(Form4-2) FINAL REPORT For Japan-Korea Joint Research Project AREA 1. Mathematics & Physics 2. Chemistry & Material Science 3. Biology 4. Informatics & Mechatronics 5. Geo-Science & Space Science 6. Medical Science 7. Humanities & Social Sciences 1. Research Title: Physiological roles of extracellular proton on inflammasome functions using TDAG8 KO mouse 2. Term of Research: From July, 2010 To March, 2012 3. Total Budget a. Financial Support by JSPS: Total amount: 2,400 thousand yen 1 st Year 1,200 thousand yen 2 nd Year 1,200 thousand yen 3 rd Year 0 thousand yen b. Other Financial Support : Total amount: 0 thousand yen 4. Project Organization a. Japanese Principal Researcher Fumikazu Okajima Name Institution / Department Position Professor b. Korean Principal Researcher Name Dong-Soon Im Institution / Department Position College of Parmacy, Pusan University Associate Professor 1
c. List of Japanese-side Participants (Except for Principal Researcher) Name Institution/Department Position Hideaki Tomura Koichi Sato Chihiro Mogi Associate Professor Assistant Professor Assistant Professor Mayumi Komachi COE fellow Research Takashi Nakakura COE fellow Research d. List of Korean-side Participants (Except for Principal Researcher) Name Institution/Department Position Soo-Jin Pak College of Parmacy, Pusan University Pusan National University Graduate student 2
5. Number of Exchanges during the Final Fiscal Year* a. from Japan to Korea *Japanese fiscal year begins April 1. Name Home Institution Duration Host Institution Fumikazu Okajima Gunma University Feb. 22-26, 2012 Kyung Hee University (Seoul), where I had a seminar and discussed our research projects with Dr Im DS of Pusan National University (a principal researcher of this project) and Dr. Jin Y of Kyung Hee University, who is an expert in the field related to our research project. Total: 1 persons Total: 5 man-days Numbers of Exchanges during the past fiscal years FY2009: Total 0 persons FY2010: Total 3 persons b. from Korea to Japan Name Home Institution Duration Host Institution Dong-Soon Im Pusan University National Feb. 1-4 Gunma Univerisity Soo-Jin Pak Pusan University National Feb. 1-4 Gunma University Total: 2 persons Total: 8 man-days Numbers of Exchanges during the past fiscal years FY2009: Total 0 persons FY2010: Total 1 persons 3
6. Objective of Research Korean principal researcher Dr. Im first reported that TDAG8 is a receptor for lysolipid psychosine in 2001 (Im, DS et al, J Cell Biol. 153, 429-33, 2001). On the other hand, we found in 2004 that TDAG8 senses extracellular proton or ph resulting in increase in intracellular camp accumulation through Gs-proteins (Wang, JQ et al, J. Biol. Chem. 279:45626-33 2004). Extracellular acidification has been shown to exert a variety of cellular responses; however, their molecular mechanisms remain poorly understood. Increase in extracellular proton or reduction of extracellular proton has been shown to occur in the sites under the states linked to deterioration of the blood supply like inflammation, ischaemia and tumor. We have recently observed the inhibitory effect of extracellular proton on cytokine release in macrophages using TDAG8 knock-out mice (Mogi, C et al, J. Immunol. 182, 3243-51, 2009). We also found that extracellular acidification is associated with inhibition of IL-1b production in macrophages and microglia. Korean side investigator has researched on atherosclerosis at University of Connecticut and found involvement of inflammasome, which has been shown to be involved in the activation of caspase 1 and subsequent cleavage of proil-1b to mature Il-1b, in macrophages (Skoura, A., 2009). Thus, both principal researchers of Japan and Korea have contributed in the identification and characterization of the receptors. We therefore planned to elucidate the molecular mechanisms of proton/tdag8-regulated inflammasome functions using TDAG8 KO mouse. The elucidation of physiological roles and signal transduction pathways of extracellular proton and TDAG8 on lipopolysaccharide-induced IL-1b and IL-18 secretion in macrophages may facilitate understanding of the molecular mechanism of ph-dependent phenomena, which are frequently observed in several experimental systems. In addition, the proton-sensing GPCR may be therapeutic targets for immune diseases and inflammation. 7. Methodology As a corresponding author of the first publication in which TDAG8 was reported to sense extracellular proton to stimulate intracellular signaling pathways, I (Okajima) will characterize signal transduction mechanisms of proton and TDAG8 on inflammasome formation and IL-1b secretion in mouse using TDAG8 knock-out mouse. On the other hand, Korean principal researcher Dr. Im will try to clarify effects of extracellular proton or ph on the inflammasome formation and subsequent secretion of IL-1b in human THP-1 monocytes/macrophages. In order to perform these collaborative studies effectively, we mutually visit each laboratory at least once a year in addition to the frequent communication by E-mail. Year 2010 Dr. Im: He will investigate effect of extracellular proton on inflammasome formation by RT-PCR and Western blot, and imflammasome function by ELISA for IL-1b in human THP-1 monocytes/ macrophages. Okajima: We will test effect of extracellular proton on lipopolysaccharide (LPS)-induced inflammasome formation and function by Western blot and ELISA of IL-1b and IL-18 in mouse bone marrow-derived macrophages, thioglycollate-elicitated macrophages, and microglia in central nervous system. Futhermore, they can show involvement of TDAG8 by using TDAG8 KO mouse. Year 2011 Dr. Im: He will test TDAG8 involvement in extracellular proton-induecd inhibition on inflammasome function in human THP-1 monocytes/machrophages by using sirna strategy. We'll investigate effect of extracellular proton and TDAG8 on expression and interactions of 4
caspase 1, caspase 5, pycard (PYD nad CARD containing or ASC), and NALP3. Okajima: By investigating signal transduction pathways of TDAG8 as like camp formation and Rho activation, Japanese side will characterize signal transduction pathways involved in modulation of LPS-induced inflammasome formation and IL-1b secretion at the molecular level.