Project/Area Number |
17590434
|
Research Category |
Grant-in-Aid for Scientific Research (C)
|
Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
Immunology
|
Research Institution | Osaka University |
Principal Investigator |
OHISHI Kazuhito Osaka University, Graduate School of Medicine, Assistant, 医学系研究科, 助手 (60273702)
|
Project Period (FY) |
2005 – 2006
|
Project Status |
Completed (Fiscal Year 2006)
|
Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
Keywords | Necrosis / TNF-α / IL-1β / Rhomboid proteases / Stress-activated kinases / IκB-α / Reactive oxygen species / IL-1 / Rhomboidセリンプロテアーゼ / TNF / Fas |
Research Abstract |
Cell death can be broadly separated in two categories based on accompanying morphological and biochemical changes, apoptosis and necrosis. To identify the genes which confer resistance to necrosis, the cyclic packaging rescue screenings were performed. L929 cells which preferentially die through necrosis upon TNF-α stimulation were infected with retrovirus cDNA libraries then the cells were killed by TNF-α. The integrated retroviruses in the resistant cells were converted to virus particles by the infection of two adenoviruses bearing retroviral gag-pol and env genes. This virus-containing culture medium was used for the subsequent infection. After six rounds of screening, integrated cDNA was amplified by PCR and sequenced. In addition to cFlip, two partial rhomboid genes lacking 5' terminus were isolated from two independent retrovirus cDNA libraries derived from mouse embryo and mouse 3T3 cells. The appearance of multiple rhomboid genes using different libraries is convincing data that these genes may be broadly applicable to cell survival. Overexpression of rhomboids in L929-hFas cells conferred resistant to TNF-α but not to FasL. In this cell, production of reactive oxygen species, activation of JNK and p38 kinases and degradation of IκB-α were impaired whereas activation of ERK-or AKT was not suppressed. Stimulation by IL-1β showed that overexpression of rhomboids did not affect these signal transduction pathways. These results indicated that rhomboids specifically inhibit TNF-α pathway. The rhomboids also inhibited the complex formation upon TNF-α stimulation of TNFR1-TRADD-RIP-TRAF2-IKKα/β in the raft fraction. Immunofluorescence staining of tagged version of rhomboids revealed non-raft distribution of rhomboids. Taken together, rhomboids probably function in the transport and distribution of TNFR1 on the cell surface.
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