ROLE OF SENESCENCE MARKER PROTEIN-30 (SMP30) IN HEPATOCYTE APOPTOSIS

我们以前鉴定了一种新的Ca ~（2+）结合蛋白SMP 30，其优先在肝细胞和肾小管上皮细胞的胞质溶胶中表达（Biochim Biophys Acta 1116，122-128，1992）。SMP 30的独特之处在于其表达在整个组织成熟过程中高度维持，但随着衰老而降低。最近，我们报道了SMP 30增强质膜Ca^2+泵活性，从而通过调节细胞内Ca^2+稳态来挽救Ca^2+离子载体诱导的细胞凋亡。有趣的是，SMP 30对Ca^2+泵活性的这种作用也依赖于钙调蛋白（CaM）。由于CaM激酶级联反应已被报道通过激活Akt/PKB通路来保护细胞免于凋亡，因此我们进一步研究了SMP 30在TNF-α加放线菌素D（Act-D）和血清剥夺诱导的细胞死亡中的保护作用。方法：如先前报道的（Biochem Biophys Res Commun 250，374-380）获得SMP 30转染子和模拟转染子 关于我们 ，1998年）。将人肝癌细胞系（Hep G2细胞）培养在含10%热灭活胎牛血清的DMEM培养基中，37℃，5%CO2和空气中，用脂质体转染pcDNA 3/SMP 30，或用不含SMP 30 cDNA插入片段的pcDNA 3作为对照。用MTT [3-（4，5-二甲基噻唑-2，5-二苯基溴化四氮唑]比色法测定总细胞的活力。TUNEL法检测凋亡细胞。DNA合成通过BrdU掺入法测定。结果如下：当细胞暴露于20 ng/ml TNF-α和10 ng/ml Act-D 12 h时，MTT法测定的细胞活力在SMP 30转染子和模拟转染子中均降低，而TNF-α或Act-D单独对两种类型细胞的活力无影响。TUNEL法检测细胞凋亡。然而，当细胞暴露于TNF-α加Act-D时，细胞的活力是SMP 30转染子比模拟转染子高3倍。三氟拉嗪（一种CaM抑制剂）的存在以剂量依赖性方式（1-100 μM）显著减弱了两种类型细胞之间的活力差异。此外，在单独的实验中，当简单地从培养基中除去血清时，两种类型的细胞的细胞活力都降低，但在SMP 30转染子中的细胞活力显著高于模拟转染子。DNA合成在两种类型的细胞之间没有差异。结论：如前所述，转染SMP 30可保护Hep G2细胞免受TNF-α和Act-D以及人工Ca^2+超载诱导的凋亡。SMP 30的这种作用也被CaM抑制剂三氟拉嗪抑制，表明SMP 30的保护作用依赖于CaM。此外，SMP 30的转染减弱了去除生长刺激诱导的细胞凋亡，而DNA合成不受影响。这些结果表明，SMP 30可能作为一种新的肝细胞存活因子，因此，年龄相关的SMP 30的减少有助于增加老年肝脏对损伤的易感性。少

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 … More , 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