| Co-Investigator(Kenkyū-buntansha) |
FUJITA Toshiko Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, Research Associate, 分子病理, 助手 (00100131)
HIROSE Miyoko Juntendo University School of Medicine, Research Associate, 医学部, 助手 (70266039)
IKEJIMA Kenichi Juntendo University School of Medicine, Research Associate, 医学部, 助手 (20317382)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
We previously identified a novel Ca^<2+> binding protein SMP30, which is preferentially expressed in cytosol of hepatocytes and renal tubular epithelia (Biochim Biophys Acta 1116, 122-128, 1992) SMP30 is unique in that its expression is highly maintained throughout the tissue maturing process, but decreases with aging. Recently, we reported that SMP30 enhances plasma membrane Ca^<2+>-pumping activity and thereby rescues Ca^<2+> ionophore-induced apoptosis through modulating intracellular Ca^<2+> homeostasis. Interestingly, this effect of SMP30 on Ca^<2+>-pumping activity was also dependent on calmodulin (CaM). Since CaM-kinase cascade has been reported to protect cells from apoptosis through activation of the Akt/PKB pathway, we further investigated the protective role of SMP30 in cell death induced by TNF-α plus actinomycin-D (Act-D) and serum deprivation. Methods : SMP30 transfectants and mock transfectants were obtained as previously reported (Biochem Biophys Res Commun 250, 374-380
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, 1998). Briefly, human hepatocellular carcinoma cell lines (Hep G2 cells) were cultured in DMEM supplemented with 10% heat inactivated fetal calf serum at 37℃ in 5% CO2 and air, and transfected with pcDNA3/SMP30, or as a control with pcDNA3, which didnot contain the SMP30 cDNA insert by using Lipofectin. The viability of total cells was assessed with a MTT [3-(4, 5-dimethylthiazol-2, 5-diphenyl tetrazolium bromide] colorimetric assay system. Apoptotic cells were determined by TUNEL assay. DNA synthesis was determined by BrdUincorporation. Results : When cells were exposed to 20 ng/ml TNF-α plus 10 ng/ml Act-D for 12 hr, the viability of cells determined by MTT assay was decreased in both SMP30 transfectants and mock transfectants, while TNF-α or Act-D alone did not affect the viability in both types of cells. Cell death was confirmed as apotosis by TUNEL assay. However, the viability of cells was 3-fold higher SMP30 transfectants than mock transfectants, when cells were exposed to TNF-α plus Act-D. The difference of viability between both types of cells was significantly attenuated by the presence of trifluoperazine, a CaM inhibitor, in a dose dependent manner (1-100 μM). Further, in separate experiments, when serum was simply removed from media, the viability of cells was decreased in both types of cells, but it was significantly higher in SMP30 transfectants than mock transfectants. DNA synthesis was not different between both types of cells. Conclusions : Transfection of SMP30 protected Hep G2 cells from apoptosis induced by TNF-α plus Act-D as well as artificial Ca^<2+> overload as previously reported. This effect of SMP30 was also inhibited by trifluoperazine, a CaM inhibitor, indicating that the protective role of SMP30 functions dependent on CaM. Further, transfection of SMP30 attenuated apoptosis induced by removal of growth stimulation, while DNA synthesis was not affected. These results suggest that SMP30 may act as a novel survival factor for hepatocytes, and thus agerelated decrease in SMP30 contributing to increased susceptibility of aged liver to injuries. Less
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