The elucidation of the apoptosis inhibitory action of SMP30.

衰老标记蛋白-30（SMP 30）是一种34 kDa的蛋白质，其在肝、肾和肺中的组织水平随着衰老而降低。为了阐明这种蛋白质的生理作用，我们将SMP 30基因的无效突变引入小鼠的生殖系，然后使用原代培养的肝细胞研究肿瘤坏死因子-α（TNF-α）诱导的细胞凋亡的组织易感性。因此，完全缺乏SMP 30的细胞（SMP 30-/-）比SMP 30 +/+肝细胞更易受TNF-α加放线菌素D（Act-D）诱导的细胞凋亡的影响，表明SMP 30可以保护细胞免受TNF-α加Act-D诱导的细胞凋亡。然而，SMP 30的抗凋亡作用的负责机制尚未完全理解。因此，本研究旨在阐明SMP 30的凋亡抑制作用。将SMP 30基因转染人肝癌细胞株，并与pcDNA 3基因转染人肝癌细胞株作对照。当细胞暴露于20 ng/ml肿瘤坏死因子-α（TNF-α）加10 ng/ml放线菌素D（Act-D）15 h时，在SMP 30和模拟转染子中细胞的活力均降低。然而，细胞的活力是三倍高的SMP 30转染比模拟转染。TUNEL法检测细胞凋亡。钙调蛋白（CaM）抑制剂三氟拉嗪的存在下，减弱了两个转染子中的SMP 30的抗凋亡作用，但该作用在SMP 30转染子中更为突出。蛋白质印迹分析显示，Akt，作为细胞中的存活因子，在存在或不存在TNF-α加Act-D的情况下，在SMP 30中被激活，而不是模拟转染子。此外，三氟拉嗪抑制SMP 30转染子中的Akt活化。因此，我们提出，钙调素和SMP 30之间的相互作用调节Akt的活性，因此SMP 30作为肝细胞中的存活因子。

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.