Wang M, Zhao X R, Wang P, Li L, Dai Y, Huang H, Lei P, Zhu H F, Shen G X
Department of Immunology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China.
Int J Biochem Cell Biol. 2007;39(11):2076-82. doi: 10.1016/j.biocel.2007.05.022. Epub 2007 Jun 8.
Endoplasmic reticulum stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cell, and contributes to the development of type 1 diabetes. The chaperone molecule, glucose regulated proteins 78 (GRP78), is required to maintain ER function during toxic insults. In this study, we investigated the effect of GRP78 on the beta-cell apoptosis. We first measured GRP78 protein expression in different phase of streptozotocin-affected beta-cell by immunoblotting analysis. An insulinoma cell line, NIT-1, transfected with GRP78 was established, named NIT-GRP78, and used to study apoptosis, which was induced by streptozotocin or inflammatory cytokines. Apoptosis of NIT-1 or NIT-GRP78 cells was detected by flow cytometry, the transcription of C/EBP homologous protein (CHOP) was monitored by real-time PCR, the concentration of nitric oxide and the activity of superoxide dismutase were measured by colorimetric method. We found that, in comparison to NIT-1 cells, NIT-GRP78 cells responded to the streptozotocin or cytokines treatments with decreased concentration of nitric oxide, but increased activity of superoxide dismutase. In addition, the level of CHOP was also decreased in the NIT-GRP78 cells, which may mediate the resistance of the GRP78 overexpressed NIT-1 cells from apoptosis. Finally, we found that NIT-GRP78 cells were also more resistant than NIT-1 cells to cytotoxic T lymphocyte (CTL) specific killing detected by flow cytometry through target cells expressing green fluorescent protein cultured with effector cells and finally stained with propidium iodide. The data suggest that modulating GRP78 expression could be useful in preventing pancreatic beta-cell from the immunological destruction in type 1 diabetes individuals.
内质网应激介导的细胞凋亡在胰腺β细胞破坏中起重要作用，并促进1型糖尿病的发展。伴侣分子葡萄糖调节蛋白78（GRP78）在毒性损伤期间维持内质网功能是必需的。在本研究中，我们研究了GRP78对β细胞凋亡的影响。我们首先通过免疫印迹分析测量链脲佐菌素影响的β细胞不同阶段的GRP78蛋白表达。建立了用GRP78转染的胰岛素瘤细胞系NIT-1，命名为NIT-GRP78，并用于研究由链脲佐菌素或炎性细胞因子诱导的细胞凋亡。通过流式细胞术检测NIT-1或NIT-GRP78细胞的凋亡，通过实时PCR监测C/EBP同源蛋白（CHOP）的转录，通过比色法测量一氧化氮浓度和超氧化物歧化酶活性。我们发现，与NIT-1细胞相比，NIT-GRP78细胞对链脲佐菌素或细胞因子处理的反应是一氧化氮浓度降低，但超氧化物歧化酶活性增加。此外，NIT-GRP78细胞中CHOP水平也降低，这可能介导了过表达GRP78的NIT-1细胞对凋亡的抗性。最后，我们发现NIT-GRP78细胞也比NIT-1细胞对细胞毒性T淋巴细胞（CTL）特异性杀伤更具抗性，通过与效应细胞共培养并最终用碘化丙啶染色的表达绿色荧光蛋白的靶细胞进行流式细胞术检测。数据表明，调节GRP78表达可能有助于预防1型糖尿病个体的胰腺β细胞受到免疫破坏。
