Larsen C M, Døssing M G, Papa S, Franzoso G, Billestrup N, Mandrup-Poulsen T
Steno Diabetes Center, Niels Steensens Vej 2, 2820, Gentofte, Denmark.
Diabetologia. 2006 May;49(5):980-9. doi: 10.1007/s00125-006-0164-0. Epub 2006 Mar 10.
AIMS/HYPOTHESIS: IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1beta-induced beta cell MAPK signalling and apoptosis.
Rat insulinoma INS-1E cells and mouse beta-TC3 cells stably expressing Gadd45b were generated. The effects of Gadd45b expression on signalling by JNK, ERK and p38 were assessed by Western blotting and kinase assays. Apoptosis rate was measured by terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling (TUNEL) and an ELISA designed to detect apoptotic nucleosomes. Expression of endogenous Gadd45b mRNA was measured by RT-PCR.
In INS-1E and beta-TC3 cells, expression of Gadd45b inhibited IL-1beta-induced activation of JNK and ERK, but augmented IL-1beta-mediated p38 activity. IL-1beta-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45beta. IL-1beta-induced apoptosis was reduced by GADD45beta by up to 77%. Although IL-1beta stimulated the time-dependent induction of endogenous Gadd45b in INS-1E cells and rat islets, expression levels were lower in these cells than in IL-1beta-exposed NIH-3T3 and 3DO T cells.
CONCLUSIONS/INTERPRETATION: Inadequate induction of Gadd45b, which encodes a novel beta cell JNK and ERK inhibitor, may in part explain the pro-apoptotic response of beta cells to IL-1beta.
目的/假设：白细胞介素-1β（IL-1β）是凋亡性β细胞破坏的候选介质，这一过程会导致1型糖尿病和2型糖尿病的进展。IL-1β激活β细胞c-Jun氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）和p38，它们均为丝裂原活化蛋白激酶（MAPK）家族成员。抑制JNK可防止IL-1β介导的β细胞破坏。在小鼠胚胎成纤维细胞和3DO T细胞中，编码生长停滞和DNA损伤诱导蛋白45β（Gadd45b）的基因过表达可下调促凋亡JNK信号通路。本研究的目的是探讨Gadd45b是否能预防IL-1β诱导的β细胞MAPK信号通路激活和细胞凋亡。
构建了稳定表达Gadd45b的大鼠胰岛素瘤INS-1E细胞和小鼠β-TC3细胞。通过蛋白质免疫印迹法和激酶分析评估Gadd45b表达对JNK、ERK和p38信号传导的影响。采用末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记法（TUNEL）和一种用于检测凋亡核小体的酶联免疫吸附测定法（ELISA）测量细胞凋亡率。通过逆转录聚合酶链反应（RT-PCR）检测内源性Gadd45b mRNA的表达。
在INS-1E细胞和β-TC3细胞中，Gadd45b的表达抑制了IL-1β诱导的JNK和ERK激活，但增强了IL-1β介导的p38活性。GADD45β降低了IL-1β诱导的一氧化氮生成以及胰岛素含量和分泌的减少。GADD45β使IL-1β诱导的细胞凋亡减少了77%。尽管IL-1β刺激了INS-1E细胞和大鼠胰岛中内源性Gadd45b的时间依赖性诱导，但这些细胞中的表达水平低于暴露于IL-1β的NIH-3T3细胞和3DO T细胞。
结论/解读：编码新型β细胞JNK和ERK抑制剂的Gadd45b诱导不足可能部分解释了β细胞对IL-1β的促凋亡反应。
