaging (an Argus image analyzer, Hamaphoto) and voltage clamping methods of Xenopus oocytes, and histological methods (in situ hybridization, immunocytochemistry etc.) . For details, please refer to our Experimental Protocols (at moment available only in Japanese).

Graduate students and postdoctoral fellows

Ambitious individuals interested in our research are welcome to apply for entrance to the graduate course in our department. Candidates should have completed a 6-year course in medicine or veterinary science or have a masters degree (M.S.) or its equivalent. English speaking and writing abilities are especially important, and will be evaluated in the Entrance Examination. The Entrance Examination is givenyearly in September, and the applications will be accepted beginning August. A few postdoctoral positions may become available during next year. Additional information can be obtained by contacting Prof. Takao Shimizu.