| Co-Investigator(Kenkyū-buntansha) |
MARUYAMA Naoki Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Vice director, 東京都老人総合研究所, 副所長 (00115940)
ISHIGAMI Akihiko Tokyo Metropolitan Foundation for Research on Aging and Promotion of Human Welfare, Research scientist, 東京都老人総合研究所, 主任研究員 (50270658)
久保 幸穂 財団法人東京都高齢者研究, 福祉振興財団・東京都老人総合研究所, 助手 (00280769)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Senescence marker protein-30 (SMP30) is a 34-kDa protein whose tissue levels in the liver, kidney, and lung decrease with aging. To elucidate the physiological role of this protein, we introduced a null mutation of the SMP30 gene into the germ line of mice, and then investigated the tissue susceptibility for apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) using primary cultured hepatocytes. Consequently, cells that are completely lacking SMP30 (SMP30-/-) were more susceptible to apoptosis induced by TNF-alpha plus actinomycin D (Act-D) than SMP30+/+ hepatocytes, indicating that SMP30 can protect cells from apoptosis induced by TNF-alpha plus Act-D. However, the responsible mechanism(s) for anti-apoptotic effect of SMP30 has not been fully understood. Therefore we aimed in this study that the elucidation of the apoptosis inhibitory action of SMP30.
Human hepatocellular carcinoma cell line was transfected with pcDNA3/SMP30 (SMP30 transfectants), or as a control with pcDNA3 (mock transfectants). When cells were exposed to 20 ng/ml tumor necrosis factor-alpha (TNF-alpha) plus 10 ng/ml actinomycin D (Act-D) for 15 h, the viability of cells was decreased in both SMP30 and mock transfectants. However, the viability of cells was threefold higher in SMP30 transfectants than mock transfectants. Cell death was confirmed as apoptosis by TUNEL assay. The presence of trifluoperazine, a calmodulin (CaM) inhibitor, attenuated anti-apoptotic effect of SMP30 in both transfectants, but the effect was more prominent in SMP30 transfectants. Western blot analyses revealed that Akt, which acts as a survival factor in cells, was activated in SMP30, but not mock, transfectants either in the presence or absence of TNF-alpha plus Act-D. Further, trifluoperazine inhibited Akt activation in SMP30 transfectants. We therefore propose that interplay between CaM and SMP30 regulates Akt activity, and thus SMP30 acts as a survival factor in hepatocytes.
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